Diabetes mellitus (DM), a metabolic disorder is one of the most prevalent chronic diseases worldwide across developed as well as developing nations. Hyperglycemia is the core feature of the type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM), following insulin deficiency and impaired insulin secretion or sensitivity leads insulin resistance (IR), respectively. Genetic and environmental factors attributed to the pathogenesis of DM and various therapeutic strategies are available for the prevention and treatment of T2DM. Among the numerous therapeutic approaches, the health effects of dietary/nutraceutical approach due to the presence of bioactive constituents, popularly termed phytochemicals are receiving special interest for pharmacological effects and therapeutic benefits. The phytochemicals classes, in particular sesquiterpenes received attention because of potent antioxidant, anti-inflammatory, and antihyperglycemic effects and health benefits mediating modulation of enzymes, receptors, and signaling pathways deranged in DM and its complications. One of the terpene compounds, β-caryophyllene (BCP), received enormous attention because of its abundant occurrence, non-psychoactive nature, and dietary availability through consumption of edible plants including spices. BCP exhibit selective full agonism on cannabinoid receptor type 2 (CB2R), an important component of endocannabinoid system, and plays a role in glucose and lipid metabolism and represents the newest drug target for chronic inflammatory diseases. BCP also showed agonist action on peroxisome proliferated activated receptor subtypes, PPAR-α and PPAR-γ, the main target of currently used fibrates and imidazolidinones for dyslipidemia and IR, respectively. Many studies demonstrated its antioxidant, anti-inflammatory, organoprotective, and antihyperglycemic properties. In the present review, the plausible therapeutic potential of BCP in diabetes and associated complications has been comprehensively elaborated based on experimental and a few clinical studies available. Further, the pharmacological and molecular mechanisms of BCP in diabetes and its complications have been represented using synoptic tables and schemes. Given the safe status, abundant natural occurrence, oral bioavailability, dietary use and pleiotropic properties modulating receptors and enzymes, BCP appears as a promising molecule for diabetes and its complications. Diabetes mellitus (DM) is the most prevalent metabolic disorder and continuing to affect a large number of people worldwide. The International Diabetic Federation reported that the prevalence of diabetes is increasing worldwide to epidemic proportions; in 2017, 425 million people were affected by DM and the number is expected to rise to 629 million in 2045 . The exaGlycemic control is one of the main aims of treatment in the management of DM and to delay the onset and progression of diabetic complications ,6. ThougMounting evidence indicate that various fruits, vegetables, and whole grains including fermented products, legumes, nuts, green tea, spices, propolis, olive oil, red wine (resveratrol), herbs, and spices are beneficial in T2DM, obesity, and metabolic syndrome due to the presence of non-nutritive secondary metabolites termed as phytochemicals ,16,17,18Z)-β-BCP also known as isocaryophyllene; a cis-double bond isomer or α-humulene, also known as α-caryophyllene, a ring-opened isomer or in a mixture with β-caryophyllene oxide, an oxidation product of BCP. The cyclobutene ring in the chemical structure of BCP imparts chemical stability and high lipophilicity [Among numerous phytochemical classes, specifically, terpenes commonly consumed in plants including edible and medicinal garnered attention because of their potent bioactivities, safety, druggable properties, and potential to target enzymes and receptors that influence glucose and lipid metabolism . In sesqhilicity . It recehilicity . Sharma hilicity .Gertsch and colleague for the first time demonstrated a selective and full agonist activity of BCP on cannabinoid type 2 receptors (CB2R), a component of the endocannabinoid system consisting of endocannabinoid ligands that exert their action by binding to two well-characterized cannabinoid receptors type 1 and type 2 (CB1R and CB2R) or endocannabinoid metabolizing enzymes, fatty acid amide hydrolase (FAAH), and monoacylglycerol lipase (MAGL) . BCP shoRecently, numerous experimental studies revealed that activation of CB2R by agonists have significant pharmacological effects such as anti-inflammatory, immunomodulatory, antioxidant, cardioprotective, hepatoprotective, gastroprotective, neuroprotective, nephroprotective, and atheroprotective and found to be beneficial in controlling hyperglycemia, IR, and dyslipidemia ,26. In tBCP is a molecule of special interest with high presence in cannabis as well as the non-cannabis plants including more than 2000 plants and termed as a dietary cannabinoid ,23. BCP In the past few years, the number of studies demonstrated that BCP possesses a wide range of pharmacological properties including anti-inflammatory and antioxidant and the potential to correct hyperglycemia and improve insulin secretion and sensitivity, thus appearing as a promising molecule for diabetes and its complications . Given tConvincing number of preclinical studies and few clinical studies demonstrated its pharmacological effects and molecular mechanisms, redox and immune-inflammatory axis, and its beneficial effects in low grade chronic inflammatory diseases including DM and its complications ,22,23. TPancreatic β-cells releases insulin to control the sugar levels in the blood when blood glucose concentration increases. BCP has been shown to influence dysfunctional pancreatic β-cells and insulin secretion. Suijun, et al.  have demThe trafficking and fusion of insulin secretory vesicle to the plasma membrane are regulated by coordinated signaling events. Previous studies have demonstrated that small G-proteins e.g., ADP-ribosylation factor 6 (Arf6), cell division cycle 42 (Cdc42), and Ras-related C3 botulinum toxin substrate 1 (Rac1) play an important role in regulating signaling events involved in insulin trafficking . It has In addition, Kumawat and Kaur  evaluateBasha and Sankaranarayanan  assessedBasha and Sankaranarayanan  further The effect of BCP on hyperglycemia-mediated oxidative stress and inflammation in STZ-induced DM in rats was evaluated. Oxidative stress is a common hallmark for the crucial pathways involved in the development and progression of DM . DysfuncA pivotal step in the pathogenesis of T2DM is the amplified hepatic gluconeogenesis, which is regulated mainly by gluconeogenic enzymes including G6pase, F1, 6Bpase, PEPCK, and PC. In diabetic rats, enhanced activities of gluconeogenic enzymes were detected in hepatic and peripheral tissues. In the diabetic conditions, insulin deficiency augments the activities of gluconeogenic enzymes that results in increased production of endogenous glucose . GlycogeIn a previous preclinical study Kumawat and Kaur , evaluatCopaifera duckei extract has been attributed to attenuate hyperglycemia and hypercholesteremia in STZ-induced diabetic rats [Cinnamomum tamala (Indian bay leaf or Indian cassia) has been screened in an in silico reverse screening approach to characterize the therapeutic targets of its components against targets involved in chronic disease treatment [Cinnamomum tamala oil attributed to exhibit antidiabetic, antioxidant, and hypolipidemic activity in STZ-induced DM in rats [BCP present in tic rats . It alsoreatment . BCP was in rats .Eugenia punicifolia has been given as adjuvant for three months in T2DM [In a non-controlled pilot clinical study, powdered dried leaves of  in T2DM . The extOverall, these in vivo studies summarized in Zheng et al.  demonstrAmong the three different isoforms, PPAR-α, mainly expressed in heart, liver, intestine, and macrophages is activated by polyunsaturated fatty acids and leukotrienes. Whereas PPAR-γ, mainly expressed in adipocytes is activated by polyunsaturated fatty acids and 15d-prostaglandin J2 and plays a vital role in adipocyte differentiation and lipid accumulation. Functionally, the PPAR isoforms resembles to those of the steroid receptors and linked to multiple functions initiated by nutrients, nutraceuticals, and phytochemicals. Ligand-binding to CB1R and CB2R enhances the activity of MAPK that further regulates the activation of PPARs through direct phosphorylation and PPAR activation provides a basis by which cannabinoids like BCP can regulate gene transcription triggered by dietary intervention. Several studies provide evidence that cannabinoids exert their anti-inflammatory properties, at least in part by activation of PPAR-α and PPAR-γ pathway ,66.Cannabinoids are known to interact or crosstalk with a family of peroxisome proliferated activator receptors (PPARs) including three distinct subtypes namely PPAR-α, PPAR-β/δ, and PPAR-γ that are encoded by distinct genes and are regulated by steroids and lipid metabolites and control lipid and glucose homeostasis and inflammatory responses ,65,67,6850 value of 3.2 μM [The activation of CB2R have shown to directly interact and activate PPAR-α and PPAR-γ ,59,71. Bf 3.2 μM . The clif 3.2 μM . BCP hasf 3.2 μM . SubsequIn regards to its pharmacokinetic profile, BCP is highly lipophilic, possessing hydrophobic core in its structure, and a relatively small molecular weight of 204 g/moL compared with other conventional ligands targeting the PPAR isoforms. Moreover, the relatively small molecular weight, lipophilicity, and the absence of adverse effects, e.g., edema, osteoporosis, obesity, associated with synthetic ligands of PPARs are the advantages of BCP over conventional agonists of PPAR-γ which are clinically available for therapeutic management of DM. Additionally, BCP is a highly lipophilic compound, which renders it permeable enough to cross the cell membrane and showed its potential benefits and pharmacological actions by regulating the intracellular pathways involved in inflammation. However, the exact mechanism of this interaction remains elusive and this interaction needs to be explored for further exploration in therapeutic targeting.Interestingly, BCP by activating CB2R as well as both of the PPAR isoforms (PPAR-α and PPAR-γ) offers a promising polypharmacological multitargeted approach for its pharmaceutical development and clinical application. Many studies demonstrated that dual agonist action on PPAR-α and PPAR-α are superior for metabolic control and dyslipidemia ,72. ConsNotably, in the majority of the experimental models involving chronic low grade inflammatory states similar to DM the principal pharmacological and molecular mechanism observed with BCP treatment is an improvement of immune dysregulation, inhibition of pro-inflammatory cytokines such as TNF-α, IL-1β, IL-6, NF-κβ, adhesion molecules and chemokines and subsequent modulation of signaling pathways mainly, toll-like receptors, opioid receptors, SIRT1/PGC-1α, AMPK/CREB, MAPK/ERK, Nrf2/Keap1/HO-1, and activation of nuclear peroxisome proliferator-activated receptors  involving CB2R-dependent mechanism .Further, BCP treatment to differentiated C2C12 myotubes stimulated SIRT1 deacetylase activity by increasing the phosphorylation of CREB, thus leading to enhanced deacetylation of PGC-1α . BCP alsA high-calorie diet is usually associated with obesity and IR, which predispose more diseases that are as serious as diabetes, cardiovascular diseases, and increase the risk of different neurological disorders. As claimed by various studies, diet-induced obese animals show higher anxiety and depressive-like behaviors . Many meIn another study, Youssef et al.  showed tIn addition, it appears that BCP being PPAR-γ agonist leads to the reduction in fat mass and triglycerides and increases in high-density lipoprotein (HDL) . These eThe down-regulation of BDNF was implicated in the pathology of neurodegenerative diseases and many inflammatory conditions, such as acute coronary syndrome and T2DM . It was Kumawat and Kaur  in an exInsulin signal transduction in normal physiological conditions involves complex sequential steps including many enzymes and mediators, resulting in facilitated glucose entry into the adipocytes, muscles, and myocardial cells via GLUT-4 (glucose transporter-4) transporters . The insThere are several types of insulin-dependent kinases such as ERK1/2 , PKC (protein kinase C), S6K1 , SIK2 (serine/- threonine-protein kinase 2), Akt (protein kinase B), mTOR , and ROCK1 (rho-associated protein kinase 1) and other types of kinases such as AMPK (AMP-activated protein kinase) and GSK-3 (glycogen synthase kinase 3) can phosphorylate and activate IRSs . After tBCP was also found to regulate the expression of several cytokines e.g., TNF-α, IL-1β, IFN-γ, IL-4, IL-5, IL-6, and IL-10, chemokines e.g., CINC-1/CXCL1, CXCL1/KC, MIP-2, MCP-1, growth factors e.g., vascular endothelial growth factor (VEGF), transforming growth factor (TGF), fibroblast growth factor (FGF), brain-derived neurotrophic factor (BDNF), enzymes e.g., acetylcholinesterase (AChE), butyrylcholinesterase (BChE), nitric oxide synthase (iNOS), NADPH oxidase (NOX), fatty acid transport protein (FATP4), ACC synthase (ACS), carnitine palmitoyltransferase 1 (CPT1), peroxisomal acyl-coenzyme A oxidase 1 (ACOX), cyclooxygenase-2 (COX-2), lipoxygenase (LOX), matrix metalloproteinase-9 (MMP9), myeloperoxidase (MPO), N-acetyl glutamate (NAG), mitogen-activated protein kinases (MAPK), signaling molecules , adhesion molecules e.g., ICAM-1 and VCAM-1, apoptosis-related proteins e.g., Bcl-2, mdm2, cmyb, bax, bak1, Apaf-1, caspases, p53, p38, ATM, DR and Fas, proteins; cell cycle proteins e.g., cyclin D1, g-proteins e.g., Arf6, Rac1, Cdc42, heat shock proteins 60, and P-gP, genes e.g., Col1a1, Tgfb1, Timp1, SIR-2.1, SKN-1, DAF-16, mev-1, daf-16, SREBP-1c and SCD1 and receptors; CB2, μ-opioid, PPAR-α, PPAR-γ and α-nAchR. Additionally, BCP also alters the activity of several transcription factors and cell-signaling proteins  and their signaling pathways . The the®-FL), containing high content of BCP have been assessed on glucose uptake and GLUT4 membrane translocation in C2C12 skeletal myotubes. PipeNig®-FL treatment to C2C12 myotubes stimulated cellular uptake of glucose and induced translocation of GLUT4. The effect of BCP rich extract in stimulating glucose uptake is due to improvement in GLUT4 trafficking to the plasma membrane. Consequently, GLUT4 storage vesicles translocation to the plasma membrane, largely in skeletal muscle and adipocyte, is directly associated with the ability to reduce raised blood glucose level [The metabolic effects of black pepper extract  in intestine are essential for carbohydrate digestion and involved in converting oligosaccharides and disaccharides to corresponding monosaccharides, which are then absorbed . After aRhaponticum acaule (L.) DC [Additionally, many plants such as Afromomum melegueta and Afromomum danielli , Phlomis (L.) DC , Acacia  (L.) DC  and Neol (L.) DC  and Senn (L.) DC  containi (L.) DC . These p (L.) DC .R,5S)-configuration products. The synthesized compounds were tested for their activity on α-glucosidase. Three derivatives with the molecular formula  = 413.1883), , and  formed potent inhibition of α-glucosidase and provided novel scaffolds for lead molecules in drug discovery for diabetes with promising inhibition (IC50 < 10 μM). The improvement inhibitory activities were due to the formation of a flexible connection between BCP and 4-hydroxycoumarin, and the presence of hydroxyl group which facilitates the binding. Linking rearranged BCP with aromatic ketones via an oxygen bridge generated compounds with better activity than acarbose, a standard drug. The presence of the para-substituted carbonyls and the hydrophobic moiety of BCP contributed to the formation of the compounds with higher inhibition [Recently, the scaffolds of BCP and 4-hydroxycourmarin were used as a starting compound for the synthesis of novel scaffolds with better stability and improved bioactivities for α-glucosidase inhibitory activity . Accordihibition .In another study, the β-lactam motif of BCP following remodeling into different types of compounds and one of the derivatives showed potent inhibitory activity against α-glucosidase and it was found superior to the standard drug acarbose . FurtherAfromomum melegueta and Afromomum danielli [Phlomis armeniaca WILLD., Phlomis nissolii L., and Phlomis pungens WILLD. var. pungens [Senna podocarpa (Guill. and Perr.) [Citrus macroptera Montr fruits containing BCP as one of the major ingredient showed α-amylase inhibitory activity and hypoglycemic activity in normal and glucose-induced hyperglycemic rats [Further, BCP has been attributed to exert inhibitory activity on pancreatic α-amylase, facilitates carbohydrate metabolism by interrupting α-1,4-glycosidic bonds to form oligosaccharides, that convert in to glucose . Thus indanielli , Phlomis pungens , and Send Perr.)  containimic rats .Ferulago nodosa (L.) Boiss. [Rhaponticum acaule (L.) DC [Artemisia annua [Ocimum kilimandscharicum [Pinus massoniana L. [Pancreatic lipase, an enzyme, plays a significant role in the metabolism of dietary fat, thus pharmacological inhibitors of lipase have a therapeutic effect on obesity, protective effect in diabetes due to reduced absorption of lipids and restoration of insulin along with correction of lipid profile. BCP present in numerous plants such as ) Boiss. , Rhapont (L.) DC , Artemisia annua , Ocimum charicum , Pinus mniana L.  has beenniana L. . HoweverSalvia amarissima Ortega [Blumea balsamifera (L.) DC has been attributed to exhibit inhibitory activity PTP1B [The effect of BCP has been demonstrated on another enzyme, protein tyrosine phosphatase 1B (PTP1B) found in important insulin-targeted tissues such as liver, muscle, and fat and play a regulatory role in insulin signal transduction, by dephosphorylating activated insulin receptors or insulin receptor substrates. Excessive PTP1B activities impairs insulin sensitivity and signal transduction, thus participates in T2DM and regulate energy in brain mediating leptin receptors and contribute to obesity . The inha Ortega , Blumea ty PTP1B . The higThe available data indicate that BCP and its derivatives have the potential to inhibit the activity of α-glucosidase, α-amylase, lipase, and PTP1B that is suggestive of its underlying mechanisms of the usefulness of BCP in diabetes. BCP appears to be more potent than acarbose that further demonstrates its use as an adjuvant with other agents in T2DM and obesity. BCP and congeners could be developed as potent antihyperglycemic agents particularly to be useful in T2DM and obesity due to polypharmacological properties on fat and carbohydrate-metabolizing enzymes. The mechanism of the antihyperglycemic action of BCP involves increasing glucose utilization and uptake, affecting glucose metabolism, and increasing insulin secretion are represented in The complications of both T1DM and T2DM constitute retinopathy, nephropathy, neuropathy, cardiomyopathy, and other associated diseases which affect the quality of life and are a main reason for morbidity and mortality ,26. DiabκB signaling pathway and inhibiting release of pro-inflammatory cytokines production and promotes polarization of M1/M2 phenotypic anti-inflammatory properties [In several diseases involving immune dysregulation including obesity, DM and its complications, the activation of toll-like receptors (TLRs), particularly toll-like receptor 4 (TLR4), among many isoforms showed to garner great therapeutic interest, thus its pharmacological manipulation received interest in recent years . TLR4 geoperties ,111.α enhances oxidative stress, inflammation, and cell death that leads to tissue damage in diabetic complications. The inhibitory effects of BCP on TLR4/RAGE, HMGB1-TLR4, NF-kB, PI3K/Akt, AMPK/CREB, ERK1/2/JNK1/2, SIRT1/PGC-1α, TLR4/NF-kB pathways are suggestive of its pharmacological and molecular mechanism of its potential usefulness in DM and its complications. The molecular targets of BCP are represented in Additionally, BCP has been shown to attenuate neuroinflammation and neuron death by mitigating high mobility group box 1 (HMGB1)-TLR4 signaling pathways . HMGB1 fNeuropathic pain is considered a diabetes consequence and presents a high impact on patient’s quality of life, showing painful thermal, electrical, and sharp sensations ,113. DiaA dietary supplement containing BCP, myrrh, carnosic acid, and PEA evaluated in twenty-five patients with the diagnosis of DM and painful distal symmetric polyneuropathy (PDSPN) showed a reduction of polyneuropathy with enhanced amplitude and reduced pain . PDSPN iBCP role in diabetic nephropathy, a major cause of end-stage renal disease in diabetic patients involving structural and functional alteration in podocytes has been demonstrated using an in vitro model of high glucose-induced glomerular mesangial cells . BCP treAdditionally, hyperglycemia a common manifestation in T2DM is also related with a higher risk of developing colorectal cancer evidenced by increased arginine-specific mono-ADP-ribosyltransferase 1 (ART1) levels in colorectal tissues from patients with T2DM compared to non-diabetic patients. The role of BCP on ART1 on glycolysis and energy metabolism has been shown in CT26 cells cultured and exposed to high-glucose conditions and in STZ-induced BALB/c mice transplanted CT26 tumor cell . BCP treCaenorhabditis elegans [Caenorhabditis elegans. BCP enhanced the expression level of stress and longevity-promoting genes such as daf-16, skn-1, sod-2, hsp-70, gst-4, gst-7, sir2.1, and sod-3, the anti-oxidative enzymes induced in response to oxidative stress. Ageing as well as diabetes also enhances cognitive decline. BCP showed to enhance lifespan, memory performance in young as well as aged mice mediating potent anti-inflammatory properties on pro-inflammatory cytokine [Kaempferia parviflora rhizome extracts based on its positive effects on swimming tests of mice [BCP showed potential in ageing and age-related disorders by improving the mean lifespan, reduced feeding behavior, induced dietary restriction like effects, and enhanced longevity-promoting stress resistance ability in  elegans . BCP alscytokine . BCP has of mice .Along with ageing, diabetes and obesity are the risk factors for osteoporosis characterized by reduced bone mass through decreased osteoblastic osteogenesis and increased osteoclastic bone resorption. BCP was found to enhance osteoblastic mineralization and suppress adipogenesis and osteoclastogenesis in cultured mouse bone marrow cells . ObesityArtemisia montana Pampan essential oil containing BCP has been shown to promote skin regeneration in human keratinocytes and wound healing by increasing phosphorylation of Akt and ERK 1/2 and induced the synthesis of type IV collagen [In diabetic patients, increased incidence of impaired wound healing severely affects the quality of life leading to prolonged hospitalization and lower limb amputation . Numeroucollagen . In anotcollagen .® oil). Essential oils of many other plants such as Eugenia dysenterica DC leaves containing BCP was found to enhance in vitro skin cell migration and promoted angiogenesis with no cytotoxicity [Aspilia africana (Pers.) C.D. Adams rich in BCP showed wound healing ability by reducing wound bleeding, enhancing wound contraction, increasing concentration of basic fibroblast growth factor and platelet-derived growth factor along with increasing white and red blood cells [BCP alone is an unstable compound as it oxidizes rapidly and is unable to permeate but the nano-emulsion formulation was found to exhibit improved stability for 60 days, superior bio adhesiveness and facilitated skin permeation than BCP alone. The nano-emulsion was found to exert a healing effect in the dorsal wound model by reducing lesions, oxidative stress, and inflammation comparable to a standard cutaneous healing agent , popularly known as Indian camphorweed used as a tea and health tonic in Southeast Asia was effective in mitigating hyperglycemia and dyslipidemia induced by high fat diet in mice. It showed to attenuate hyperglycemia and dyslipidemia by correcting altered lipid and hematological profile, restoring liver enzymes and renal function, and improving oral glucose tolerance along with histological salvage of kidney and liver. The benefits of this plant were ascribed to the high content of BCP and advocated to be dietary supplement/nutraceutical for hyperlipidemia, obesity, and T2DM.Sirichaiwetchakoon et al.  reportedBaldissera, et al.  investigHarb, et al.  have exa2+ signaling pathway. The studies are suggestive of that BCP could be helpful in preventing and ameliorating fatty liver diseases including NAFLD.In recent years, AMPK activation appears promising as a therapeutic target for the prevention and treatment of obesity, DM, and steatosis . AMPK, aArizuka, et al.  have invRecently, BCP structure was utilized as a template for drug discovery . FollowiThe alcoholic extract of clove containing BCP showed to inhibit fatty acid synthase, an enzyme for de novo lipogenesis, inhibited weight gain, abdominal adipose tissue, lowered lipid accumulation in the liver by regulating the content of total triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), and epididymal adipose tissue in HFD-induced obesity . The ext2+ signaling pathway leading to the downregulation of FAS and upregulation of ATGL. Additionally, BCP could be a promising therapeutic drug in treating hypercholesterolemia and fatty liver disease.Altogether, these studies indicate that BCP has an antioxidant activity leading to the reduction of ROS, therefore, may be associated with deactivation of HMG-CoA reductase and decreased cholesterol synthesis, leading to hypolipidemia are represented in 50 in rats were found greater than 5000 mg/kg [BCP is classified as a substance of category five (toxic at doses greater than 2000 mg/kg) in accordance with OECD (Organization for Economic Co-operation and Development) guideline 423 . BCP adm00 mg/kg . BCP or 00 mg/kg . Also, B00 mg/kg , and fou00 mg/kg . In chro00 mg/kg .In rodents, doses ranging from 20 to 300 mg/kg have been used to study pharmacological effects in drugs, xenobiotics or chemical toxicants-induced animal models of the heart ,154, liv50 values of BCP on midazolam 1′-hydroxylation in HLM was 1.28 mM. This study indicated the potential of BCP drug interaction with other drugs metabolized by CYP3A4. BCP inhibited CYP3A activities in rats as well as in human hepatic microsomes rat and human hepatic subcellular fractions [BCP  was found antigenotoxic in benzo(a)pyrene-induced toxicity in mice by inhibiting the number of sister-chromatid exchanges and chromosomal aberrations . The antractions . Thereforactions  and founractions . Based oOverall, the data from preclinical studies demonstrate the underlying mechanisms of BCP particularly in skeletal muscles, adipose tissues, liver, and pancreatic β-cells. The available studies demonstrate that BCP shows the capability to increase insulin secretion, insulin sensitivity, glucose uptake and reduce glucose absorption. In addition, it also reduces the levels of triglycerides and cholesterol, increases fatty acid oxidation and lipid homeostasis along with hypolipidemic effects. Moreover, BCP has insulinotropic effects mediating CB2R- and PPARs-dependent mechanism. BCP is one of the important components of essentials oils of many plants used in diet and traditional medicines for their well-being and health benefits. BCP is unique due to its selective full agonist property on CB2Rs as well as modulating other receptors and enzymes play significant role in glucose and lipid metabolism. However, further studies are required to translate these mechanistic studies into therapeutic benefits in humans.Based on the health benefits, wide natural occurrence, dietary availability, low toxicity, relatively safe in humans use, and organoprotective properties with a plausible pharmacological affinity, selectivity and potency on different receptors and enzyme and molecular mechanisms targeting signaling pathways involved in glucose and lipid homeostasis, BCP appears to be a promising candidate for use in IR, T2DM, obesity, hyperlipidemia, and diabetic complications. The available literature reveals that BCP can be used as an adjuvant and reduce the doses of the currently used drugs and minimize their adverse effect and synergistically enhance therapeutic effects. Though, in perspective of pharmaceutical and nutraceutical development more pharmacokinetic and regulatory toxicity studies are required to ascertain the safety and efficacy for use in diabetes and associated complications.


NLRP1 cause inflammatory skin syndromes and a predisposition for the development of skin cancer. SNPs (single nucleotide polymorphisms) of NLRP1 are associated with several (auto)inflammatory diseases with a major skin phenotype, such as psoriasis or vitiligo. Here, we summarize knowledge about NLRP1 with emphasis on its role in human keratinocytes and skin. Due to its accessibility, pharmacological targeting of NLRP1 activation in epidermal keratinocytes represents a promising strategy for the treatment of the numerous patients suffering from NLRP1-dependent inflammatory skin conditions and cancer.Inflammasomes represent a group of protein complexes that contribute to host defense against pathogens and repair processes upon the induction of inflammation. However, aberrant and chronic inflammasome activation underlies the pathology of numerous common inflammatory diseases. Inflammasome assembly causes activation of the protease caspase-1 which in turn activates proinflammatory cytokines and induces a lytic type of cell death termed pyroptosis. Although NLRP1  was the first inflammasome sensor, described almost 20 years ago, the molecular mechanisms underlying its activation and the resulting downstream events are incompletely understood. This is partially a consequence of the poor conservation of the NLRP1 pathway between human and mice. Moreover, recent evidence demonstrates a complex and multi-stage mechanism of NLRP1 inflammasome activation. In contrast to other inflammasome sensors, NLRP1 possesses protease activity required for proteolytic self-cleavage and activation mediated by the function-to-find domain (FIIND). CARD8 is a second FIIND protein and is expressed in humans but not in mice. In immune cells and AML (acute myeloid leukemia) cells, the anti-cancer drug talabostat induces CARD8 activation and causes caspase-1-dependent pyroptosis. In contrast, in human keratinocytes talabostat induces NLRP1 activation and massive proinflammatory cytokine activation. NLRP1 is regarded as the principal inflammasome sensor in human keratinocytes and UVB radiation induces its activation, which is believed to underlie the induction of sunburn. Moreover, gain-of-function mutations of  Skin represents the essential outer barrier of the human body and is in permanent contact with the environment . Its outIn the skin, but also in other tissues, the detection of stress factors can mount a highly complex response, termed inflammation . InflammNOD-like receptors (NLRs) form oligomers. These complexes, termed inflammasomes, cause a series of downstream events and ultimately inducing inflammation ,7650,76]poptosis , and as tivation . In conttivation . Splicintivation . The 55 tivation . Interestivation . It diretivation ,83,84. Ctivation . Mutatedtivation . Periphetivation . CARD8 itivation .Although talabostat induces CARD8-dependent pyroptosis in human AML cells, such as THP-1 cells, and NLRP1b-dependent pyroptosis in murine immune cells ,76, humaDeath Effector Filament-forming Ced-4-like Apoptosis Protein) [nucleotide-binding domain and CARD) [In 2001, NLRP1 was identified as a novel Ced-4 family member and termed DEFCAP (Protein)  or NAC (nd CARD) . The 166nd CARD) . DEFCAP-nd CARD) . The funnd CARD)  induces nd CARD) . Furthernd CARD) . NLRP1 ind CARD)  and, intnd CARD) .Toxoplasma gondii in monocytic cells [2+ and reactive oxygen species (ROS) are likely contributing factors [In addition to NLRP1, human primary keratinocytes (HPKs) express other inflammasome components such as ASC and caspase-1, as well as proIL-1β and -18 . Most imic cells , UVB radic cells ,105. Howic cells . Most liic cells . Moreoveic cells ,107. The factors ,99.Upon treatment of HPKs with IFNγ or TNFα (priming), expression of other inflammasome sensors is induced ,108. NexAlthough caspase-1 expression is required for the induction of UVB-induced skin inflammation in mice , UVB irrNLRP1 have been described in patients who suffer from different inflammatory diseases with a skin phenotype, demonstrating that NLRP1 plays a particularly important role in the skin  and FKLC  are two rare monogenic inflammatory skin diseases, which are caused by gain-of-function (GoF) mutations of NLRP1 . The mutNLRP1 . KeratinNLRP1 ,111. MosNLRP1 . FurtherNLRP1 . Althouginocytes ,106), doinocytes . MSPC prinocytes , a majorinocytes ,116,117.inocytes ,119. In inocytes ,116,117.elopment , is suppelopment  supportsNLRP1 occurring either between the NACHT and LRRs or within the FIIND, close to the autoprocessing site [Recently, a novel autoimmune-inflammatory disease termed NAIAD (NLRP1-associated autoinflammation with arthritis and dyskeratosis) with a clear skin phenotype has been described. It is caused by mutations of ing site . The serNLRP1 are associated with an increased risk for the development of several diseases with partial involvement of the skin [NLRP1 gene were found to be associated with vitiligo [NLRP1 gene [Furthermore, several other SNPs of the skin . Vitiligthe skin . Severalvitiligo ,122,123.RP1 gene ,121. Up RP1 gene . Other cRP1 gene , leprosyRP1 gene , juvenilRP1 gene , and sevRP1 gene ,127.NLRP1 cause skin inflammation via hyperactivation of the inflammasome in epidermal keratinocytes. This demonstrates a particularly important role of NLRP1 in keratinocytes and in the skin. In conclusion, NLRP1 is the only inflammasome sensor expressed by keratinocytes in vitro and in vivo in the skin under normal and homeostatic conditions . FurtherNLRP1 have mainly a skin phenotype, systemic treatment of patients suffering from NLRP1-dependent skin conditions with NLRP1 targeting molecules also might perhaps represent a possible approach with tolerable side-effects. Inhibition of NLRP1 might be effective for patients suffering from inflammatory skin conditions such as vitiligo or psoriasis. Furthermore, as the NLRP1 pathway is silenced in skin cancer [Pharmacological targeting of NLRP1 activation represents an attractive strategy for treatment of the many patients suffering from NLRP1-dependent skin conditions. Due to its accessibility, the skin is predestined for topical application of small hydrophobic molecules able to penetrate into the tissue and into epidermal keratinocytes as well as skin-resident immune cells. Moreover, as patients with GoF mutations of n cancer , pharmacn cancer , and itsn cancer .Several open points revolve around the molecular mechanisms underlying talabostat-induced inflammasome activation and particularly the cell type-specific consequences. What are the DPP8/9 substrates that regulate activation of human NLRP1, CARD8, and murine NLRP1b? Why does NLRP1 sense talabostat in keratinocytes, whereas CARD8 expression is required for caspase-1 activation in AML cells, although both cell types express NLRP1 and CARD8? Talabostat-induced CARD8 and downstream caspase-1 activation  causCARD8 is not only able to cause caspase-1 activation  but can also inhibit NLRP3 upon direct interaction ,83,84. TSince its discovery almost 20 years ago, we learned a lot about the molecular mechanisms underlying upstream and downstream pathways of NLRP1 and its physiologic roles in humans and mice. NLRP1 activation is regulated in a very complex manner, sharing certain aspects with other inflammasome sensors. In addition, NLRP1 activation is characterized by very specific features, such as proteolytic self-activation and functional degradation. In the near future, we can expect a better understanding of the mechanisms governing NLRP1 activation and its species-specific roles in the skin and beyond.


Several members of the Transient Receptor Potential (TRP) channel superfamily are expressed and mediate Ca2+-dependent functions in vascular endothelial cells and in ECFCs, the only known truly endothelial precursor. TRP Vanilloid 1 (TRPV1), a polymodal cation channel, is emerging as an important player in endothelial cell migration, proliferation, and tubulogenesis, through the integration of several chemical stimuli. Herein, we first summarize TRPV1 structure and gating mechanisms. Next, we illustrate the physiological roles of TRPV1 in vascular endothelium, focusing our attention on how endothelial TRPV1 promotes angiogenesis. In particular, we describe a recent strategy to stimulate TRPV1-mediated pro-angiogenic activity in ECFCs, in the presence of a photosensitive conjugated polymer. Taken together, these observations suggest that TRPV1 represents a useful target in the treatment of ischemic diseases.Therapeutic angiogenesis represents an emerging strategy to treat ischemic diseases by stimulating blood vessel growth to rescue local blood perfusion. Therefore, injured microvasculature may be repaired by stimulating resident endothelial cells or circulating endothelial colony forming cells (ECFCs) or by autologous cell-based therapy. Endothelial Ca V,9. V3,9]Embryonal vasculogenesis represents one of the earliest events during organogenesis, as it starts already during gastrulation ,51. VascOnce the primitive vascular labyrinth has been built up, blood vessels are finely remodeled, to define tissues structures based on metabolic need, in a hierarchical tree-like network of arteries, arterioles, capillary beds, venules, and veins ,49,55. TIt has long been thought that sprouting angiogenesis represents the most important mechanism responsible for vessel growth and remodeling in postnatal life ,9. This i-protein coupled receptor, CXCR4 mediates SDF-1α-induced endothelial motility by enrolling a wide array of intracellular signaling pathways, including PLCβ and PLCγ2, RAS-mitogen-activated protein kinase (MAPK), and PI3K-Akt-mTOR i) i) ,32,35,81igration ,75. A nu calpain ,75. VEGFgnatures ,27,32,81ogenesis ,35. For al cells , HUVECs al cells , and umbal cells , whereasal cells . Additioal cells , whereasal cells . In this2-, and COOH-tails and present a cation-permeable region formed by a loop between TM5 and TM6 i i ,122,123. neurons . TRPV1 e neurons  and brea neurons  and pros neurons ,128 canc neurons . It is, N--nonamide), a synthetized molecule deriving from capsaicin, was endothelium-dependent i, thereby inducing to Ca2+-dependent cell death . I. I2+-depand eNOS . This siogenesis ,165,166.−/− mice . Indeed,−/− mice . Finally−/− mice . Of note−/− mice . These s2+]i in Human Mammary Epithelial Cells (HMECs). The Ca2+ response to simvastatin was inhibited by two structurally unrelated TRPV1 inhibitors, such as capsazepine and SB366791; furthermore, simvastatin-induced intracellular Ca2+ signals were potentiated in HEK293 cells overexpressing a full-length TRPV1 WT, while it was inhibited by transducing the cells with TRPV1-Y671K (a TRPV1 mutant which is not able to conduct Ca2+) [2+-dependent assembly of the TRPV1/Akt/CaMKII/AMPK/eNOS complex (−/− mice) blockade of TRPV1 [2+]i that disappeared in the absence of extracellular Ca2+ and upon pharmacological blockade of TRPV1 with capsazepine or SB366791 in HMEC -enoic acid , and a recent investigation disclosed that GRP40 activation resulted in a Ca2+ response similar to that evoked by 14,15-EET i i . Erythro release , thereby−/− mice . Of note−/− mice ) and genrylation . These f−/− mice or the effect of Waon therapy in the presence of TRPV1 blockers are, however, required to assess TRPV1 involvement to the angiogenic switch observed in response to temperature increases.TRPV channels could confer temperature-sensing properties to vascular endothelial cells. For instance, TRPV4 may be activate by a temperature increase (from 19 to 38 °C) in mouse aortic endothelial cells . A more 2+ signaling in circulating and umbilical-cord-blood-derived ECFCs [2+ signals lie, indeed, at the center of an intricate network of signaling cascades that finely tune ECFC proliferation, migration, and neovessel formation [2+ oscillations and promotes angiogenesis in circulating [2+ signal [2+ signatures by inducing a concerted interplay between InsP3-induced ER Ca2+ release and SOCE [2+ response to VEGF in umbilical-cord-blood-derived ECFCs [2+ signaling could be targeted to boost the regenerative potential of autologous ECFCs for regenerative purposes [2+ entry in ECFCs [2+ response to a specific TRPV4 agonist (GSK1016790A) was not sensitive to capsazepine [2+-dependent and independent manner [A growing number of studies revealed the crucial role of Caed ECFCs ,196. Intormation ,28,32,81culating , whereas+ signal . VEGF anand SOCE ,32. Howeed ECFCs . In addied ECFCs , may alsed ECFCs . The coned ECFCs . In thised ECFCs . It has purposes ,47. It hin ECFCs ,120. Unlin ECFCs , TRPV1 isazepine . Of notet manner ,120; in t manner , thereby2+-independent manner [2+, which seemingly ruled out the involvement of intracellular Ca2+ signaling in the downstream effects of TRPV1 [Anandamide is an endogenous endocannabinoid which may confer protection against multiple cardiovascular disorders, including hypertension and ischemia/reperfusion injury . Earliert manner   theThree different photostimulation mechanisms that are active at the polymer/cell interface have been proposed so far . These i2+ entry. This caused the downstream activation of Ca2+-dependent transcriptional factor NF-κB, thus leading to the expression of multiple pro-angiogenic genes that converted the initial optical excitation into an angiogenic response (2+-dependent decoder that translates VEGF-induced intracellular Ca2+ oscillations into a pro-angiogenic output [2O2), which can permeate the plasma membrane through specific aquaporins  and thereby activate TRPV1 [Recently, conjugated polymer semiconductors have also been used to gain optical control of cell fate via TRPV1 activation . Lodola response D. Of notc output . Finallyte TRPV1 ,207,217.2+ entry. The available evidence hints at vascular endothelial cells as the final actuators of agonists-induced TRPV1-dependent Ca2+ signals. Nevertheless, the evidence that TRPV1 is also expressed and mediates angiogenesis in circulating ECFCs strongly suggests that systemic or local administration of these small molecule drugs might also stimulate vasculogenesis. In this regard, a novel approach demonstrated that optical stimulation of TRPV1 could stimulate ECFCs with unprecedented spatial and temporal resolution and with no need of viral infection. Future work is necessary to assess whether this approach is also useful to stimulate capillary endothelial cells and to design a therapeutically relevant approach. This requires, e.g., the design of a strategy to precisely deliver visible light to the injured tissue or to exploit an infrared-sensitive photosensitive conjugated polymer, as well as the production of biocompatible and photosensitive nanoparticles to be injected within ischemic organs. Additional issues that remain to be clarified to better exploit TRPV1 for regenerative purposes are the following. First, it should be assessed whether TRPV1 supports VEGF-induced endothelial Ca2+ signals in vascular districts other than bovine retina. Likewise, TRPV1 contribution to the pro-angiogenic effect of other growth factors, such as bFGF and PDGF, should be evaluated. Second, it should be evaluated whether the intracellular distribution of TRPV1 is restricted to bovine RMECs or is a common feature in vascular endothelial cells and ECFCs. If so, the contribution of this intracellular TRPV1 pool to endothelial Ca2+ signaling and its Ca2+-dependent interaction with InsP3- and/or ryanodine-receptors should be investigated. Third, future work will have to show whether pharmacological or optoceutical stimulation of TRPV1 leads to revascularization also in the ischemic brain/heart. This issue is quite relevant due to the increasing incidence of brain stroke and AMI in the aging population. Fourth, TRPV1 may assemble with another pro-angiogenic Ca2+ entry pathway, such as TRPC1 [2+ depletion. It is, however, still unclear whether this signaling complex stimulates angiogenesis, an issue that surely deserves future investigation.Therapeutic angiogenesis represents one of the most promising strategies to rescue local blood flow in ischemic organs, when pharmacological treatment is ineffective and surgical vascularization is not feasible. Manipulating the intracellular signaling pathways, which drive endothelial proliferation, migration, and neovessel formation, could be crucial to enhance the clinical outcome of regenerative medicine. Therefore, unravelling novel pro-angiogenic mediators will enrich the arsenal of signaling pathways that can be targeted for therapeutic purposes. The polymodal TRPV1 channel is emerging as a crucial regulator of angiogenesis, due to its ability to integrate multiple chemical and physical stimuli, ranging from dietary agonists  and the arachidonic acid-derivative 14,15-EET to a moderate increase in microenvironmental temperature or ROS levels. Furthermore, recent work demonstrated that some clinically available drugs, such as simvastatin or recombinant erythropoietin, could be repurposed to stimulate angiogenesis by activating TRPV1-mediated extracellular Caas TRPC1 ,218, in


H19, which has been known for more than 30 years now, is still controversially discussed. H19 was described to work either as a tumor suppressor in vitro and in vivo, or to have oncogenic features. This review attempts to survey the conflicting study results and tries to elucidate the potential reasons for the contrary findings, i.e., different methods, models, or readout parameters. This review encompasses in vitro and in vivo models as well as studies on human patient samples. Although the function of H19 in HCC remains elusive, a short outlook summarizes some ideas of using the H19 locus as a novel target for liver cancer therapy.Hepatocellular carcinoma (HCC), the most common primary liver cancer, is challenging to treat due to its typical late diagnosis, mostly at an advanced stage. Therefore, there is a particular need for research in diagnostic and prognostic biomarkers and therapeutic targets for HCC. The use of long noncoding (lnc) RNAs can widen the list of novel molecular targets improving cancer therapy. In hepatocarcinogenesis, the role of the lncRNA  H19 has been a matter of prolonged debate. The biological functions of H19 in hepatocarcinogenesis remain controversial. It has been a major challenge to interpret the data on H19 expression in HCC given the small number of samples in most of the available studies and the different experimental settings [H19, which might represent a therapeutic target in HCC.Hepatocellular carcinoma (HCC) is the sixth most prevalent cancer worldwide and the fourth most frequent cause of cancer-related deaths, with about 841,000 new cases and 782,000 deaths in 2018 . In bothsettings . TherefoOur liver can regenerate in both volume and function after chemical or physical damage . But malThe limited treatment options available range from chemoembolization therapy to systemic therapy. Sorafenib, as an inhibitor of tumor cell proliferation and angiogenesis ,12 is efH19 locus encodes various transcripts: the main transcript lncRNA H19, the encoded microRNA, miR-675, and the two antisense transcripts 91H and H19 opposite tumor suppressor (HOTS) [The r (HOTS)   conservation between mouse and human, but a high overall DNA sequence identity [H19 was first examined during development in the mouse [IGF2) gene. Both genes share the same enhancer elements, which bind and thereby activate the H19 promotor [pression ,15,16. Hidentity . The genidentity . The exphe mouse . It is hpromotor .H19 underlies the genomic imprinting: Under imprinted conditions, H19 is expressed from the maternal allele and IGF2 from the paternal allele [IGF2 [IGF2. On the paternal allele, the DMR is methylated, thereby blocking the CTCF insulator activity and enabling IGF2 expression. Further, monoallelic expression of H19 and IGF2 is regulated in two distinct ways by a shared cis-acting element [H19 has been reviewed by Gabory et al. in more detail [l allele ,19. Bothle [IGF2 . In the  element . Thus, tH19 is highly expressed [H19 inhibits the proliferation of fetal liver cells and the activity of the Wnt/β-catenin signaling pathway [H19 is strongly down-regulated in all tissues, except for the skeletal muscle [H19 is reactivated in different tumor types, including HCC [H19 as a tumor-suppressor or as an oncogene is strongly debated [H19 in adult livers, the imprinting can be lost in HCC [Especially during the development of the fetal liver, xpressed ,17,22. Il muscle ,23. H19 ding HCC ,25,26,27 debated ,29,30,31t in HCC . This sot in HCC ,33.H19 observed in tumorigenesis might be explained by independent functions of a microRNA (miRNA) encoded by H19 [miR-675 is detectable at all stages of embryonic development, although the H19 transcript is much more abundant [miR-675 has various targets like the receptor insulin-like growth factor 1 receptor (IGF1R), which is involved in proliferation and migration [RB) [IGF-1R, SMAD family member 1 (SMAD1), SMAD family member 5 (SMAD5), and cell division cycle 6 (CDC6), Cadherin-11, Cadherin-13, Rb, runt-related transcription factor 1 (Runx1), Nodal Modulator 1, transforming growth factor, beta-induced (TGFBI), calcium-binding protein 8 (CALN1), and microphthalmia-associated transcription factor (MITF) as reviewed by Raveh et al. [miR-675 promotes carcinogenesis through the downregulation of its target RB [miR-675 processing by binding H19 [miR-675-3p is downregulated [miR-675-5p is almost not detectable in HCC [H19 in cancer, there are conflicting opinions about the role of miR-675 as well [miR-675 might be easily resolved by carefully investigating both miRNAs or by clearly stating which miRNA was analyzed.Part of the controversial roles of d by H19 . In the abundant . In lateigration  or the tion [RB) . It can h et al. . Interesarget RB . Howeverding H19 . HuR1/EL as well ,37. HoweH19 can act as a molecular sponge, i.e., competing endogenous RNA (ceRNA). Thereby, H19 can affect activity of miRNAs and their respective target mRNA levels [H19 sequesters let-7, which targets genes inhibiting the insulin-phosphointositol-3 (PI3K)-mammalian target of rapamycin (mTOR) pathway [H19 and lower let-7a levels, and an H19 knockdown restores chemosensitivity in breast cancer cells [H19 and let-7 [let-7, also interactions with other miRNAs are mentioned, such as miR-200b and miRNA-200c [mir-675 by H19 in ovarian cancer [Interestingly, A levels . CeRNAs  pathway . In vitrer cells . Interesnd let-7 . BesidesRNA-200c ,40. A hin cancer ,27.H19 locus have identified an H19 antisense RNA named 91H in humans and mice [91H is a single 120-kb transcript, which is nuclear and short-lived. It is predominantly expressed from the maternal allele in human and mouse within the H19 gene region. In HCC tissue and cells, 91H is overexpressed [91H expression is associated with a poor prognosis in HCC patients: both disease-free survival time and the overall survival time was shorter compared to patients with low 91H expressions [91H in Hep3B cells down-regulated the expression of insulin-like growth factor 2 (IGF2) suggesting a co-regulation of 91H and IGF2 [Studies on the and mice . 91H is xpressed . High 91ressions . In vitrand IGF2 .H19 locus encodes another maternally expressed translated gene in primates, which is antisense to the H19 transcript [In 2011, Onyango and Feinberg showed that the human anscript . For thianscript . Howeveranscript . The expanscript , while fH19 as functionally-validated lncRNA has a poor ORF of 256 nucleotides. But compared to the definition of lncRNAs with a threshold set to 300 nucleotides, H19 has a large ORF. Therefore, it is impossible to exclude the possibility that H19 might be translated into functional proteins, perhaps in cell-type-specific contexts or at low levels [As mentioned above, w levels .H19 in hepatic function and liver diseases has been comprehensively reviewed by Pope et al. [Hepatocarcinogenesis usually evolves after years of chronic inflammatory liver disease. Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease and is expected to be the top indication for liver transplantation within the next decade. NAFLD is characterized by the accumulation of lipids in the liver. This so-called steatosis can progress to an inflammatory disease, which is then termed non-alcoholic steatohepatitis (NASH). NASH patients, as well as alcoholic steatohepatitis (ASH) patients, may develop liver fibrosis leading to cirrhosis, which is the main risk factor for hepatocarcinogenesis. The role of e et al. . Thus, wIGF2 has been shown to induce hepatic lipid deposition [IGF2 mRNA binding protein (IGF2BP2/IMP2), leading to upregulated expression of H19, induces steatosis [H19 in this disease context had remained elusive until a very recent study by Wang and colleagues. These authors observed increased expression of H19 in steatosis and high-fat diet (HFD)-induced fatty liver [H19 has been reported to induce numerous genes involved in lipid metabolism, whereby Carbohydrate-responsive element-binding protein (ChREBP) is suggested to be mechanistically important [H19 overexpression up-regulates mammalian Target of Rapamycin (mTOR) complex 1 (mTORC1) signaling complex in hepatocytes, which is inhibited by silencing H19 [H19 stabilizes the nuclear transcriptional activity of the lipogenic master regulator sterol regulatory element-binding protein 1c (SREBP1c) by interacting with polypyrimidine tract binding protein 1 (PTBP1). Vice versa, H19 is induced by fatty acids in hepatocytes, and ectopic expression of H19 induces steatosis [H19 might further act via its ceRNA function. With this, H19 was reported to target miR-130a, thereby affecting Peroxisome proliferator-activated receptor (PPAR) γ associated lipid deposition [H19 in hepatic stellate cells (HSCs) [H19 expression is highly altered [H19 is downregulated in livers of db/db mice [The co-regulated gene position . Furtherteatosis  and promteatosis ,48. Howety liver . H19 hasmportant . H19 ovecing H19 . H19 stateatosis . H19 migposition . Lipid ds (HSCs) . In conts (HSCs) , H19 exp altered . In line/db mice .H19 to be altered in cirrhotic livers compared to normal healthy tissue [H19 is increased in the classical mouse model of liver fibrosis, treatment with CCl4, and seems to play a role, especially in cholestatic liver fibrosis. In the latter condition, knockdown of H19 still partially rescues B-cell lymphoma 2 (BCL2)-induced liver injury [H19 prevents the inhibition of epithelial cell adhesion molecule (EpCAM) by zinc finger E-box-binding homeobox 1 (ZEB1) through repression of promoter activity [The progression of NASH is marked by hepatic fibrosis, the scarring of the liver. Fibrosis occurs due to increased deposition of extracellular matrix in the hepatic tissue. Whole transcriptome RNA-sequencing analysis revealed y tissue . Furtherr injury . In thatactivity .miR-874 [H19 [H19 is miR-148a. MiR-148a targets the protease of ubiquitin specific protease 4 (USP4), which stabilizes transforming growth factor beta (TGF) β receptor 1. Thus, H19 promotes TGFβ signaling through the miR-148a/USP4 axis in both HSCs and hepatocytes [H19 was reported to be negatively associated with phosphorylation of extracellular signal-regulated kinases 1/2 (ERK1/2) in HSCs [Hepatic stellate cells (HSCs) are the predominant cell type producing extracellular matrix, and their activation promotes fibrogenesis. Activated HSCs show an induction of miR-874 , which c874 [H19 . Anotheratocytes . H19 was in HSCs . This mi in HSCs .H19 levels [−/−Mdr2 mice and CCl4 treated mice, H19 from cholangiocyte derived exosomes promotes cholestatic liver injury and correlates with disease progression [H19 can induce the expression and secretion of chemokine ligand 2 (CCL2) and interleukin 6 (IL6) in Kupffer cells. Further, it is associated with M1 polarization of Kupffer cells and the recruitment and differentiation of bone marrow-derived macrophages [Due to its multicellular composition of hepatocytes, liver sinusoidal endothelial cells, Kupffer cells, lymphocytes, biliary cells, and HSCs, the liver needs a complex intercellular communication network. Besides autocrine, paracrine, endocrine, and cell-cell contacts, exosomes have recently gained attention as an additional mechanism of cellular communication. Therefore, exosomes contain various bioactive molecules, such as lipids, proteins, RNA, and DNA. Interestingly, serum exosomes from human patients with cirrhosis contain elevated 9 levels . In Mdr2gression . The chorophages .H19 plays a role in premalignant stages of liver disease. Although, some observations are controversial, most of the studies point towards a disease-promoting action of H19 in this context. Still, only a few potential mechanisms shedding light on the role of H19 in premalignant liver diseases are described so far. Future studies investigating the exact mechanisms by which H19 affects the development of simple steatosis and NASH progression towards fibrosis will help to assess H19’s role in the early steps of hepatocarcinogenesis.Taken together, H19 being a tumor suppressor to H19 being an oncogene in HCC  gene and a decreased accumulation of doxorubicin [H19 in R-HepG2 cells suppresses MDR1 expression, increased cellular doxorubicin accumulation level, and sensitizes towards doxorubicin toxicity [H19 might induce the MDR1-associated drug resistance in liver cancer cells. In contrast, Schultheiss et al. investigated the function of H19 in HCC in vitro and in vivo and observed opposite results compared to the latter studies [H19 in three different human liver cancer cell lines  resistant to either doxorubicin or sorafenib. H19 was down-regulated in all six chemoresistant cell lines [H19, established by plasmid transfection, were treated with doxorubicin or sorafenib. H19 overexpression decreases colonoy formation, indicating a tumor cell survival suppressing and chemotherapy-sensitizing action of H19 [H19-induced apoptosis in HepG2 cells is regulated by autophagy through the phosphoinositol-3 kinase (PI3K) signaling pathway [H19 knockdown [H19 and treatment with chemotherapeutics, cell survival significantly increases compared to the control, especially in Bel-7402 and QGY-7703 cells. In addition, in Bel-7402 cells, the apoptosis rate decreases [H19 works as a tumor suppressor. Furthermore, overexpression of H19 in SMMC-7721 cells inhibits tumor cell invasion by the reversion of EMT [Supporting the role of c stress . Hep3B cnditions . These eup study , in whicc stress . Consistnhibitor . In doxoorubicin . Knockdotoxicity . These r pathway . Interesnockdown . Therefonockdown . The stuecreases  again sun of EMT . SKHEP1 n of EMT . Furthern of EMT . Still, n of EMT . In summn of EMT . Thus, fn of EMT ,80,81,82n of EMT .H19 knockdown Hep3B cells into the dorsal flank of athymic female CD-1 nude mice [H19 knockdown significantly inhibited tumor growth, and, in some cases, tumors did not form at all, suggesting an oncogenic role for H19 in HCC. In contrast, Schultheiss et al. examined the role of H19 in a hepatocarcinogenesis model [H19 knockout mice were injected with 5 mg/kg body weight diethylnitrosamine (DEN), an inflammation-inducing carcinogen, and sacrificed 24 weeks after injection [H19 knockout leads to increased tumor development and tumor multiplicity upon DEN, thus showing a tumor-suppressive effect of H19 in vivo and supporting their previous conclusions from in vitro models. Interestingly, this was independent of the reciprocally imprinted IGF2. Another H19 knockout mouse model study from Yoshimizu et al. used a transgenic SV40 hepatocarcinoma model [H19 leads to acceleration in the development of liver tumors and larger tumors [IGF2 levels [In vivo models also clearly show involvement of H19 in HCC. In addition to the in vitro data, Matouk et al. investigated tumor growth in vivo in CD-1 nude mice by subcutaneously injecting ude mice . H19 knois model . H19 knoma model . Males or tumors . In line2 levels .H19 is a tumor suppressor was obtained from a xenograft study by Zhang et al. [H19 HCCLM3 cells, the number of intrahepatic tumor metastases significantly increased [Further, support for the hypothesis that g et al. . In thisncreased .H19 appears to function as a tumor suppressor rather than an oncogene in hepatocarcinogenesis. The main differences between these studies are again in the conditions of the experiments. Matouk et al. used a xenograft model to investigate their hypothesis [Regarding the latter four studies, pothesis . One haspothesis . For tumpothesis . Furtherpothesis .H19 and IGF2 are significantly upregulated in Phb1 deficient livers [H19 or IGF2 by Phb1 knockout leads to tumor development.Another interesting aspect was highlighted by the study by Ramani et al. . Mice lat livers . HoweverH19 [Taken together, most in vivo studies support a tumor suppressor function for H19 ,16,70, wH19 .H19 is a tumor suppressor in hepatocarcinogenesis rather than an oncogene. Nevertheless, it is essential to compare these results with human HCC samples. Most human studies show that H19 is overexpressed in HCC [H19 in human HCC samples [n = 18–80) as well as different methods, such as in situ hybridization (ISH) or qPCR. Besides, H19 was not expressed in all of the analyzed HCC samples. Therefore, the number of samples should have been even higher in order to achieve statistical significance. Yang et al. investigated a cohort with a significant sample size of 240 tumor tissues from primary HCC patients [H19 expression correlated with HCC aggressiveness and poor disease outcome. However, the expression of H19 in healthy tissue was not available in this study. In contrast, Zhang and colleagues described downregulation of H19 in HCC tissue compared to adjacent non-tumorous tissue [H19 in different regions of the non-tumorous tissue defined by the distance to the tumor [H19 expression was highest in the peritumoral area, meaning that the sample collection, especially of the non-tumorous adjacent tissue, has a significant impact on the outcome of the study. Therefore, future studies should be thoroughly conducted regarding sample collection. In line with the findings by Zhang et al. [H19 expression compared to adjacent non-tumorous tissue [H19 expression in tumor tissue. One explanation for these contradictory findings and of specific HCC subgroups with high H19 levels that show a more aggressive phenotype, has been reported by Iizuka and colleagues [H19 nor IGF2 expression levels alone are prognostic, patients with imbalanced, anti-correlated expression levels of both genes are associated with poor survival [H19 and IGF2 are generally tightly co-regulated, it is important to study bith genes in parallel in future studies. Furthermore, contradictory findings of former studies might be due to different cell composition in sample subsets of the patient cohorts. Interestingly, a recent study showed that tumor-associated macrophages highly express H19 and thereby promote HCC aggressiveness [H19 are needed to clarify this controversy. A promising approach could be single-cell analysis of HCC samples instead of using bulk tissue samples to gain insight into the cell-specifc expression of H19.In vivo models suggest that d in HCC ,89,90,91 samples ,89,90,91patients . In thiss tissue . The lathe tumor . Interesg et al. , Schulths tissue . Howeverlleagues . The autsurvival . Since Hsiveness . Thus, fH19 and IGF2 in HCC samples [H19 has been shown for several tumors [H19 [IGF2 locus in non-tumorous tissue of Hepatitis C virus (HCV) infected livers is predictive for cancer risk or disease progression [H19/IGF2 locus in the healthy adult liver is complex due to the fact that while H19 expression is monoallelic, expression of IGF2 is biallelic [H19 or H19 expression independent of the DNA methylation status plays a role in hepatocarcinogenesis. In fact, in colorectal cancer, IGF2 expression is unrelated to its imprinting status [H19.Wu et al. screened for polymorphisms of  samples . They idl tumors ,95, incll tumors ,96. In ggression . It shouiallelic ,99. In ag status , which mH19 polymorphisms and the susceptibility as well as prognosis on tumorigenesis [H19, is associated with a reduced risk of glioma. Interestingly, this region encodes for the antisense transcript HOTS. But so far, there is no evidence for a co-expression of H19 and HOTS in vivo [H19 rs217727 polymorphism, which is associated with decreased H19 expression, enhanced the survival rate of patients with HCC [H19 gene polymorphisms and the susceptibility to develop late-stage tumors [H19 showed an increased susceptibility to HCC. So far, the authors did not find any correlation between H19 and serological markers of HCC, such as AFP, alanine transaminase (ALT), or aspartate transaminase (AST). Beside the increased risk to develop HCC when carrying the rs2839698 polymorphism, an association with poor prognosis exists in smoking patients [H19 gene polymorphisms could have either tumor-suppressing or promoting effects. Thus, H19 polymorphisms might be an explanation for different study outcomes.Two other exciting studies investigate the association between different igenesis ,87. A stigenesis . The rs3 in vivo . In HCC,with HCC . Howeverpatients . HoweverH19 locus offer many possibilities for novel diagnostics or therapeutic targets in HCC. One possibility was mentioned by Ariel et al. [H19 could be performed. They showed that the expression of H19 was detected with ISH in 13 of 18 samples with HCC. AFP, a commonly used tumor marker in HCC, was found only in nine of 18 samples [H19 could be a useful diagnostic method.Although the literature is still confusing, the various interactions of the l et al. : for the samples . TherefoH19 and IGF2 are often co-expressed in tumor cells. Therefore, Amit et al. examined the therapeutic potential of a double promoter toxin vector H19-DTA-(IGF2)-P4-DTA leading to, expressioned of the diphtheria toxin [As mentioned before, ia toxin . The vecia toxin . This woH19, designed a methylated oligonucleotide (MON1) [IGF2 P4 promoter. It specifically inhibited IGF2 mRNA accumulation in vitro in Hep3B cells. In vivo, animals bearing engrafted tumors had prolonged survival upon treatment with MON1. Thus, a methylated sense oligonucleotide, complementary to H19, may lead to enhanced survival in vivo [Another study with a focus on IGF2, but potentially translatable for e (MON1) . MON1 is in vivo .H19 as a therapeutic target was only investigated under the hypothesis of H19 being an oncogene. With respect to most of the in vivo and ex vivo studies the tumor suppressor role seems to be more plausible. Of course, re-activation of expression is far more challenging in therapy than inhibiting expression. Still, increasing knowledge about the whole H19 locus and the transcript-specific functions in HCC could lead to more potential therapeutic strategies for the treatment of HCC or the modulation of the hepatic inflammatory environment in the pre-malignant phase of liver disease.Taken together, however, H19 locus is a complex gene locus and the transcript specific effects have not always been investigated in parallel in the same studies. Data from in vivo models point towards a tumor suppressor effect of H19 in HCC. Recent human data raise the possibility that H19 gene polymorphisms and cell-type specific expression could explain many of the contradictory findings in the past. Therefore, a deeper understanding of this gene locus is vital for defining a potential use of H19 in new diagnostic and therapeutic strategies.The


Human papillomavirus (HPV) antigens had been found in colorectal cancer (CRC) tissue, but little evidence demonstrates the association of HPV with oncogene mutations in CRC. We aim to elucidate the mutated genes that link HPV infection and CRC carcinogenesis.  Cancerous and adjacent noncancerous tissues were obtained from CRC patients. HPV antigen was measured by using the immunohistochemical (IHC) technique. The differentially expressed genes (DEGs) in HPV-positive and HPV-negative tumor tissues were measured by using TaqMan Array Plates. The target genes were validated with the qPCR method.  15 (31.9%) cases of CRC patients were observed to be HPV positive, in which HPV antigen was expressed in most tumor tissues rather than in adjacent noncancerous tissues. With TaqMan Array Plates analyses, we found that 39 differentially expressed genes (DEGs) were upregulated, while 17 DEGs were downregulated in HPV-positive CRC tissues compared with HPV-negative tissues. Four DEGs  were upregulated in tumor vs. normal tissues, or adenoma vs. normal tissue in TCGA, which was overlapped with our data. In the confirmation test, MMP-7, MYC, WNT-5A, and AXIN2 were upregulated in cancerous tissue compared with adjacent noncancerous tissue. MYC, WNT-5A, and AXIN2 were shown to be upregulated in HPV-positive CRC tissues when compared to HPV-negative tissues.  HPV-encoding genome may integrate into the tumor genomes that involved in multiple signaling pathways. Further genomic and proteomic investigation is necessary for obtaining a more comprehensive knowledge of signaling pathways associated with the CRC carcinogenesis. Colorectal cancer (CRC) is the primary cause of cancer mortality. Although progress has been made, the long-term survival of patients with metastatic disease is still poor . Both geHPV carries out its life cycle in either the mucosal or the cutaneous stratified squamous epithelia. An oncogenic HPV infection is associated with several malignancies, including cervical cancer , 15 and Gene analysis with microarray technology has shown great potential in discriminating sophisticated gene profiling, simultaneously mapping thousands of genes in a single sample, and giving a measurement of articulated gene expression patterns . To clarThe Ethics Committee approved this project of the Shantou University Medical College Ethics Board. This study was conducted under the Declaration of Helsinki.All clinical samples were obtained from patients admitted to the First Affiliated Hospital of Shantou University Medical College. Written informed consent was obtained from all patients. The CRC patients were selected based on histologically confirmed CRC at TNM stage I-IV disease with or without distant metastases. An individual with a history of polyps, adenomas, or other diseases related to cancer was excluded. Tumor types were evaluated according to histology, immunohistochemistry (IHC), and chromatin assays. Clinical and histopathologic staging at diagnosis was determined in all patients by combining histopathologic findings with surgical records and operative imaging. All selected tissues were based on histopathological diagnoses; adjacent noncancerous tissue was collected from an area that was 15 cm distal from the tumor. Fresh frozen tissues were obtained from CRC patients before they underwent curative surgical resection. Tumor and the nontumor regions were distinguished using H&E matched slides and microdissected to pinpoint the tumor as well as nontumor areas. Samples were kept at -80°C until measurement. The demographic, clinical, and histopathological data of patients were recorded. Clinical data including race, gender, associated past medical history, medication use, family history of colonic cancer, and laboratory tests were collected for all patients.μm thick. The sections were cut, dewaxed in xylene, and rehydrated in decreasing concentrations of ethanol. HPV was measured in the tumor tissues and tumor-adjacent tissues using an IHC technique according to the manufacturer's instructions. Considering the HPV's pathogenesis, we had chosen the anti-HPV high-risk subtype antibodies, which is a monoclonal antibody, reacting with HPV subtypes 6, 11, 16, 18, 31, 33, 42, 51, 52, 56, and 58. The details of antibody are HPV mouse monoclonal antibody IgG1, ab75574, Lot. GR207368-5, Abcam, U.K. In negative control sections, the primary antibody was omitted. Virus-infected monocytes or lymphocytes from the same patient and cervical squamous cell carcinoma were used as the positive control for staining. The sections were graded based on the estimated percentages of virus protein-positive cells. IHC staining was evaluated and graded by two pathologists in isolation from the investigator who performed the real-time PCR.Immunohistochemical staining was performed on paraffin sections 4 Genes expressed in HPV-positive and HPV-negative tumor tissues were measured by using TaqMan Array Plates. Fresh specimens were obtained from the initial diagnosis of CRC patients who underwent curative surgical resection; adjacent noncancerous tissue was taken far mostly from the tumor. Total RNA was extracted from the tissue using a PureLink RNA Mini Kit  according to the manufacturer's instructions. The RNA concentration was measured using the NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific), and RNA quality was evaluated by using the BioAnalyzer 2100 microcapillary electrophoresis system . The RNA concentration was adjusted 75 ng/L, so that the RNA concentration of each sample was consistent, and then, reverse transcription was performed. RNA was converted into cDNA by using a High-Capacity cDNA Reverse Transcription Kit . The cDNA microarray analysis was performed using the TaqMan® Array Human Colorectal Cancer Metastasis 96-well plate that contains 92 assays for CRC metastasis-associated genes and four assays to candidate endogenous control genes. Each plate includes predefined probes and endogenous controls, ready for accurate assessment of an entire gene signature in one simple experiment (Thermo Fisher Scientific). Each patient's sample was measured in the tumor tissues and tumor-adjacent tissues simultaneously, and the gene expression level was calculated by gene level in tumor tissue minus gene level in tumor-adjacent tissue. Each cDNA was analyzed using real-time PCR on the “mixed” TaqMan Array Cards, and Ct values for target genes were obtained. All Ct values were normalized with an inner control (GAPDH) gene, and ΔCt values were calculated. Relative gene expression was computed using the relative expression level for each gene. The 2ΔCt values were calculated by SDS Software v2.3 and exported. Relative expression values were mean-centered, and heat maps were generated using the “Cluster” and “TreeView” software tools.http://www.ncbi.nlm.nih.gov/geo/). The GSE20916 dataset contained 145 samples, including 105 CRC tumor tissues and 40 normal colon epitheliums. The hierarchical clustering analysis was used to categorize the data into two groups that had similar expression patterns in CRC and normal colon epitheliums. We used a classical t-test to identify DEGs with a change ≥ twofold and defined P < 0.05 to be statistically significant. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed to analyze the DEGs at the functional level. The DEGs were screened using Expression Console Affymetrix.To avoid the limitation of a small sample size that may not provide a robust prediction, we utilized the TCGA database. The gene expression profile of GSE20916-RAW.tar was downloaded from Gene Expression Omnibus , MMP-7 (ID. Hs01042796_m1), MYC (ID. Hs99999003_m1), WNT5A (ID. Hs00998537_m1), and GAPDH (ID. Hs99999905_m1), Thermo Fisher Scientific, USA). Specifically, amplified RNA from each sample was reverse-transcribed using SuperScript II reverse transcriptase and random primers (Invitrogen). cDNA from each reverse-transcribed sample was PCR amplified using TaqMan quantitative PCR. The quantification of the gene copy number, together with the human albumin gene, was performed with the 7500 Real-Time PCR System . The expression values for each gene were normalized with GAPDH.U test (two groups). DEGs were determined by Welch's t-test, and precise targets were selected from the DEGs using the prediction analysis for microarrays (PAM) software package. Significance was established when the statistical tests returned P values < 0.05. Software R (v3.1.1) was used to determine the significance in the TaqMan Array Plates, comparing genes expressed in CRC-HPV+ vs. CRC HPV tumor tissues (heat map).Statistical analyses were performed using SPSS (Version 10.0). Pearson's chi-squared analysis and Fisher's exact test were employed to compare the differences in categorical variables between patient groups. The relative gene expression levels were compared between various subgroups using the Kruskal–Wallis test (42 groups) and Mann–Whitney To understand whether CRC patients associated with HPV would have unique features, we investigated their clinicopathological characteristics in this study; the parameters included age, gender, tumor location, histological grading, lymphatic metastasis, and clinical staging. The 47 patients included 4 cases of mucinous adenocarcinoma and 43 cases of nonmucinous adenocarcinoma. Males comprised a more significant proportion of CRC patients compared to females. The majority of tumor regions were located in the sigmoid colon and rectum .IHC staining showed that 15 (31.9%) cases were HPV positive, while 68.1% were HPV negative. HPV antigen was expressed in most cancerous tissues but not in adjacent noncancerous tissues in some CRC patients . At TNM APC, CASP3, CDKN1A, IFNG, MAPK3, MAP2K4, NOS2, PTGS2, PTGER4, SMAD2, and TP53. Twelve genes increased between onefold and twofold, namely, ARRB1, CASP9, DCC, EGF, FZD1, IFNGR1, KRAS, JAK1, MAP2K2, MAP2K6, NFKB1, and TNF. Sixteen genes increased between 0.5-fold and onefold, namely, AKT1, GNA5, GNB3, IL-6R, KRAS, NFKB2, PRKACA, PTGS2, RELA, SMAD4, STAT1, TGF4, TGFB1, TGFBR1, TLR4, and WNT5A. On the contrary, In the HPV-positive group, seventeen genes were downregulated between 0-fold and 0.5-fold, namely, BAX, BCL2L1, CCND1, CCND2, CDH1, CTNNB1, DVL1, GRB2, LRP5, MLH1, MMP7, MMP9, MYC, PIK3R1, PIK3R2, SRC, and TCF3. Five genes decreased between 0.5-fold and onefold, namely, AXIN2, MAPK1, MSH2, PIK3CA, and LRP6. The heat map of DEG expression (upregulated and downregulated genes) is shown in We randomly selected four CRC patients, including two patients with HPV positive and two patients with HPV negative, analyzing ninety-two genes with the TaqMan Array Plates method. Cancerous and adjacent noncancerous tissues (paired samples) were used for the case-matched studies. Gene expression profiles in case-matched groups were analyzed using TaqMan Array Plates to screen DEGs  include IL-6R, TLR3, PIGER2, MAP2K6, ARRB1, PTGER4, CDKN1A, MAPK3, and TLR7. The DEG expression heat map is shown in MMP1, MMP7, WNT5A, MYC, AXIN2, and TLR4, while six genes increased between 0.5-fold and onefold: MSH2, TP53, NOS2, CCND2, BAX, and PTGER2. The downregulated genes include IL-6R, TLR3, ARRB1, and TLR7.Additionally, we downloaded 145 raw CEL files from the NCBI Gene Expression Omnibus (GEO) database and normalized them with GeneChip RMA (GCRMA). Gene expression data were normalized for each tissue type by computing the Robust Multichip Average (RMA) directly from the Affymetrix. CEL were files obtained for tumors, adenoma, and healthy samples. A total of 3500 genes were identified, and the oncogenes associated with CRC were in the range. From these data, we found 16 oncogenes with a significantly different expression between the tumor and healthy tissues. Compared with healthy tissue, nine genes were upregulated between onefold and twofold in the CRC group: MMP7, WNT5A, MYC, and AXIN2 genes upregulated in tumor vs. healthy tissues, adenoma vs. healthy tissue in TCGA; these genes were overlapped between the TaqMan Array Plates data and TCGA data. To validate the results, we detected them in the 47 samples of CRC using the qRT-PCR assay. For all the four genes, the results were concordant with the TaqMan Array Plates data. Initially, a comparison was made between cancerous tissue and adjacent noncancerous tissue. The results showed that MMP-7, MYC, WNT-5A, and AXIN2 were upregulated in cancerous tissue compared to adjacent noncancerous tissue, but only MMP-7 and WNT-5A reached statistical significance (P < 0.05)   . Then, w > 0.05) .We then applied the hierarchical clustering analysis and principal component analysis (PCA) to categorize all the genes into different groups. Unsupervised two-dimensional hierarchical cluster analysis of relative gene expression data (2DCt method) reveals distinct clusters of cancer samples and healthy control samples. With PCA analyses, the 92-gene signature discriminated four samples into two significantly separated areas: viral-positive and viral-negative groups. Our results revealed that isolated areas considerably accurately classify the patterns of viral-positive and viral-negative CRC . The twoFusobacterium nucleatum, can signal epithelial cells via E-cadherin, a cell-surface molecule essential for CRC metastasis as well as a component of the WNT/β-catenin signaling pathway, the most commonly mutated pathway in CRC [The human microbiota has implicated in the etiology of CRC; moreover, the microbiome has proven to be an active contributor to CRC . FadA pry in CRC , 35. Nowβ-catenin, phosphoinositide 3-kinase (PI3K), mitogen-activated protein kinase (MAPK), mammalian target of rapamycin (mTOR) signaling, and inhibitors of TGFβ and TP53 signaling, which were also the most frequent changes in CRC progression. The DEGs are also involved in cell invasion, adhesion, and tissue remodeling which may play an essential role in the carcinogenesis and progression of metastases in CRC [Comparative analysis of the DEGs reported by independent studies shows a relatively limited degree of overlap due to variations in population and technical methods , 37. Thes in CRC , 39–41. β-catenin signaling pathway controls cell proliferation and differentiation in the intestinal crypt microenvironment [β-catenin signaling were associated with CRC [APC gene, plays a vital role in maintaining homeostasis of the gut epithelium and thus, when mutated, can impart a functional consequence capable of driving cancerous cell proliferation [β-catenin signaling pathway. Evidence from the previous study also supports that microbes play a role in dysregulating the Wnt/β-catenin signaling pathway as a means of tumor initiation [β-catenin–dependent Wnt signaling, which acts as a scaffold factor in the β-catenin destruction complex. The two AXIN proteins have similarity in dysregulation of the Wnt pathway that believed to be an essential first step in the genesis of CRCs, while aberrant Wnt pathway activation is in roughly 90% of sporadic CRCs [The Wnt/ironment . In the ironment . Deregulwith CRC , 45. Theferation . In thisitiation . AXIN pritiation . AXIN2 idic CRCs , 49.β-catenin signaling pathway include the c-MYC proto-oncogene (MYC) [In our study, we found that WNT5A was upregulated while AXIN2 was downregulated in the HPV-positive group as compared to the HPV-negative group. However, both AXIN2 and WNT5A were upregulated in the HPV-positive group when we verified the results with qPCR in additional samples. The data from TCGA showed that AXIN2 was upregulated in CRC tumor tissues. Our results revealed that differences in gene expression might be extended in individual patients with CRC; therefore, a large cohort is required to eliminate a distinctive difference in CRC oncogenes. The differences between microarray and q-PCR should be carefully examined to reveal the role of AXIN and Wnt signaling in HPV-associated CRC pathogenesis. Genes mutations in the Wnt/ne (MYC) . c-Myc ine (MYC) . With thne (MYC) .IFNG, INFGR1, and TNF genes upregulated in HPV-positive CRC tissues as compared to HPV-negative CRC tissues in this study. The upregulated IFNG and INFGR1 may trigger the STAT  proteins that mediate some biological functions and form part of the signaling cascades [IFN/TNF/IL-6R/STAT1expression, and the associated signaling pathway is valuable in elucidating the mechanism of virus-associating CRC.Interferon (IFN) has a wide range of biological functions, including antiviral, antiproliferative, and immunomodulatory properties. IFN-induced cellular antiviral response is the primary defense mechanism against viral infections. Consequently, viral infection may disrupt the secretion of interferon, cascades . TherefoκB [MMPs, VEGFA, JAK1, EGF, KRAS, AKT1, MAPK3, and MAP2K4 were upregulated in HPV-positive CRC. The functions of these genes in virus-associated CRC need further investigation.Striking experimental data have shown that gut microbes play a crucial role in vascularization of the intestinal mucosa and wound-healing processes . MMPs anκB . PTEN isκB . In thisCurrently, traditional clinical and pathological parameters are not always sufficient to discriminate high-risk from low-risk CRC, and validated molecular markers with prognostic value are not yet available . A recenThe main limitation of this study is that no significance was observed between deregulated genes from HPV-positive vs. HPV-negative CRC cases of the cohort. We could not exclude the reason for the small sample size. However, there may be a more complicated mechanism involved. Further study with large sample size is needed to elucidate the HPV-associated CRC.HPV-induced cervical cancer has been widely confirmed clinically, and the anti-HPV vaccine has made significant progress in the prevention and treatment of cervical cancer. At present, studies have found that HPV viral antigens also exist in colon cancer tissues. The relationship between HPV infection and CRC carcinogenesis is very worth studying. In this study, we demonstrated that tumor tissues had higher levels of HPV antigen than adjacent nonneoplastic tissue. Moreover, we found that 39 differentially expressed genes (DEGs) were upregulated, while 17 DEGs were downregulated when compared to HPV-positive CRC tissues with HPV-negative tissues. Combined with the database of the Cancer Genome Atlas (TCGA), we screen four DEGs and tested with the PCR method, and there was some overlap between our data and TCGA data. Our data revealed that HPV could take part in CRC carcinogenesis through gene mutation. The further genomic and proteomic investigation is necessary for obtaining a more comprehensive knowledge of signaling pathways associated with CRC carcinogenesis.


Sophora flavescens, was suggested to exert potent antioxidant and immunosuppressive effects. However, the underlying mechanisms remain unclear. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a key transcription factor that regulates the antioxidant defense system with anti-inflammatory activity. In the present study, we demonstrated that kurarinone activated Nrf2 and increased the expression of antioxidant enzymes, including heme oxygenase-1 (HO-1). Mechanistically, kurarinone downregulated the expression of kelch-like ECH-associated protein 1 (KEAP1), subsequently leading to the activation of Nrf2. Kurarinone also inhibited the expression of the inflammatory cytokine, interleukin (IL)-1β, and inducible nitric oxide synthase (iNos) in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. The overexpression of HO-1 suppressed the LPS-induced production of inflammatory mediators in RAW264.7 cells, and the immunosuppressive effects of kurarinone were partially inhibited by a treatment with Tin Protomorphyrin IX (TinPPIX), an inhibitor of HO-1. These results indicate that kurarinone activates the KEAP1/Nrf2 pathway to induce HO-1 expression, thereby exerting immunosuppressive effects.Kurarinone, a flavonoid isolated from the roots of  Sophora flavescens (Fabaceae), also known as “Kushen” in Chinese and “Kujin” in Japanese, were traditionally used in combination with other herbal medicines to treat asthma, bronchitis, bacterial and fungal infections, skin disorders, and inflammation . C. CHO-1 gWe examined Nrf2 protein levels in cytoplasmic and nuclear extracts of PC3 cells, treated with different concentrations of kurarinone for 6 h. As shown in KEAP1 mRNA levels were reduced by the kurarinone treatment, reaching approximately 50% after 6 h, and then recovered -1β and inducible nitric oxide synthase (iNos) in RAW264.7 cells. However, the overexpression of HO-1, suppressed these increases   were reported to target KEAP1 mRNA by binding to its 3′-untranslated region sequence sites [KEAP1 mRNA might be that kurarinone represses KEAP1 transcription, or that the induction of these miRNAs might reduce KEAP1 mRNA. In addition, kurarinone reduced the KEAP1 protein levels at a later stage, but it is unclear whether kurarinone chemically modified the KEAP1 protein. Taguchi et al. showed that KEAP1 degradation was accelerated in electrophilic stress conditions [The majority of compounds that activate the KEAP1/Nrf2 pathway were electrophiles, which modify specific cysteine residues within KEAP1 . Kurarince sites . We specnditions . Thus, tThe in vivo antioxidant system centered on Nrf2 plays an important role in protecting cells from various stresses, including oxidative stress . In respSince oxidative stress is associated with many pathological conditions, such as cancer, neurodegenerative diseases, cardiovascular diseases, and metabolic diseases ,37, clinDrug discovery research to activate the Nrf2 pathway is now actively conducted. Although bardoxolone methyl exhibits Nrf2-inducing, antioxidant, and anti-inflammatory activities and is expected to have clinical applications ,43, clinS. flavescens extract-treated rats (2.5 g/kg) [S. flavescens in rats [S. flavescens extract induced hepatic lipid accumulation and liver injury. In contrast, Yang et al. demonstrated that kurarinone showed no detectable toxicity in mice, since there were no significant effects on body weight, behavior, and appearance between the kurarinone-treated mice and the control [In this study, we demonstrated that kurarinone represented antioxidant and anti-inflammatory properties via Nrf2 activation. Kurarinone-induced HO-1 expression was observed at a concentration of 20 μM. Although there are no reports in humans, there are few reports examining the pharmacokinetics of kurarinone in rats. Jiang et al. analyzed kurarinone levels in fluids and tissues of .5 g/kg) . After a.5 g/kg) . Therefo in rats ,49. Alth control . Nrf2 is control ,52. TherIn conclusion, kurarinone is expected to become a seed compound for new drugs that activate Nrf2. It might be used for clinical applications and has the potential to provide new therapeutic options for chronic inflammatory diseases. Further studies are needed to establish the precise mechanisms of action of kurarinone on the KEAP1/Nrf2 pathway.


Although estrogens alleviate hot flushes in these patients, they also cause adverse systemic side effects. Because only estrogens can provide mitigation of hot flushes on the basis of current clinical practices, there is an unmet need for an effective and safe pharmacotherapeutic intervention that would also greatly enhance patient adherence. To this end, we evaluated treatment of orchidectomized (ORDX) rats with 10β, 17β-dihydroxyestra-1,4-dien-3-one (DHED), a brain-selective bioprecursor prodrug of E2. A pilot pharmacokinetic study using oral administration of DHED to these animals revealed the formation of E2 in the brain without the appearance of the hormone in the circulation. Therefore, DHED treatment alleviated androgen deprivation-associated hot flushes without peripheral impact in the ORDX rat model. Concomitantly, we showed that DHED-derived E2 induced progesterone receptor gene expression in the hypothalamus without stimulating galanin expression in the anterior pituitary, further indicating the lack of systemic estrogen exposure upon oral treatment with DHED.Hot flushes are best-known for affecting menopausal women, but men who undergo life-saving castration due to androgen-sensitive prostate cancer also suffer from these vasomotor symptoms. Estrogen deficiency in these patients is a direct consequence of androgen deprivation, because estrogens (notably 17β-estradiol, E Men lacking functional nuclear estrogen receptors (ERs) or aromatase exhibit symptoms similar to menopausal women, including hot flushes [Hot flushes often negatively impact quality of life because they are associated with acute physical discomfort, as well as sleep disturbances resulting in fatigue, irritability, and forgetfulness ,2. Althotization ,9. Amonghe brain ,11 and,  flushes ,12, rein2 patch and the oral synthetic ethinylestradiol (EE)) have been successfully used in men receiving androgen-deprivation therapy (ADT) for the alleviation of hot flushes, cognitive deficits, and depression/anxiety associated with hypoandrogenic conditions [Estrogens , as shown in 2 level and associated adverse estrogenic effects in the periphery [To overcome these obstacles, we have developed a CNS-selective estrogen therapy and unequivocally showed the target-selective formation of Eeriphery ,17. We heriphery .Although hot flushes are characteristic events of primates, only a limited number of studies have been published using a handful of non-human primates actually ,22,23. R2 formation in the ORDX rat brain  [2 concentration apparently increased in the hypothalamus, as shown in 2 reached its maximum value around 30–60 min post drug treatment, which then declined and was no longer quantifiable 8 h after DHED administration.To provide evidence for the DHED-derived Eat brain , we condC–MS/MS) . This brC–MS/MS) . When OR2 concentrations were detected from serum samples  compared to the untreated control levels that were at or below our assay’s ≥10 pg/mL limit of detection. These findings confirmed that DHED administration to ORDX rats also produced central formation of E2 without peripheral hormonal liability, similar to OVX rats [Importantly, no increase in circulating EOVX rats ,17,18,19Our chosen animal model was validated with the clinically used EE to confirm the desired effect of exogenous estrogen on lowering TST ,25,26,27p < 0.05) decrease in TST in relation to the control in our paradigm. This effect was not significantly different from that of EE treatment at higher concentration (1 mg/kg/day) by the same route and treatment schedule , however, keep men away from estrogens or discourage adherence to continued therapy among those who initially decide to take it ,15. Men 2 [2 is estimated to originate from direct testicular secretion, with the rest resulting from aromatization of testosterone, particularly in adipose tissue, muscle, bone, and brain, where it also may be synthesized de novo [The idea of using estrogen in men looks surprising at first sight, but it has been shown that many actions of estrogen in males are mediated by the aromatization of testosterone to E2 ,9. About de novo ,12. Estr2 selectively in the brain [2 at the target site [The use of estrogen in men is scientifically sound, but it requires an approach that avoids peripheral side effects. On the basis of our initial preclinical assessment presented here, DHED could fulfill this expectation. As DHED is converted to Ehe brain , centralget site ,18,19. Aget site ,19.2 formation in the hypothalamus . DHED was synthesized in our laboratory, as described previously [13C6 (13C6-E2) was supplied by Cambridge Isotope Laboratories .EE, Eeviously . MorphinAll experimental protocols involving animals, including orchidectomy, were approved by the institutional animal care and use committee at the School of Medicine, University of Maryland, Baltimore, MD . Male Sprague-Dawley (SD) rats, 3 months old (≈300 g body weight), were obtained from Charles River Laboratories . The animals were housed in pairs under a 12 h light/dark cycle and given a phytoestrogen-free diet. Water and food were available ad libitum. All animals were cared for according to the National Academy of Sciences guidelines for compassionate use and care of laboratory animals.n = 5–7) were treated with DHED  and then euthanized 0.25, 0.5, 1, 2, 4, and 8 h after treatment. The hypothalami were dissected from the harvested brains, followed by tissue homogenization  and adding 13C-labeled internal standard (IS)  for E2 quantitation, as reported previously [Drug quantification by LC–MS/MS was based on the principles of isotope dilution . ORDX SDeviously ,19,28. Lv/v) dimethyl sulfoxide. The drug treatment protocol of this model is summarized in In this morphine-addicted male rat pharmacological hot flush model, we administered saline vehicle as the negative control, EE (1 mg/kg/day) as the positive control, and DHED (300 µg/kg/day), by oral gavage, respectively, b.i.d. Test agents were dissolved in saline vehicle containing 2%  probe complementary to rat PR sequence according to previously published protocol . At the −ΔΔCT method [Galanin gene expressions in the AP were measured with qRT-PCR protocol following the procedures reported previously ,17,32. Bn = 12 for DHED treatment and 10 for EE) and control (n = 8) groups, we used Tukey’s multiple comparison analyses to compare the distribution of AUC values among the treatment groups, along with ANOVA. For the analysis of treatments on gene expression and E2-concentration measurements, we used one- or two-way ANOVA in evaluating simple experiments, followed by appropriate post hoc (Tukey or Tukey–Kramer) tests to compare differences among groups. In all analyses, p < 0.05 was considered as statistically significant. Data are displayed as mean ± standard error of the mean (SEM).AUC for each subject was calculated with a trapezoidal method in SAS version 9.4. Because of the unequal group sizes among the experimental (