Cardiotonic steroids (CTS) were first documented by ancient Egyptians more than 3000 years ago. Cardiotonic steroids are a group of steroid hormones that circulate in the blood of amphibians and toads and can also be extracted from natural products such as plants, herbs, and marines. It is well known that cardiotonic steroids reveal effects against congestive heart failure and atrial fibrillation; therefore, the term "cardiotonic" has been coined. Cardiotonic steroids are divided into two distinct groups: cardenolides (plant-derived) and bufadienolides . Cardenolides have an unsaturated five-membered lactone ring attached to the steroid nucleus at position 17; bufadienolides have a doubly unsaturated six-membered lactone ring. Cancer is a leading cause of mortality in humans all over the world. In 2040, the global cancer load is expected to be 28.4 million cases, which would be a 47% increase from 2020. Moreover, viruses and inflammations also have a very nebative impact on human health and lead to mortality. In the current review, we focus on the chemistry, antiviral and anti-cancer activities of cardiotonic steroids from the naturally derived (toads) venom to combat these chronic devastating health problems. The databases of different research engines  were screened using different combinations of the following terms: “cardiotonic steroids”, “anti-inflammatory”, “antiviral”, “anticancer”, “toad venom”, “bufadienolides”, and “poison chemical composition”. Various cardiotonic steroids were isolated from diverse toad species and exhibited superior anti-inflammatory, anticancer, and antiviral activities in in vivo and in vitro models such as marinobufagenin, gammabufotalin, resibufogenin, and bufalin. These steroids are especially difficult to identify. However, several compounds and their bioactivities were identified by using different molecular and biotechnological techniques. Biotechnology is a new tool to fully or partially generate upscaled quantities of natural products, which are otherwise only available at trace amounts in organisms. It has also been reported that both pregnenolone and mevalonic acid were poor precursors in winter , but mevalonic acid was involved in the summer (active life of the toad), while cholesterol was potent in both seasons of the year. The outcomes also indicated that the double unsaturated δ-lactone ring of the bufadienolides was directly derived from the cholesterol side chain without the compound’s prior conversion into pregnenolone .,53.α, β-bon atom . This glt muscle ,55. Henct muscle ,56.Nevertheless, terrestrial plants and animal CTS are still underestimated and, consequently, less medicinally explored. The well-known CGs include those isolated from frog venoms. These compounds are mainly bufadienolides.Toads’ (34 genera and 410 species ) CTS areα either alone as in gamabufagin isolated from Bufo japonicus Temminck & Schlegel and 11α-hydroxytelocinobufagin in Rhinella marina L. or at 19β, as in 11α, 19-dihydroxy-telocinobufagin in R. marina. Other structural modifications may also involve 14β, 15β-epoxy ring formations as in marinobufagenin and resibufogenin found in Bufo rubescens (now Rhinella rubescens Lutz) and Bufo gargarizans Cantor, respectively [These toxins are found in the skin of parotoid glands and, consequently, can be extracted from dried secretion of the auricular and skin glands of toads. With the aid of metabolomics, specifically with metabolite profiling with ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) of toad secretions, 31 bufadienolide metabolites have been identified . Some exectively .β-hydroxyl group, 11α-hydroxyl substituent, and aldehyde group at 19 have been shown to increase the antitumor effect, while 5β-hydroxyl, 16β-acetoxy, and 19-CH2-OH substituents decreased the activity [Alternaria alternata (Fr.) Keissl. (Fr.), explained by the production of 12β-hydroxyl and 3-oxo-12α-hydroxyl derivatives. The products exhibited decreased but still significant in vitro cytotoxic activities [Based on Kamano et al. and Gopalakrishnakone et al., structural modifications in the BF molecule can increase or decrease the cytotoxic activity against primary liver carcinoma cells PLC/PRF/5. For example, the acetylation of the 3activity ,59. In ativities .+/K+ ATPase protein affects cell host intracellular signaling processes and viral life at different levels, resulting in inhibition of the viral genome expression, replication, protein translation, release, and entry. Nevertheless, these activities have been thoroughly investigated for CTS [In particular, the antiviral effects have recently attracted interest as compared with the well-documented antitumor activity ,62,63,64 for CTS ,66,67,6850) against enterovirus 71 (EV71) infection in vitro at 10.9 ± 2.4 and 218 ± 31 nM, respectively. EV71 is a pathogen affecting hand, foot, and mouth disease (HFMD) that induces CNS inflammation and life-threatening systemic complications, i.e., cardiorespiratory failure. Viral protein synthesis are likely to be targeted by both compounds [90 at 15 and 40 nM, respectively [The CTS with antiviral activity are reported in ompounds . BF and ectively .+/K+-ATPase. The altered expression of the sodium pump subunits in different cancers strongly suggests that targeting Na+/K+-ATPase represents a novel means to fight the growing number of cancers [+ followed by a decrease of K+ efflux in heart cells. Increases in Ca2+ ions raise the cardiac contractile force, since more Ca2+ is available for the involved proteins. CGs trigger the accumulation of intracellular Ca2+ levels leading to an increase in muscle tone and the circulating blood volume per minute, leading to the control of heart rate and stroke volume [+/K+-ATPase activity [+/K+-ATPase activity are already present at very early stages of tumorigenesis, even before the morphological alteration and the abnormal appearance of tumors [The anticancer activities of cardiotonic steroids (CTS) from different toad species are shown in  cancers . Cardiot cancers . Inhibite volume . Numerouactivity ,78,79,80f tumors ,82. CTS f tumors ,86,87,88f tumors . Additiof tumors ,91.+ levels and depletes the K+ levels. Consequently, the intracytoplasmic Ca2+ levels increase owing to exacerbation of mitochondrial Na+/Ca2+ exchange. Other theories have suggested that CGs may interact with the plasma membrane via the steroid nucleus, causing a change in membrane fluidity and indirectly affecting the function of several membrane proteins and receptors [The antiproliferative activity of CGs on different types of cancer cell lines, including those of breast cancer, prostate cancer, pancreatic cancer, leukemia, neuroblastoma, and melanoma, have been previously described ,92. The eceptors ,94. The eceptors ,95,96. Ieceptors .2/M phases; inhibition of interleukin (IL)-8 production [CGs can induce several signal transduction pathways such as inhibition of DNA topoisomerase II ; inhibitoduction ; and mododuction ,100. CGsoduction . Furtheroduction . Gamabufoduction .BF-induced apoptosis in human leukemia cells  is mediaBF exerts an antitumor effect by inducing apoptosis and triggering autophagic cell death in various human cancer cells. The anti-inflammatory activities of BF are also important for its antitumor function . BF has BF was able to suppress the migration and invasion of prostate cancer cells through HOTAIR, the sponge of miR-520b . In addiThe bufadienolides are a group of steroid compounds belonging to CGs, a class of circulating substances ,130,131.Bufo gargarizans Cantor. This compound is known for its analgesic, anti-inflammatory, anticancer, and anti-radiation properties, as well as its cardio protective and anesthetic activities. RB effectively inhibits cell viability, and induces apoptosis, caspase-3, and caspase-8 activities in MGC-803 cells by suppressing the PI3K/AKT/GSK3β signaling pathway. Therefore, it may be considered as a possible treatment for gastric carcinoma [Resibufogenin (RB) is a commonly used natural medicinal compound extracted from Asiatic toad arcinoma . RB supparcinoma . RB supparcinoma . RB supparcinoma .Some research has suggested that cardiotonic steroids, including bufalin, have immunomodulatory properties , as theyβ [Bufalin has a long history of being used as an anti-inflammatory medicinal drug in China and other Asian countries . Wen et β . NF-κB sβ ,162. Ye β  revealedβ  demonstrβ .The potential pharmaceutical properties of bufadienolides isolated from toad venom have been investigated recently. Anticancer activity of bufalin has been seen against breast, liver, and gastric and leukemia cancer cells, as listed in Anti-inflammatory and anticancer effects of bufalin have been illustrated through the inhibiting NF-B pathway, which is an important pathway in both anti-inflammation and cancer ,169. Buf+/K+-ATPase. Targeting Na+/K+-ATPase and altering the expression of the sodium pump subunits represents a novel means to fight the growing number of cancers. For instance, marinobufagenin has been shown to inhibit the growth of glioma cancer in vivo and in vitro via the sodium pump α1 subunit and ERK signaling-mediated mitochondrial apoptotic pathways. Additionally, γ bufotalin has been reported to suppress vascular endothelial growth factor (VEGF)-triggered proliferation, migration, and invasion by suppressing the VEGF receptor-2 signaling pathway in vitro. Similarly, resibufogenin (RB) is a known anticancer agent that acts via the inhibition of cell viability, induces apoptosis, caspase 3, and caspase 8 activities in MGC 803 cells by suppressing the PI3K/AKT/GSK3β signaling pathway. Finally, bufalin (BF) is active against corona viruses, i.e., SARS-CoV-2 and MERS-CoV, via inhibition of Src signaling mediated by ATP1A1, and thus, blocking entry of the virus into the host cells. As well, BF and cinobufagin inhibit mRNA expression of the hepatitis B virus (HBV) and HIV-1. Furthermore, cinobufagin and resibufogenin have been shown to prevent 71 (EV71) infections from enterovirous. In the current review, we illustrate the significance of cardiotonic steroids as potential anticancer, anti-inflammatory, and antiviral candidates.Using natural products as therapeutic agents has gained much attention, therefore, it is noteworthy to document the recent updates in the literature with particular emphasis on the possible applications of cardiotonic steroids in the promotion of anticancer and antiviral strategies. Limited numbers of CTS have been expected to have anticancer activity. Cardenolides, mainly attributed to digitalis and digoxin, have potential effects in the treatment of congestive heart failure and atrial fibrillation by binding to the extracellular membrane and activating NaFurther studies are suggested to screen the efficacy of CTS isolated from toad species from different geographical regions, and under different environmental conditions to ensure stability of their chemical composition. Additional molecular mechanisms and modeling, drug safety, and quality control are needed to motivate the future implications of CTS as novel therapeutic agents that will certainly contribute to the development of pharmaceutical industry.


The major active form of vitamin D, 1,25-dihydroxyvitamin D3 , is known for its wide bioactivity in periodontal tissues. Although the exact mechanisms underlying its protective action against periodontitis remain unclear, recent studies have shown that 1,25D3 regulates autophagy. Autophagy is vital for intracellular pathogen invasion control, inflammation regulation, and bone metabolic balance in periodontal tissue homeostasis, and its regulation could be an interesting pathway for future periodontal studies. Since vitamin D deficiency is a worldwide health problem, its role as a potential regulator of autophagy provides new insights into periodontal diseases. Based on this premise, this narrative literature review aimed to investigate the possible connection between 1,25D3 and autophagy in periodontitis. A comprehensive literature search was conducted on PubMed using the following keywords . In this review, the latest studies on the protective action of 1,25D3 against periodontitis and the regulation of autophagy by 1,25D3 are summarized, and the potential role of 1,25D3-activated autophagy in the pathogenesis of periodontitis is analyzed. 1,25D3 can exert a protective effect against periodontitis through different signaling pathways in the pathogenesis of periodontitis, and at least part of this regulatory effect is achieved through the activation of the autophagic response. This review will help clarify the relationship between 1,25D3 and autophagy in the homeostasis of periodontal tissues and provide perspectives for researchers to optimize prevention and treatment strategies in the future. Periodontitis is a complex infectious disease that destroys periodontal tissues and has various etiological and contributing factors. It is highly prevalent in populations worldwide . The dynVD is a fat-soluble vitamin and precursor of steroid hormones. After two hydroxylations mainly in the liver and kidney (or some other tissues) , it is cAutophagy, a highly conserved lysosomal degradation process, is central to maintaining organismal homeostasis. Alterations in autophagy have been associated with various diseases, including periodontitis . Its potCompared to antibiotics that may lead to bacterial resistance and some allergic reactions, 1,25D3 has a high safety profile because it modulates innate immunity  and autophagy) to exert antimicrobial effects and may also directly act on bacteria  might also result from 1,25D3-induced LL-37 [AMPs, including cathelicidin, β-defensins, and S100 proteins, are mainly produced by immune and epithelial cells . LL-37, ed LL-37 . When thed LL-37 , and saled LL-37 . These rFusobacterium nucleatum (F. nucleatum), A. actinomycetemcomitans, Solobacterium moorei, and Streptococcus mutans (S. mutans) at high concentrations (≥ 100 μg/mL), whereas 1,25D3 has been found to exert specific antibacterial activity against Porphyromonas gingivalis  at very low concentrations . In addition, 1,25D3 can significantly reduce the gene expression of virulence factors involved in bacterial colonization  and factors involved in tissue destruction  [P. gingivalis when combined with metronidazole. In combination with tetracycline, 1,25D3 showed an additive effect [Recently, a few studies have reported a direct effect of 1,25D3 on some bacterial cells. Due to its strong lipid solubility, cell membrane integrity might be altered, and permeability to other substances, such as antibiotics, might be increased , 23. 1,2and kgp) . Unlike e effect .The oral epithelial barrier separates the host from the oral environment, and the body’s natural physiological barrier prevents pathogens and exogenous substances from entering the deep tissues. In the gingival epithelium, oral keratinocytes are the primary cell type connected by various transmembrane proteins with special structures and functions, such as tight junctions, adherens junctions, and gap junctions . Tight jEscherichia coli LPS, including phospho-p65 (p-p65) and active caspase 3/9, were also reduced by 1,25D3 [Increased apoptosis of oral epithelial cells can disrupt the mucosal barrier and accelerate bacterial invasion. 1,25D3/VDR reduces oral keratinocyte apoptosis by inhibiting the activation of NF-κB-dependent p53-upregulated modulator of apoptosis (PUMA), which is a key pro-apoptotic regulator. In addition, other apoptogenic factors induced by y 1,25D3 .P. gingivalis, while in other periodontal tissue cells, such as HGFs and HPDLCs, 1,25D3 was also found to reduce inflammatory levels [1,25D3/VDR can reduce the inflammatory response in both von Hippel-Lindau (VHL)- and NF-κB-dependent pathways , 30. Humy levels , 33.These findings suggest that the inhibition of NF-κB signaling by 1,25D3 plays an important role in enhancing intercellular junctions, reducing apoptosis, and relieving the inflammatory response in oral epithelial cells  and increase the expression of osteogenesis-related factors  , 50. HowAutophagy is a major intracellular degradation process in which cytoplasmic components  are delivered to lysosomes for degradation . AutophaIn recent years, many studies have found that in addition to affecting calcium and phosphorus metabolism and regulating immunity and infection, 1,25D3 also mediates autophagy via genomic and non-genomic signaling pathways to influence the physiological functions of different organs . VD defi2+ release [Mycobacterium tuberculosis (Mtb)-infected monocytes. Cathelicidin LL-37 is a downstream target gene that promotes the fusion of autophagosomes and lysosomes to form autolysosome [MAP1LC31B) which is constitutively repressed by VDR [In detail, 1,25D3/VDR can induce autophagy by increasing the levels of cytosolic free calcium and downregulating the expression of mammalian target of rapamycin (mTOR) and Bcl-2 which represses Ca release . Regulat release , 56. Bec release . Additiolysosome . Moreovelysosome . Anotherlysosome . In addilysosome . Treatmed by VDR  d by VDR , which iIt was found that compared to healthy gingival tissues, inflammatory sites from naturally occurring periodontitis of rhesus monkeys showed significant alterations in the expression of some autophagy-related genes, suggesting that autophagy may be impaired in periodontal lesions and involved in the pathogenesis of periodontitis . Other hSalmonella colitis [Although the specific mechanism remains unclear, there have been already some in vivo and in vitro studies supporting the hypothesis of the involvement of autophagy regulation in the protective effects of vitamin D in other infectious and inflammatory diseases such as  colitis , UV-medi colitis , allergi colitis , and ost colitis . The pot colitis . HoweverSalmonella clearance in intestinal epithelia and appears to be a promising treatment strategy for the control of Mtb infection [P. gingivalis, a major opportunistic pathogen, can induce autophagy with different functions in phagocytic (macrophages and dendritic cells) and non-phagocytic cells  after internalization [P. gingivalis internalized by macrophages and dendritic cells. However, to avoid clearance by the host immune system, P. gingivalis has developed specific survival strategies against GECs. In GECs and human coronary artery endothelial cells (HCAECs), P. gingivalis impairs the formation of autolysosomes to escape lysosomal degradation and replicate inside autophagosome vacuoles for persistent intracellular survival [P. gingivalis-induced autophagy provides a favorable microenvironment for replication, survival, and dissemination in the GECs and HCAECs, indicting its significant role in the progression of periodontitis and atherosclerosis [P. gingivalis in epithelial cells could become effective via increased number of autophagosome vacuoles and promoted fusion of autophagosomes and lysosomes. 1,25D3 significantly reduced the number of live P. gingivalis internalized into HeLa cell subline KB cells and U937 cells by promoting autophagy in a dose-dependent manner ; this process inhibits the intracellular survival of the bacteria and significantly reduces the number of JEKs undergoing cell death [A. actinomycetemcomitans in cultured GEC [Autophagy activated in infected cells is involved in intracellular antimicrobial defense mechanisms via a lysosomal degradation pathway . Active nfection . In perilization –75. Autosurvival , 75. P. clerosis , 76. Intner Fig. A. The anll death . 1,25D3 ured GEC . HoweverInterestingly, excessive autophagy or inadequate activation of autophagy may lead to cellular damage or even death . ButyratP. gingivalis and A. actinomycetemcomitans is inhibited by enhanced autophagy [P. gingivalis in U937-derived macrophages decreased in a dose-dependent manner. Its mechanism of action may be related to the degradation of live P. gingivalis due to the 1,25D3-promoted co-localization of P. gingivalis with autophagosome and lysosomal markers [P. gingivalis within dendritic cells is impaired by rapamycin-induced autophagy [P. gingivalis by dendritic cells results in two scenarios: blocking autophagy for survival and promoting autophagy for degradation. Using autophagy promoters could help promote the killing of pathogens and periodontitis resolution, thus providing insights into a novel therapeutic approach [1,25D3 plays a pivotal role in regulating immunity through autophagy, providing an antimicrobial defense mechanism against pathogens that invade immune cells. 1,25D3-induced autophagy is critical for the elimination of intracellular Mtb in human monocytes/macrophages , and catutophagy , 85. It  markers . Moreoveutophagy . The recapproach .P. gingivalis [In addition, autophagy has become more interrelated with TLRs signaling. TLR signaling stimulated by TLR ligands is important for the initiation and regulation of autophagy activation . Additiongivalis , 92, 93.+ T cell counts and a decrease in CD8+ T cell counts [Autophagy is also considered a regulator of T cells, affecting T cell function, differentiation, and metabolism . In patil counts .Autophagy activation can limit excessive inflammation in periodontal tissue by inhibiting IL-1β secretion, NLRP3 inflammasome formation, and reactive oxygen species (ROS) accumulation , 95–97, IL-1β amplifies periodontal inflammation and plays an important role in tissue destruction. LPS-induced p-p65 activates the NLRP3 inflammasome in immune cells by binding to NF-κB sites in the promoter region of NLRP3 . The NLRIn addition, 1,25D3 is known to induce autophagy to inhibit apoptosis in some diseases. For instance, 1,25D3 prevents influenza virus-induced cellular apoptosis by restoring autophagic flux, providing a therapeutic strategy for viral infection  Fig. 4CC4C.Since VDR exists widely in vascular endothelial cells and smooth muscle cells, the regulatory role of 1,25D3 in angiogenesis and vascular cell activity has been reported . StudiesAlveolar bone homeostasis is tightly controlled by the balance between osteoclastogenesis and osteoblastogenesis. In periodontitis, an imbalance favoring bone resorption leads to pathological resorption of alveolar bone . AutophaThe above findings suggest that the regulation of autophagy in bone cells may have therapeutic implications . 1,25D3,The protective role of 1,25D3 in the pathogenesis of periodontitis, including the clearance of periodontal pathogens, maintenance of the epithelial barrier, relief from inflammation, and reduction of alveolar bone loss, may be achieved, in part, through the regulation of autophagy. 1,25D3 signaling regulates autophagy, and that the regulation of autophagy is important for periodontal health. Given that autophagy is involved in the protective effect of 1,25D3 on infection, inflammation and bone metabolism in various diseases, further studies on the connection between 1,25D3 and autophagy in periodontitis may reveal the therapeutic potential of 1,25D3 and new strategies for periodontal prevention and treatment.


Thus, any change in oxygen pressure leads to modulated activation of the hypoxia pathway, which affects numerous physiological and pathological processes, including hematopoiesis, inflammation, and tumor development. The Hypoxia Inducible Factors (HIFs) are essential transcription factors and the driving force of the hypoxia pathway; whereas, their inhibitors, HIF prolyl hydroxylase domain (PHDs) proteins are the true oxygen sensors that critically regulate this response. Recently, we and others have described the central role of the PHD/HIF axis in various compartments of the adrenal gland and its potential influence in associated tumors, including pheochromocytomas and paragangliomas. Here, we provide an overview of the most recent findings on the hypoxia signaling pathway  Oxygen is essential for the functioning and survival of cells and tissues because it is required for cellular energy production and as a cofactor/substrate for various enzymes; thus, the absence of sufficient oxygen pressure results in hypoxia. However, the term hypoxia is relative and should be used in the correct context as normal oxygen levels vary among tissues, e.g., from ~13% in arterial blood to ~4% in the brain , 3. FurtStructurally, each functional HIF transcription factor is a heterodimer of two subunits, an oxygen- sensitive HIF-α subunit and a constitutively expressed HIF-1β subunit, and both these subunits belong to the basic helix–loop-helix (HLH)-PER-ARNT-SIM (bHLH-PAS) protein family , 7. The Another oxygen-dependent mechanism of HIF regulation involves Factor inhibiting HIF-1 (FIH1), an asparaginyl hydroxylase, which hydroxylates the HIF-α subunit at the asparagine in the C-terminal activation domain (N-803 in human HIF-1α) under normoxia and mild hypoxia. Such hydroxylation prevents activation of HIF as it inhibits interactions between HIF-1α and its co-activators p300/CBP . FIH1 alWhile the above pertained to oxygen-dependent regulation of HIFs, various tumor suppressor and oncogenic pathways (such as MAPK/ERK and PI3K/AKT) non-specifically regulate HIFs in an oxygen-independent manner. Tumor suppressors such as p53 and GSK3β decrease HIF-1α stability or transcriptional activity and thereby interfere with HIF function ; in contNotably, even though both HIF-1α and HIF-2α contain similar binding and dimerization domains, they have different transactivation domains . TherefoSolid tumors are characterized by hypoxic or even anoxic regions within the tumor mass because existing blood vessels fail to meet the oxygen requirements of the rapidly proliferating cancer cells . The conEndocrine tumors can originate in any of the hormone-producing endocrine organs— thyroid, pituitary glands, pancreas, and adrenal gland, and their nomenclature reflects both origin and location. Thyroid cancer is considered the most common malignancy among all endocrine tumors and anaplastic thyroid carcinomas (ATCs) are predominantly aggressive tumors with an average survival of 3–4 months , 41. On Processes and factors such as signaling pathways, metabolic reprogramming, extracellular matrix remodeling, and epigenetic changes regulate the development and propagation of endocrine tumors , 43. As Pheochromocytomas (PCCs) and paragangliomas  are unique neuroendocrine tumors that make up less than 1% of all endocrine neoplasia. PCCs arise from chromaffin cells of the adrenal medulla whereas PGLs are extra-adrenal, neural crest-derived, neuroendocrine tumors (NETs) of the sympathetic and parasympathetic ganglia , 49. PGLSDH, VHL, fumarate hydratase (FH) and EPAS1, whereas cluster 2 PPGLs bears mutations in NF1, rearranged during transfection (RET) proto-oncogene, TMEM127, and MAX. While cluster 1 PPGLs are characterized by an immature catecholamine phenotype (noradrenergic phenotype) and higher aggressiveness, cluster 2 PPGLs have more mature phenotype (adrenergic phenotype) and are mostly non-metastatic  and recurrent fusions involving MAML3 and a cortical admixture subtype , which is required for controlling apoptosis in PCC cells (Approximately 35–40% of PCCs and PGLs have a hereditary predisposition that is attributable to germline pathogenic variants (PVs) in over twenty susceptibility genes –55. Ratetastatic , 58. Howntiation . It has CC cells , 61.VHL1, the SDH complex  and occasionally, the egl-9 family hypoxia-inducible factor 1/Prolyl hydroxylase domain 2 protein (EGLN1/PHD2) (EPAS1) (EGLN2)  , 63. Rec (EPAS1) , 65, FH  (EPAS1) , and PHD (EGLN2) , suggestVHL) disease is an autosomal dominant neoplastic disorder that is characterized by multiple benign and malignant tumors, including cysts, that develop in the central nervous system and visceral organs  in the adrenal medulla , erythropoietin (EPO) and EPO-receptor, cyclin D1 (CCND1), and other genes involved in extracellular matrix reorganization that facilitate tumor development  or type - 2, which is linked to missense mutations . About 2tastasis , 74. Typtastasis , 76. Addtastasis , 78. Nevtastasis , 80. As  medulla , 81–83. elopment , 84. Nonelopment , and som3 levels , 86, 87.SDHx) family comprising of 4 SDH subunits   , 88. Mutf HIF-2α –91.SDHx-associated PCCs/PGLs also have a norepinephrine/normetanephrine predominant profile   , 95. FurEGLN1, is a crucial oxygen sensor that regulates HIFα levels  in the absence of polycythemia  TH-restricted Cre mouse line, resembles a combination of pseudohypoxic PGL and a PNMT negative noradrenergic phenotype  represent a rare, aggressive, and heterogeneous tumors that arises from the cortex of the adrenal gland. The 5-year survival rate for ACC ranges from 16% to 47% . As mentKCNJ5, ATP1A1, ATP2B3, CACNA1D, CACNA1H, CLCN2, and CTNNB1  are benign aldosterone producing tumors associated with Primary aldosteronism (PA) causing secondary hypertension . These ad CTNNB1 , 129. HoMixed corticomedullary tumor (MCT), an extremely rare condition with unclear tumorigenesis, is an adrenal tumor with cortical and medullary cells. Adrenal MCT was first described by Mathison and Water-House in 1969 and only 30 cases have been reported to date. Adrenal tumors, including MCTs, exhibit different stemness expression , e.g., aAs with many types of solid tumors, hypoxia due to inadequate vascularization is a prominent micro-environmental component in thyroid cancer . Differe), VEGF, and VEGFR2 in thyroid cancer  is 0–5.3 mmHg (0–0.7%); in contrast, the pO2 of the adjacent normal pancreatic tissue is 24.3–92.7 mmHg (3.2–12.3%)  . Thus, p2–12.3%) . More si2–12.3%) . Patient2–12.3%) .TCF7L2/TCF4) plays a vital role in the Wnt/β-catenin signaling pathway, is responsible for PHD1 (EGLN2) silencing, leads to upregulation of HIF-1α, and affects glycolysis reprogramming  are noncoding, about 20–22 nucleotides in length, and regulate gene expression by binding to 3′ UTRs of their related parental mRNAs. MiRNAs have been shown to control many physiological and pathophysiological processes, such as proliferation, differentiation, metabolism, and apoptosis by modulating target gene expression. Altered miRNA expression has been identified in several endocrine diseases, including tumors. For example, the 14q32 miRNA cluster is frequently dysregulated in human diseases and has been implicated in tumorigenesis of multiple endocrine glands . These 1SDHB-mutated tumors, respectively (SDHD-mutant malignant PCC tissues (Several miRNA expression profiling studies have been performed on different endocrine tumors including PCC/PGL, and repression of 2 miRs, namely, miR-15a and miR-16, has been reported in pituitary adenomas and prostate cancer . Furtherectively . Increasectively . Moreove tissues , and miR tissues , 157.VHL and SDHB germline mutations (SDHx germline mutations, indicating that the HIF-1α/miR-210/SDHB axis may play a role in the pathogenesis of PGLs (MiR-210, a recent discovery in the field of hypoxia, is the most consistently and predominantly upregulated miRNA in response to hypoxia. Functional studies have demonstrated that miR-210 is a central gene that regulates many aspects of the hypoxia pathway, both under physiological and malignant conditions . MiR-210utations . However of PGLs . Indeed, of PGLs  (Figure in vitro and in vivo (Targeting tumor hypoxia and HIFs is an extremely interesting therapeutic approach as tumor hypoxia mediates the aggressive, the metastatic, and the resistant phenotypes. Different approaches have been developed to target hypoxic cancer cells, such as gene therapy, hypoxia-activated prodrugs, recombinant anaerobic bacteria, pathways important in hypoxic cells like mTOR and UPR, and specific targeting of HIFs –165. How in vivo  and a ph in vivo .VHL germline mutations. Another inhibitor of HIF-2α, PT2977, was found to have increased potency and better pharmacokinetic profile than PT2385 and PT1977 (HIF-2α inhibitors are thought to possess great prospects for the treatment of advanced PPGL  and thesd PT1977 . Previoud PT1977 , 173 andd PT1977 . Thus, HVHL, SDHx, EPAS1 and EGLN1 are associated with endocrine tumor initiation, many of these mutations directly or indirectly lead to stabilization of HIFs and consequently triggering the hypoxia pathway. This contributes to tumor development either by upregulation of oncogenes or genes facilitating tumor progression such as angiogenesis, skewed glycolysis or suppression of apoptosis in tumor cells. Therefore, considering the essential role of hypoxia signaling in endocrine tumors, the development of HIF inhibitors as therapeutic agents would be a potential anti-tumor strategy. Considering the upregulation of HIF-2α in many PCC/PGLs, HIF-2α inhibitors are potentially most promising, although more research is certainly warranted.Taken together, this review underlines that the hypoxia pathway proteins are essential contributors in the development and progression of endocrine tumors. Even though several mutations such as DW and MJ, wrote the manuscript. NB and BW contributed to the discussion and edited the manuscript. All authors contributed to the article and approved the submitted version.


The airway epithelial glycocalyx plays an important role in preventing severe acute respiratory syndrome coronavirus 2 entry into the epithelial cells, while the endothelial glycocalyx contributes to vascular permeability and tone, as well as modulating immune, inflammatory, and coagulation responses. With ample evidence in the scientific literature that coronavirus disease 2019 (COVID-19) is related to epithelial and endothelial dysfunction, preserving the glycocalyx should be the main focus of any COVID-19 treatment protocol. The most studied functional unit of the glycocalyx is the glycosaminoglycan heparan sulfate, where the degree and position of the sulfate groups determine the biological activity. N-acetylcysteine (NAC) and other sulfur donors contribute to the inorganic sulfate pool, the rate-limiting molecule in sulfation. NAC is not only a precursor to glutathione but also converts to hydrogen sulfide, inorganic sulfate, taurine, Coenzyme A, and albumin. By optimising inorganic sulfate availability, and therefore sulfation, it is proposed that COVID-19 can be prevented or at least most of the symptoms attenuated. A comprehensive COVID-19 treatment protocol is needed to preserve the glycocalyx in both the prevention and treatment of COVID-19. The use of NAC at a dosage of 600 mg bid for the prevention of COVID-19 is proposed, but a higher dosage of NAC (1200 mg bid) should be administered upon the first onset of symptoms. In the severe to critically ill, it is advised that IV NAC should be administered immediately upon hospital admission, and in the late stage of the disease, IV sodium thiosulfate should be considered. Doxycycline as a protease inhibitor will prevent shedding and further degradation of the glycocalyx. The pandemic surrounding coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, has been associated with immense economic and social disruption around the globe, apart from high mortality rates. More than two years later, there is still a need to find effective therapeutic solutions to prevent and treat established disease. Because of their various physiological functions in viral infection and inflammation, the use of N-acetylcysteine (NAC) and sodium thiosulfate (STS) [We have recently reviewed the pathogenesis of COVID-19  that proSince the GL surrounds all eukaryotic cells, the EpGL serves as the interface between our internal cells and the external world. It therefore serves as the first defence of the innate immune system, and if compromised, we are vulnerable to the onslaught of various pathogens, viruses, and environmental toxins . The GL Sulfation occurs in all tissues and involves the transfer of a sulfate group to various substrates, such as proteins, GAGs, lipids, hormones, various drugs, and toxins –18, 3, , 3, 19. Pre-COVID-19 vaccination, the elderly were most susceptible to SARS-CoV-2, where the typical profile of the critically ill patient was 65 years and older, presenting with comorbidities and ARDS , 31. TheSince we are all exposed to SARS-CoV-2, the main question is why more than 80% of the world population did not become infected before the introduction of the COVID-19 vaccine, were asymptomatic, or showed only very mild symptoms –46, compThe extracellular matrix that surrounds all cells is a complex external layer of PGs, glycoproteins bound to sialic acid, GAGs, glycolipids, and proteins such as collagen , 48. ThiGlycosaminoglycan chains that bind to PGs are long linear, hydrophilic , negativIn mammalian cells, the sulfation process begins with the uptake of inorganic sulfate from the extracellular milieu , where tUnder normal healthy physiological conditions, HSPGs interact with and modulate the activity of several molecules , includiOf relevance to COVID-19, the primary role of the EpGL is the ability of a highly sulfated intact, negatively charged GL, to repel SARS-CoV-2 through electrostatic forces, thereby preventing cell invasion . Most viIn COVID-19 patients that present with severe symptoms, sepsis has set in, which is a complicated condition that can be defined as severe organ dysfunction caused by an uncontrollable host response to infection . After ePulmonary EnGL degradation increases the availability of En surface adhesion molecules to circulating microspheres. It contributes to neutrophil adhesion, leading to diffuse alveolar damage, interstitial lung oedema, and clot formation during acute lung injury in sepsis and ARDS , 68, 78.α (TNFα) [β, IL-6, and IL-10 [Apart from viral infections , multiplα (TNFα) , 85, intnd IL-10 , 57, 58,nd IL-10 , 65, 87,Preserving the integrity of the GL is paramount both in the prevention and treatment of infectious diseases. Since most of the physiological properties of the GL are determined by the degree of GAG sulfation, ways to optimise inorganic sulfate, the rate-limiting substrate for sulfation, should be the primary focus of treatment. The intact Ep- and EnGL, with adequate sulfated GAGs, prevent viral infection and replication , determi2S) [Most of the literature to date focuses on NAC as a precursor to glutathione (GSH) , 90–93. 2S) , 96, 97,2S) , taurine2S) , 98, Coe2S)  and albu2S)  Figure S [FigureSince SARS-CoV-2 infection has been demonstrated to cause rapid depletion of sulfur amino acids (SAAs) due to oxidative stress or inflammation-induced proteolysis , amino aα receptor activity towards a proinflammatory state [−/− genotype or Ile 105Val glutathione S-transferase P1 (GSTP1) polymorphism, were more susceptible to infection and had higher mortality rates [Since Cys is the rate-limiting substrate in GSH and inorganic sulfate intracellular synthesis, low Cys levels will therefore result in lower GSH and inorganic sulfate levels. Through a series of redox reactions in the plasma, NAC directly affects the amino acid pool of extracellular cystine and intracellular Cys , 31. NACry state , 31, 95.ry state , 105. Thry state , 37, higry state , 106. Erry state , while dry state , 109, onry state  and consry state , 110. Bary state , 111. Evry state , 113 andty rates , 115, thty rates .2S supports basal, physiological, cellular bioenergetic functions, while the activity of this metabolic support decreases with physiological ageing [2S as an endogenous gaseous transmitter have been established over the last decade [2S is produced, respectively, by cystathionine-β-synthase (CBS), CSE, and 3-mercaptopyruvate sulfurtransferase (3-MST) [2S serves as a stimulator of electron transport in mammalian cells, by acting as a mitochondrial electron donor [2S as an “emergency” substrate that balances and complements the electron-donating effect of Krebs cycle-derived electron donors [2S concentration is likely to be constantly in dynamic equilibrium with a much larger pool of bound forms of sulfur, including thiol groups of proteins. Mitochondrial production of H2S starts with Cys [2S as electron donor [2S catabolic pathways in the mitochondria leads to the formation of thiosulfate, which is ultimately converted into sulfate [2S would be favoured as an electron donor under hypoxic and stress conditions. This will affect the degree of HS sulfation [2S and ROS levels, mediated through hypoxic conditions [Endogenous Hl ageing . Multiplt decade , 97, 118 (3-MST) , 96, 118on donor , 118. Iton donor  favour Hn donors . Of notewith Cys , 117, anon donor , with a on donor . Even thulfation . Moreovenditions , may synnditions . Indeed,nditions .2S is extensively studied because of the role it plays as a proinflammatory mediator at high concentrations. Inhibition of endogenous H2S alleviates the diseased inflammatory condition, whereas exogenous H2S, released from natural sulfur compounds and SAAs, has shown protective effects in biological systems [2S is stimulatory at lower concentrations, while it has an inhibitory effect on cellular bioenergetic functions at higher concentrations [2S concentration [2S at low physiological concentrations [α, and IL-6 [2S attenuates pulmonary tissue injury by inducing ACE2 upregulation, while H2S may exhibit its antiviral activity against SARS-CoV-2 by interfering with both ACE2 and transmembrane protease serine 2 [2S levels than those found in patients with severe COVID-19 pneumonia [2S concentration must remain in a homeostatic balance to exert its protective effects. Even though Cys is the rate-limiting substrate for H2S production, pyridoxal 5′-phosphate (vitamin B6) is a very important cofactor, not only supporting CBS and CSE, but also the conversion of Cys to inorganic sulfate via CDO, along with iron. When supplementing with NAC to increase Cys levels, it is imperative to ensure a deficiency of the important cofactor nutrients does not impair the metabolic conversions in the sulfur metabolism pathway.Intrinsically synthesised H systems , 119. H2trations . Inflammntration , 119. Cotrations , 97, 122trations , 123, mutrations , as welland IL-6 . Moreoveserine 2 . COVID-1neumonia , 96. It β, TNFα, and transforming growth factor-beta (TGF-β), downregulate CDO at mRNA level [Even though a small percentage of inorganic sulfate is found in foods and various sources of drinking water, the major source of inorganic sulfate for humans is from the biodegradation of dietary proteins. First, the essential sulfur amino acid methionine (Met) is converted to Cys . ThereafNA level , 124. ThNA level .2S levels and consequently membrane depolarisation [Most hospitalised COVID-19 patients in ICU are being placed on enteral nutrition . Met is risation , even morisation  and apoprisation .Cys is one of the amino acids in the hepatic precursor amino acid pool involved in both albumin and inorganic sulfate synthesis. The close arrangement between albumin synthesis and catabolic rates  suggestsκB) activation and TNFα-induced production of cytokines and ROS, thereby preventing upregulation of IL-6. It is well established that STS is a potent antioxidant and anti-inflammatory agent, and it has been used to treat cyanide poisoning [2S donor [Apart from NAC and HSA being sulfur donors, STS, methylsulfonylmethane (MSM) , and suloisoning , 142 andoisoning  with a r2S donor . Taken t2S donor .α, malondialdehyde (MDA), and myeloperoxidase (MPO) levels, and an increase in SOD, GSH, and catalase (CAT) levels in lung and liver tissues [κB signaling [κB to the nucleus, therefore minimising downstream events associated with local and systemic inflammation [β levels and protected against hepatic liver injury by decreasing TNFα and IL-6 levels [β expression. Moreover, MSM reduced caerulein-induced H2S levels by decreasing CSE expression in the lungs and pancreas, and increased CD34+ expression [As a source of organic sulfur, MSM will increase the synthesis of GSH  and inor tissues . Severalignaling , 146. Kaammation –149. Indammation , 150. Th6 levels , 150. InUnder normal physiological conditions, oncotic pressure in the vascular system is maintained by HSA . The sheHypoalbuminemia, vascular disease, and coagulopathy have all been linked to COVID-19 and have been shown to predict outcomes independent of age and morbidity . All theThere is no doubt that the binding of SARS-CoV-2 to ACE2 receptors influences various processes, such as vasoconstriction, kidney injury, cardiovascular disease, apoptosis, and oxidative processes , but thePlasma proteins, such as albumin , 68, 80 2S, GSH, and albumin demonstrate many similar physiological functions, their donation of sulfur [NAC and Cys play an essential role as the rate-limiting substrate in many vital physiological processes, and it is crucial to understand the homeostatic balance between the sulfur substrates to maintain physiological function . It seemf sulfur , 157 forVarious research studies indicated that NAC modulates the immune system , inhibitβ, IL-8, and TNFα; prevents systemic endotoxemia and inflammatory response by improving gut barrier dysfunction [Apart from being a precursor to GSH, various other mechanisms have been ascribed to NAC's antioxidant and anti-inflammatory properties. NAC downregulates inflammasome NLRP3 mRNA expression, therefore decreasing proinflammatory cytokine expression and release from activated mononuclear phagocytes; NAC inhibits the endotoxin-induced release of IL-1function , where pfunction . NAC alsfunction , 170. Thfunction . Its immfunction , 173. MaκB pathway support within host cells [κB is a mediator of SARS-CoV-2 pulmonary pathology, since it triggers the production of numerous proinflammatory cytokines [κB [κB [κB [κB and MCP-1 levels [Oxidative processes enhance viral infection by promoting replication in infected cells, inhibition of cell proliferation, and cell apoptosis induction , 95. Stust cells . In a reytokines . It was ines [κB  signific [κB [κB , 95, 105 [κB [κB , 165, he [κB [κB . In addi [κB [κB , 160. Th [κB [κB , because [κB [κB . The act [κB [κB . Since N1 levels , it demo1 levels . Further1 levels , 113. In1 levels .NAC was also shown to protect GL shedding, since HS is expected to restore the GL when it is shed from EnCs . Pecora Various case reports using NAC to treat COVID-19 patients successfully have recently appeared in the literature , 160. Inin vivo NAC modifies the function of the renin/angiotensin system, which is probably mediated by inhibition of ACE2 activity. By blocking ACE2, NAC will potentially protect patients from the deleterious effects of angiotensin-2, which seems to be a potentially useful strategy in SARS-CoV-2 infection [Even though various clinical studies reported contradicting evidence , NAC counfection , 162. NAnfection , may dranfection , 94, 160nfection .Furthermore, since proteases, such as thrombin, have been reported to support the cleavage of syndecan 1 PG ectodomains, thus facilitating shedding of the GL, the therapeutic use of protease inhibitors should be considered . Among tThe main goal of any COVID-19 treatment regimen should be the preservation of an intact well-sulfated GL , 49, 82.Even though we propose that NAC and STS can be used as first-line therapy, their application would complement any other treatment protocol as a safe adjunct intervention. COVID-19 is a complex disease that cannot be treated with a single drug approach. Various strategies to preserve the intact sulfated GL need to be applied. It is, however, essential to prescribe a sulfur donor such as NAC or STS when drugs that are administered need sulfation for their metabolism, such as steroids, aspirin, colchicine, acetaminophen, and nonsteroidal anti-inflammatory drugs , 187; 1, 189. ApμM NAC in the peripheral blood within half an hour after administration [We propose that for the prevention of COVID-19 in the general population, especially in health care workers, frontline personnel, and those at high risk with comorbidities, a cysteine derivative such as NAC , carbocistration . Oral NAstration , 95. It stration  can imprstration , 160. Asstration . Upon thstration , 160. Nostration . Other mstration , seleniustration , 196, mastration , 197, shstration , while sstration , or maristration . These sstration , or the stration , 43.2+/H2O2-dependent oxidative stress and increase superoxide radical formation [In patients experiencing moderate to severe COVID-19 symptoms, IV NAC administration upon hospital admission should be considered as a standard practice of care, if allowed within the safety protocols of the hospital. The idea is to preserve and repair the GL to prevent a cytokine storm, and the sooner proper measures can be applied, the better the outcome that could be expected . Shi & Pormation . To offsormation , 201. Itormation , 193 at ormation . Neverthormation . Since Hormation , 87, 183p < 0.05) [In the severe to critically ill COVID-19 patients, either IV NAC or IV STS should be considered, where the dosage and timeframe will be critical. STS is expected to be a better option to modulate the cytokine storm in the critically ill, since higher levels of cytokines will hamper the conversion of NAC to sulfate , 124, wh < 0.05) . During  < 0.05) .nd or 3rd day until there is no viral load present. In the paediatric group of 0–18 years, 1 mL/kg of body weight (250 mg/kg or approximately 30–40 mL/m2 of BSA) (rate of 2.5 to 5 mL/minute) should be given, not to exceed 50 mL of the total dose of STS [We propose STS dosage to be the same as the standard treatment dosage given to patients with acute cyanide poisoning  or calcie of STS .The cytokine storm experienced in the critically ill will result in severe shedding of the GL that should be attenuated at all costs. One can consider the addition of dexamethasone with NAC as a combined anti-inflammatory therapy , as wellSteroids, aspirin, colchicine, acetaminophen, and nonsteroidal anti-inflammatory drugs appear to enhance viral replication and aggravate Ep and En dysfunction and the cytokine storm , 204 thrWhey protein, high in cysteine and albumin, should be added to the diet , 134 in Administer albumin , 64, 68 Note that high dosages of vitamin C may cause hyperoxaluria through endogenous conversion of ascorbic acid to oxalate . Both viSeen that NAC has powerful anticoagulatory effects, adjust dosages of blood thinning medications accordingly.Ventilation (VT) – avoid high tidal volume that could attenuate the lung EpGL , 209. HoAvoid hypervolemia and hypernatremia, which are injurious to the EnGL , 69, 208Although Etanercept has been proposed for use in COVID-19 to reduce the mortality of toxic epidermal necrolysis (TEN) , Atef etCheck for G6PD deficiency before HCQ is administered, and if present, give IV NAC to avoid the risk of haemolysis in G6PD-deficient patients , 181.Limit the use of antibiotics unless there is a known secondary infection. Gut dysbiosis seems to be an underlying risk factor for COVID-19 , 214.It is best to avoid the use of statins in COVID-19 patients, since they may cause rhabdomyolysis as a complication .β, IL-16, and TNFα, cause injury to the BBB [Even though Ivermectin shows great potential as an inhibitor of SARS-CoV-2 replication , it is c the BBB , 220 andSeveral clinical trials are currently evaluating the efficacy of NAC as therapeutic for COVID-19 , 123, buAfter a comprehensive COVID-19 vaccine roll-out, the appearance of new variants with multiple mutations provides evidence that they are not effective; neither do they stop transmission nor infectivity, making it even more imperative to establish effective therapeutic alternatives to prevent viral infection. NAC would also appear to be a very good antidote against the possible adverse reactions experienced in susceptible individuals who received any of the COVID-19 vaccines. NAC may bind to the spike proteins, acting as a decoy to prevent binding to HS and consequent potential shedding of the GL. NAC will also facilitate detoxification of the adjuvants used in the vaccines, apart from restoring GSH levels depleted by the presence of ethylene glycol  and grapSulfated GAGs and various sulfur donor/thiol compounds regulate innate , 30 and Since NAC demonstrates multiple mechanisms of action, it is more likely to be effective than drugs having a single target. Moreover, since NAC addresses the underlying causes of COVID-19, it should have a beneficial effect on preventing and reducing long-COVID symptoms. Based on the herein discussed mechanistic premises, we propose using NAC in both the prevention and treatment of COVID-19. By better understanding the pathophysiological role of GAG sulfation , therapiThis review does not only emphasise the important clinical role of NAC in the prevention and treatment of COVID-19, but also highlights the physiological benefits of NAC as a precursor to inorganic sulfate. We believe that many of the physiological functions that have been attributed to NAC as a precursor to GSH in the literature to date, could in fact have been the action of inorganic sulfate, the rate-limiting precursor to sulfation. The application of NAC is therefore far-reaching beyond the treatment of COVID-19 or as an antidote to acetaminophen overdose.


Aggregation-induced emission (AIE) is an extraordinaryphotochemicalphenomenon described by Tang’s group in 2001, where the aggregationof some organic molecules enhances their light emission by limitingintramolecular activity in the aggregate state. This phenomenon offersnew opportunities for researchers due to its potential applicationsin optoelectronics, energy, and biophysics. Tetraphenylethylenes (TPEs)are reliable AIE luminogens with a wide range of successful applicationsin material chemistry. To expand the library of AIE-active TPEs, botha series of TPE analogues, in which the phenyl rotor has been replacedby the indole ring, and indole-substituted TPE derivatives were designedand synthesized through vinyl–aryl and aryl–aryl bondformations using the Suzuki coupling reaction. Efficient syntheticroutes that delivered indole-modified and indole-substituted TPEshave been developed, and almost all heterocyclic TPE analogues havedemonstrated AIE behavior. Furthermore, to test whether the indolering can be diversified, two title compounds were converted to a seriesof bis(indolyl)methane (BIM), and these BIM–TPE materials showedtypical AIE properties. Interestingly, two compounds indicated a solventvapor fuming reversible switch between bright blue emission and greenish-yellowemission. Upon fuming with dichloromethane, their fluorescence spectrashowed 8 and 32 nm red-shift and could return to the original stateafter fuming with hexane. Furthermore, we have explored the effectsof replacing the phenyl ring in TPE with indole together with thesubstitution of TPE with indole ring(s) on the performance of organiclight-emitting diode (OLED) device applications. In addition, densityfunctional theory calculations; the optical, electrochemical, lightemission, electroluminescence characteristics; and admittance spectroscopicanalysis of OLED devices of four representative TPEs have been investigatedin detail. As a result, the indole–TPEs are potential blueemitters with AIE features for conventional OLEDs, which is a significantcolor in displays and lighting. During the past decade,both experimental and theoretical studies have proposed that the maincause of the AIE effect is the restriction of intramolecular motion,including rotation, vibration, and so forth in the aggregates.8 Since then, many studies have reported on the design and synthesisof AIE luminogens possessing advantages such as efficient solid-stateemission, facile synthesis, and ease of functionalization.10 Till date, a large number of TPE-based AIEgens have been developed,and their potential applications have been evaluated.11 In the solution state, the rotation of phenyl rotors inTPE leads to annihilating the excitons in a nonradiative transitionmode. However, intramolecular rotation (IMR) of the rotor is restrictedin the aggregated state, resulting in the predominant radiative transitionof the excited state, so that the luminogens render emissive.12 Therefore, diversification of aromatic rotorsis important to create AIE luminogens.13 Replacement of phenyl rotors in TPE by various aromatic groups suchas naphthalene,12 anthracene,14 pyrene,15 triphenylamine,16 carbazole,9a spirobifluorene,17 and pyridine18 haveallowed a generation of new AIE luminogens , and itsdiphenylated derivative emitted blue light with a maximum luminanceof ∼1800 and 11000 cd/m2, respectively.21 TPE (1) has no good emission, however,its electroluminescence (EL) emission is shown in the deep-blue region.Modifications of TPE have provided novel AIE emitters with superiorlight output efficiency.22 Consideringapplications in chemistry and materials science in the last two decades,the tetraarylethylenes (TAEs) have been classified into five groupsbased on the aryl groups, such as [4 + 0]-, [3 + 1]-, [2 + 2]-, [2+ 1 + 1]-, and [1 + 1 + 1 + 1]-TAEs.23Luminescent materials have attracted increasingattention, in thefield of science and technology, due to their wide range of applicationssuch as fluorescent probes, sensors, bioimaging agents, and organiclight-emitting diode (OLED) devices, in recent years.fication 1.1b The minogens 1. The su24 Indole analogues are considered to be one of the most importantclasses of heterocyclic compounds due to their several applicationsin the medical fields. Also, indoles are capable of having potentialphysical properties for many different applications due to their unsaturatedand electron-rich structures. Indole-derived compounds may representphotochromism and EL; thus, some of these compounds have been commonlyused in the designs associated with nonlinear optics, two-photon fluorescentprobes, cell imaging, photothermal ablation of cancer cells, solarcells, and optoelectronic materials.25 Especially,indole-based triazatruxene materials remain attractive to chemistsdue to their various organic electronics and optoelectronic applications.26 Remarkable properties of both indole and TPEencouraged us to the synthesis of TPE-based indole derivatives. Tilldate, most studies have been restricted with more carbocyclic-basedaryl groups as the rotor. We hereby introduced a new heteroaryl-basedmolecular rotor where the phenyl ring was replaced with an indoleunit. We wondered whether this replacement caused changes in the photophysicaland EL properties of TPE, a splendid AIE-luminogen with a twisted,propeller-like conformation. Herein, we reported a series of indole-modifiedtriphenylethenes ([3 + 1]-type TAEs), including their photophysicalproperties in the aggregated state. Also, we have synthesized TPEs([4 + 0]-type TAEs) with indol substituent(s) on the TPE core andinvestigated in detail their AIE behavior. Furthermore, to investigatetheir AIE characterization, some indole derivatives were convertedto bis(indolyl)methanes (BIM). In addition, the OLED performance measurementsand admittance spectroscopy of four propeller-shaped indole-modifiedand indole-substituted TPEs were also investigated as an emissivelayer (EML) to achieve blue emission in OLEDs, which has been theimportant color in display and lighting areas.Among heterocycles, indole and its derivativesare the parent substanceof a large number of important compounds that occur in nature andexhibit a broad pharmacological profile.H-indole (2)27 anddiaryl ketones (4–7)28 were synthesized via a palladium-catalyzed cross-coupling reaction,as reported in the literature (3) and (1H-indol-5-yl) (phenyl)methanones(4–7) in the presence of n-butyllithium followed by dehydration, which failed to provide the desiredKnoevenagel condensation product. The cross-couplings of indol-5-yl-phenylmethanone(4) and benzophenone (8) in the presenceof TiCl4 and zinc dust afforded the desired indole-modifiedTPE 9 in a low yield (39%) together with TPE (1)  1b. Howev9 and 10, via Knoevenagel condensation and McMurry coupling werenot efficient and fruitful. Initially, 1,1,2-triphenyl-2-bromoethenes 11 and 12 as starting materials were synthesizedfollowing literature protocols.29 We selectedthe Suzuki coupling reaction between 1,1,2-triphenyl-2-bromoethene(11) and indolyl-5-pinacol boronate (13)as model substrates that would establish indole-modified ethene 9 (3)4 as a catalyst with aqueous Na2CO3 as a base in toluene at 110 °C afforded the desiredproduct 9 in a 70% yield. We examined the scope of thesubstrates in this catalytic system 31 –indole–TPEs 9 and 10 and alkyl bromides in the presence of NaOH in dimethylsulfoxide (DMSO) at room temperature. Additionally, nitro derivative 21 was reduced to the corresponding amine 22 inhigh yield (95%) via catalytic hydrogenation 31 2. Howevegenation 2.N,N-diphenylamine-substituted indole–TPE 29  was carried out with tri(2-furyl)phosphine as the ligand in thepresence of Pd(dba)2 as a catalyst. This reaction conditionrevealed that the choice of phosphine ligand was crucial.34 The sole product in the reaction was the desiredcross-coupled product 26 in good yield. No bis-cross-couplingproduct 30 was observed in any case. In the presenceof palladium/Na2CO3 catalysts, the cross-couplingof 26 and 27 gave the desired indole–TPE 28. Furthermore, an N-alkylated product of 29 was obtained (24 (H-indole) (30) is illustrated in 24 and indoleboronic acidpinacol ester 13 then afforded 30 in goodyields benzene (31)35 with the 5-indoleboronic acid pinacol ester(13) in 85% yield (33 and 34) were installed onto the TPE scaffold throughSuzuki cross-coupling reactions  in the presence of zinc triflate affordedBIMs 36a including TPE-core via a Friedel–Crafts-typealkylation (37a were synthesized from the reaction of 35a and TPE–indole 32 under the same reactionconditions (36a–f and 37a–f in excellent yields (89–96%)  6.9, 10, 14, 15, 21, 22, 28–30, 32–34, 36a–f, and 37a–f) were AIE active, the AIE propertiesof compounds were evaluated in THF/water solvent mixtures throughgradual increments of water (fw). Thesecompounds were well dissolved in THF but completely insoluble in water.The AIE characteristics were explored using emission and absorptionspectroscopies. As a model compound, the UV–vis absorptionspectra of 9 exhibited the main absorption band peakedat ∼279 nm , while thePL intensity started to rise swiftly for fw > 90% (9 was increasedby 750-fold at 468 nm (9 indifferent THF–water mixtures under 365 nm UV light showed theAIE behavior (fw) were also studiedwith the cooperation of dynamic light scattering (DLS) and scanningelectron microscopy (SEM) measurements. The DLS study showed the formationof nanosized particles with average sizes of 260 nm , supporting that these luminogens were also AIE-active.Almost all compounds showed similar emission behaviorin the water–THFmixture , respectively. The luminophores exhibitedbright blue emission and greenish-yellow emission before and afterfuming under UV excitation at 365 nm, respectively. Even, the colorchange of solids upon fuming operation was visible to the naked eyeunder UV excitation and daylight. The luminophores exhibited the emissionmaximum at 478 and 488 nm in the ground form before fuming. Compound 9 upon fumigation exhibited a slight bathochromic shift by8 nm in comparison with the basic form. On the other hand, 32 was remarkably red-shifted by 32 nm (from 456 to 488 nm) after fuming.We attributed that the extent of planarization (and hence conjugation)after fuming was not the same in both compounds. The fumed samplescould be completely switched into the ground state by hexane fumigation.Subsequently, the mechanochromiccharacteristics of indole–TPEs in hand were researched by PLspectroscopy. Among the indole–TPP–luminogens, indole-modifiedTPE 9 and 32–34) could be used as an EML forconventional OLED device applications. For this purpose, the optical,theoretical, and electrical properties of these TPE-based OLEDs wereinvestigated in detail.In the next stage, we focusedon whether four indole-modified and indole-substituted TPEs  at 50 °C annealing temperature.As seen in 32, 33, and 34, respectively. The absorption shoulders ofTPEs 32, 33, and 34 were at360, 367, and 362 nm, respectively. The absorption spectrum peaksof TPEs 9, 32, 33, and 34 in thin-film forms were at 352, 352, 340, and 394 nm, respectively(9),42 nm (TPE 32), 40 nm (TPE 33), and 80 nm(TPE 34), which might be attributed to the presence ofintermolecular interactions in the 2D solid state. Comparing the spectraof a spin-coated thin film of TPEs in 9 has the maximum absorptioncharacteristics, while TPE 32 has the lowest. This differencemight be ascribed to the intramolecular charge transfer (ICT) betweenthe indole substituent and TPE unit of TPE 32. In addition,large differences were observed in the absorption curves of the compounds.The TPEs 9, 32, and 34 havea broader absorption curve, while the TPE 33 showed aGaussian-like shape with a more specific peak wavelength. The absorptionvalues in the thin-film state of the TPEs 9, 32, 33, and 34 were lower than the solutionforms. These low photon absorptions could be attributed to the restrictedrotations of the aryl rings in 2D thin-film states. Finally, the opticalband gaps were calculated from the UV–vis absorption edge tobe 3.11, 3.11, 2.91, and 2.61 eV for TPEs 9, 32, 33, and 34, respectively  , whereas the value of TPE 34 was the lowest (0.30%). The order of decrease in the solid-statequantum efficiencies (9 > 32 > 33 > 34) was most likely correlated with theincreasein indole substituents on the TPE backbone. These decreases couldbe mainly due to the ICT effect of the electron-rich indole rings.Chromaticity coordinates for TPEs 9, 32, 33, and 34 were found to be , , , and , respectively, correspondingto the blueish region in CIE gamut, as shown in The solid-state absolute PL quantumyields (PLQYs) of TPEs 5–0.30%) 2. The fl9, 32, 33, and 34 materials, cyclic voltammetry (CV)and differential pulse voltammetry (DPV) were used  of indium tin oxide (ITO) anode, which can significantly decreasethe energy barrier between the emission and the hole injection layers(HILs). The lowest unoccupied molecular orbital (LUMO) energy levelswere determined from band gaps via the onset of UV–vis absorptionspectra. Finally, the LUMO levels of TPEs 9, 32, 33, and 34 were found to be −2.31,−2.27, −2,54, and −2.66 eV, respectively.The corresponding highest occupied molecular orbital(HOMO) energylevels were determined from the values of the first onset oxidationpotential concerning ferrocene as an external reference. Accordingto the formula of 9, 32, 33, and 34 were conducted via densityfunctional theory (DFT) at the B3LYP with the basis set of 6-311G.37 The four-winged propeller-likemolecular structures were linked with different torsion angles betweenthe ethylene core and the adjacent aryl rings benzene(TPBi) as electron injection layer (EIL) to inject electrons fromthe EIL to the EML due to its outstanding electron transporting andhole blocking capabilities with very deep HOMO energy level. The film-formingability of compound 34 was not good due to the inhomogeneityand roughness of its thin film. This was apparent evidence that thehigh tendency for crystallization to take a role as the solution-processedEML in the device. For compound 34, the worst film-formingability could be attributed to the following two reasons: (1) nothaving strong π–π stacking interactions betweencompound 34 and the thiophene rings on PEDOT/PSS and(2) formation of aggregates or crystallines.To assess the EL performance,materials 9, 32, 33, and 34 (50 nm)/TPBi (40 nm)/LiF (0.5 nm)/Al (110 nm) (L–V) characteristics measured for each conventional OLED device.OLED with TPE 32 had the highest luminance with 67.6cd/m2 at 12 V than others 9, 32, 33, and 34  characteristics of OLED devices with TPEs 9, 32, 33, and 34. While theOLED device with 32 has no turn-on voltage (voltage whenthe luminance of 1 cd/m2) due to poor luminance, turn-onvoltages of OLED devices with 9, 33, and 34 are 9.45, 7.80, and 6.30 V, respectively /TPEs (110 nm) 8a. The l, and 34 9a. Whileciencies 9b and hiciencies 9d. Figurectively 9c. Althot 503 nm 9e, assoct 503 nm 2. The ra33 had the best performance,its thin film employed electrochemical impedance spectroscopy (EIS)to investigate charge transport properties (33/Al] device are presented in p =1), the ohmic relation meant that the current density depended linearlyon the voltage.39 In q is the charge of the electron, pO is the free carrier density, and d is theorganic layer thicknessSince the OLED device of operties 10. DevicJ depends quadratically on the applied voltage (p = 2).40 In The space charge limited current (SCLC) regionwas observed ata higher applied voltage, where the current density J (V) and JSCLC (V) characteristics. From the J–V characteristics of the [ITO/TPE 33/Al] device, charge carrier mobility, The charge mobility and carrier densityin the device were calculatedfrom the intersection of logarithmic 41 Therefore, this method was usedto determine the dielectric features of the device [ITO/TPE 33/Al]. The behavior of the reel  and imaginary (Zimag) part with thefrequency was investigated for a device with TPE 33  and time(τ0) showing the (τ0 = 1/2π f0) presence of two relaxation processes in thesystem.42 τ0 values 9.1ms and 4.65 μs indicated a dipolar relaxation type.43 The real ε′ and the imaginary ε″parts of the dielectric constants were dependent on the frequency[ε*(ω) = ε′(ω) – ε″(ω)].The frequency-dependent dielectric constants are given in Impedance spectroscopy is a powerfulmethod to obtain the chargetransport and understand the conduction mechanism of the optoelectronicdevices.h TPE 33 10b. Thes33 werecalculated by using A is the active area of the device, t is the thickness of the thin film, and ε0 is the permittivity of free space. As seen in The dielectric constants for a devicewith TPE 44 The dielectric constantof any material is due to the dipolar, electronic, ionic, and interfacialpolarization. At low frequencies, dipolar and interfacial polarizationsare responsible for the dielectric behavior of the material. However,at higher frequencies, electronic polarization is responsible forthe dielectric, and the contribution of dipolar polarization becomesinsignificant. The decrease in dielectric constant with increasedfrequency could be explained based on the dipole relaxation phenomenon.45As seen in 33/Al] devices. At low frequencies, theσ was DC conductance and remained constant (ω →0); however, a decline of the conductivity was detected at a criticalfrequency (f = 34 KHz) which was the second relaxationzone in DC However, this behavior was opposite of the Jonscher universal dynamicresponse,46 in which the frequency–conductivityrelation can be expressed using eq DC is the dc, σAC is the ac conductivity, ω is the angular frequency, s is the exponent (0 ≤ s <≤1),and A is the dispersion parameter that determinesthe strength of polarizability. At the f = 34 kHz,the charge carriers transport from site to site, after this frequency,it could be the heat that caused the decrease in the conductivity,dependent on the capacitive reactance of TPE 33. Also,in 0 = constant permittivity of free; ε′ = real partof dielectric constant, and tan δ = loss tangent or dielectricloss.AC might decrease with frequency.In this 33 served as a blue emitter in a solution-processed OLED device. TPEs 9, 33, and 34 as an EML in OLEDdevices had a good film-forming ability. In addition, the energy levelsof molecules matched with the HIL and EIL. Moreover, a detailed impedanceanalysis and calculation of impedance, capacitance, and dielectricconstants change with the frequency of TPEs 9, 32, 33, and 34 were performed. Adetailed mathematical computation procedure was associated with voltage,current, capacitance, impedance, charge carrier mobility, and chargedensity. The TPE 33-based conventional OLED device hasthe highest luminance with 67.6 cd/m2 with ITO/PEDOT: PSS/TPEs/TPBi/LiF/Al.While OLED devices with TPEs 9, 32, and 34 showed poor performances, the OLED with TPE 33 exhibited a bright blue light emission with 0.84 cd/A current efficienciesand higher external quantum efficiency (0.41%). Therefore, to understandthe charge carrier properties of TPE 33, the ITO/TPE 33/Al device was also fabricated for impedance analysis.In summary, a series of indole-modifiedand indole-substitutedTPE derivatives have been designed and readily synthesized. The photophysicalproperties of novel TPEs have been studied in detail. Results showedthat the molecules are all typical AIE luminogens (AIEgen). Apartfrom this, two of these AIEgens have been converted to BIM derivativesas a series of new AIEgens via the indole ring. We have utilized AIEgenmolecules to fabricate conventional OLED devices, investigate theoptoelectronic properties of the indole-modified and indole-substitutedTPE derivatives in OLEDs, and obtain efficient blue color, which isstill a great important display and lighting area. TPE


Rosmarinic acid (RA) has biological and pharmaceutical properties and shows hepatoprotective potential. However, the hepatoprotective mechanism of RA needs to be further elucidated in vivo and in vitro.4)-induced liver injury and elucidate the hepatoprotective mechanism of RA in vivo and in vitro.This study was aimed to evaluate the protective effect of RA on carbon tetrachloride  was used to induce liver injury. In vitro, the big rat liver (BRL) hepatocytes were pretreated with RA  for 3 h, and then the hepatocytes were treated with CC14  for 3 h to induce cell injury. The related indexes, including hepatic function, oxidative stress, protein expression of nuclear-factor erythroid 2-related factor 2 (Nrf2) pathway, inflammation, histopathological change, hepatocyte apoptosis, and mitochondrial membrane potential, were evaluated.In vivo, the mice were orally administrated with RA  daily for 28 consecutive days, and 1% CCl4-induced elevation of serum alanine aminotransferase (ALT), alkaline phosphatase (ALP), triacylglycerols (TG), total cholesterol (TC), total bilirubin (TBIL), hepatic reactive oxygen species (ROS), malondialdehyde (MDA), nitric oxide (NO), 8-hydroxydeoxyguanosine (8-OHdG), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-8 (IL-8). RA also increased the levels of hepatic glutathione (GSH), superoxide dismutase (SOD), and catalase (CAT) and the protein expressions of Nrf2, quinine oxidoreductase (NQO1), and heme oxygenease-1 (HO-1). Histopathological examinations indicated that RA (20 and 40 mg/kg bw) alleviated the liver tissue injury induced by CCl4. Moreover, RA inhibited the hepatocyte apoptosis caused by CCl4 based on TUNEL assay. In vitro, RA pretreatment remarkably recovered the cell viability and reduced the CCl4-induced elevation of AST, ALT, lactate dehydrogenase (LDH), ROS, and 8-OHdG. Immunohistochemistry staining demonstrated that pretreatment with RA markedly inhibited the expression of IL-6, inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and Caspase-3 in CCl4-treated hepatocytes. Additionally, RA pretreatment significantly decreased the elevation of mitochondrial membrane potential in CCl4-treated hepatocytes.Oral administration of RA to mice considerably decreased the CCl4-induced liver injury in mice through activating Nrf2 signaling pathway, reducing antioxidant damage, suppressing inflammatory response, and inhibiting hepatocyte apoptosis. RA could attenuate BRL hepatocyte ROS production, DNA oxidative damage, inflammatory response, and apoptosis induced by CCl4 exposure.RA exerted a protective effect against CCl The popular scientific summary of the manuscript is as follows:The hepatoprotective activity of RA in vivo and in vitro was evaluated.4-induced liver damage via antioxidative and anti-inflammatory effects.RA protected the CCl4-induced hepatic injury in mice.RA activated the Nrf2 signaling pathway, thereby reducing CClLiver diseases, including hepatitis, cirrhosis, fibrosis, and hepatocellular carcinoma, are the most common chronic diseases in the world, which have continuously risen to become one of the foremost causes of death and illness over the past several decades . Lots of4) is a chemical agent, which is frequently used in the liver injury model. CCl4 is activated by liver microsomal cytochromes to generate trichloromethyl radical (CCl3), which induces oxidative stress by binding to cellular molecules, thereby leading to cell damage, inflammation, and apoptosis (Carbon tetrachloride CCl is a chepoptosis –8.Lamiaceae and Boraginaceae families (Rosmarinic acid (RA), a natural phenolic compound, is commonly found in plants of families , among ofamilies , 11, corfamilies , anti-infamilies , 13, antfamilies , neuroprfamilies , 11, woufamilies , hepatopfamilies –18, amon4-induced liver injury in vivo and in vitro, from the aspect of hepatic function, oxidative stress, protein expression of nuclear-factor erythroid 2-related factor 2 (Nrf2) signaling pathway, inflammation, histopathological change, hepatocyte apoptosis, and mitochondrial membrane potential.In the present study, we aimed to evaluate the hepatoprotective effects of RA against CClRA  was purchased from Aladdin . The diagnostic kits for superoxide dismutase (SOD), glutathione (GSH), catalase (CAT), malondialdehyde (MDA), 8-hydroxydeoxyguanosine (8-OHdG), reactive oxygen species (ROS), NO, interleukin-8 (IL-8), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and mitochondrial membrane potential assay were provided by the Nanjing Jiancheng Bioengineering Institute . Rabbit anti-cyclooxygenase-2 (COX-2), rabbit anti-caspase-3, rabbit anti-inducible nitric oxide synthase (iNOS) antibodies, rabbit anti-IL-6, DAPI, and Tunel kit were purchased from Servicebio Chemical Co. . Nrf2, OH-1, and quinine oxidoreductase (NQO1) antibodies were provided by Cell Signaling Technology (CST), (USA). Horseradish Peroxidase (HRP), goat anti-rabbit IgG was provided by Kirkegaard & Perry Laboratories (KPL), (USA). BRL hepatocyte was purchased from the Institutes for Biological Sciences Cell Resource Center .4-treated) were administered physiological saline daily. The ideal dose of RA was established according to the preliminary experiment. As a result, the low, medium, and high-dosage RA-treated groups supplemented with RA at 10, 20, and 40 mg/kg bw daily for 28 consecutive days, respectively. On the 29th day, all the groups except the normal group administered 1% CCl4 , while the normal group received peanut oil alone. All animals were administered by gavage. 24 h after the final CCl4,treatment animals were sacrificed. Serum was obtained by centrifugation blood samples at 3,000 rpm for 10 min at 4°C and then conserved at −20°C for further detection. The liver of each mouse was carefully dissected and immediately washed with ice cold saline and stored at −80°C for further analysis. The following equations were adopted to calculate the liver and spleen index: liver index = liver weight (mg)/body weight (g) and spleen index = spleen weight (mg)/body weight (g).Fifty male Kunming mice  were provided by the Experimental Animal Centre of Dali University. All animal operations complied with the guidelines of Chinese Council for Animal Care. After adaptation to the environment for 7 days, the mice were assigned to five groups with 10 mice in each group. Mice were housed at temperature (22°C ± 1°C) and humidity (50–60%) with a 12 h light-dark cycle. Mice were fed to a standard pelleted diet. The normal and model groups , aspartate aminotransferase (AST), alkaline phosphatase (ALP), total cholesterol (TC), triacylglycerols (TG), and total bilirubin (TBIL) were assessed using a clinical automatic biochemical analyzer , and the results were exhibited in U/L for ALT, AST, and ALP, μmol/L for TBIL, and mmol/L for TG and TC.. Additionally, the fluorescence images were also captured using a fluorescence microscope  to reflect the ROS level.Liver was mixed with frozen normal saline  and then homogenized to prepare the liver homogenate 10.0% (w/v). The liver homogenate was centrifuged at 3,000 rpm for 10 min. Then, the homogenate supernatant was used for the determination of MDA, ROS, NO, 8-OHdG, SOD, CAT, GSH, TNF-α, IL-6, and IL-8. All these metrics were measured according to the kit instructions, and the results were exhibited in ng/mg protein (prot) for 8-OHdG, fluorescence/g prot for ROS, nmol/mg prot for MDA, μmol/g prot for NO and GSH, U/mg prot for CAT and SOD, and ng/g prot for TNF-α, IL-6, and IL-8The fixation of liver tissue was done by putting liver samples in 10% neutral buffered formalin for 24 h. After dehydration by ethanol and embedding in paraffin, the paraffin masses were cut into 5 μm sections. Finally, hematoxylin-eosin (H&E) was used to stain for the observation of histopathological changes.The livers were lysed and centrifuged to gain total protein extracts. The bicinchoninic acid (BCA) protein assay kit  was used to measure protein concentrations. After separation by electro phoresis on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels and transmembrane onto polyvinylidene difluoride (PVDF) membrane, the samples were blocked with 10% skimmed milk. Then, membranes were incubated with primary antibodies at 4°C for 12 h. Subsequently, membranes were washed and then incubated with secondary antibodies at room temperature for 30 min followed by visualization using the Electrochemiluminescence system. Representative blots were chosen from three independent experiments. Optical density value of western blots was performed with the use of Alpha software. Protein levels were normalized against those of corresponding β-actin.Paraffin sections of control, model, and RA (40 mg/kg bw) pretreatment groups were used to carry out the TUNEL assay based on the kit instructions. Briefly, the paraffin sections were washed with xylene , absolute alcohol , and distilled water, in turn, to dewax. After the incubation with Proteinase K solution at 37°C for 20 min, the liver tissues were covered using 0.1% triton at room temperature for 20 min. The sections were dried, and the reaction solution  was added to cover the tissues. Then, the sections were put in a moist box and incubated at 37°C for 2 h. After that, the nucleus was counterstained with dihydrochloride (DAPI). Finally, the treated slides were sealed with anti-fade mounting medium. Images were obtained using a fluorescence microscope (400×). Ten microscopic fields in each group were randomly selected for the quantitative analysis.3 cells per well. After incubation for 12 h, cells of RA pretreatment groups were treated with 150 μL of RA  for 4 h. The control and model groups were given 150 μL of cell-culture medium. Then, 40 μL PBS was added to the control group, while the other groups were treated with 40 μL CC14 (100 mM) for 3 h to induce cell injury. Finally, microculture tetrazolium assay was used to evaluate cell viability. The results were expressed as percent cell viability.BRL hepatocytes were seeded onto 96-well plates at 2.5 × 104 cells per well. After incubation for 24 h, cells of RA pretreatment groups were treated with 750 μL of RA  for 4 h. The control and model groups were given 750 μL of cell-culture medium. Then, 200 μL PBS was added to the control group, while the other groups were treated with 200 μL CC14 (100 mM) for 3 h to induce cell injury. Finally, the AST, ALT, and LDH activities in supernatants were determined using a clinical automatic biochemical analyzer .BRL hepatocytes were seeded into 24-well plates at 2.5×10The BRL hepatocytes were digested by pancreatin. Then, the homogenates were centrifuged to get cell precipitate. Finally, the levels of ROS and 8-OHdG were determined according to the kit instructions.The cell climbing pieces were prepared, and the cells were fixed in acetone for 15 min. Then, 3% bovine serum (BSA) was used to cover the cells for 30 min. The treated slides were incubated with IL-6 (1:800), COX-2 (1:500), Caspase-3 (1:200), and iNOS (1:500) antibodies in a moist box at 4°C for 12 h, followed by incubation with HRP labeled goat anti-rabbit IgG for 50 min. DAB was used in color development and counterstained by hematoxylin. The slides were rehydrated with different concentration gradients of alcohol  once for 5 min and sealed with neutral tree gum. Images were captured by a light microscopy (400×).∆Ψm in the BRL hepatocytes. ∆Ψm was determined by the green and red fluorescence, and a confocal laser scanning microscopy  was used to capture the images.A mitochondrial membrane potential assay kit with JC-1 was used to detect the change of All the experiments were conducted in triplicates, and the experimental data were exhibited in mean ± standard deviation (SD). One-way analysis of variance (ANOVA) and Duncan’s multiple range test were conducted to analyze statistical significance between the means by SPSS (version 17.0). Spearman correlation was performed to analyze the correlation between antioxidant markers and liver damage-related indicators.4 dramatically elevated the liver and spleen index compared to the normal group (P < 0.05), indicating significant swelling of liver and spleen (4-treated mice pretreated with RA at a high dosage (40 mg/kg bw) showed a marked decrease of liver and spleen index (P < 0.05) compared to the model group, indicating a preventive effect against CCl4-induced liver and spleen swelling in mice.The administration of CCld spleen . However4-induced toxicity caused significant elevation in serum biochemical indexes, including ALT, AST, ALP, TBIL, TC, and TG. In contrast, the 4-week pretreatment of mice with RA reduced the biochemical parameters in a dose-dependent manner, particularly when the dosage was increased from 20 to 40 mg/kg bw.As shown in P < 0.05). However, the CCl4-induced increase in ROS production was significantly inhibited by pretreatment with RA. The ROS level of low-, medium-, and high-dosage RA-treated groups was 7.22, 8.36, and 5.56 fluorescence/g prot, respectively .ROS, MDA, NO, and 8-OHdG were detected to evaluate the degree of oxidative damage in liver tissue. DCFH-DA  was used as a fluorescence probe to detect the ROS level. In the normal group, the ROS level was 3.89 fluorescence/g prot. In the model group, the ROS level sharply increased to 22.46 fluorescence/g prot  decreased the level of hepatocyte apoptosis by 69.28% in comparison to the CCl4 treated group.Being consistent with the results of fluorescence images, the quantitative analysis results showed that CCl4-toxicity , indicating severe hepatocyte injury of CCl4 treatment. In contrast, pretreatment with RA markedly inhibited the rise of AST, ALT, and LDH activities induced by CCl4.The results showed hepatoprotective effect of RA in a dose-dependent manner against CCltoxicity . The cel4-induced toxicity caused significant increase in the hepatocyte levels of ROS and 8-OHdG, which were 131.32 and 8.65 times higher than that of the normal cells, respectively. However, the RA exhibited a dose-dependent inhibition effect on the levels of ROS and 8-OHdG.As exhibited in 4 treatment caused considerable increase in the expression of hepatocyte Caspase-3, IL-6, COX-2, and iNOS with brown staining  and trichloromethyl peroxy radical (OOCCl3) by hepatic cellular cytochrome P450 in the liver   . A markeand LOO)  during C4 poisoning resulted in the increase of ROS. MDA, an indicator of lipid peroxidation, and its content reflects the extent of lipid peroxidation. NO is a highly reactive oxidant and a multifunctional free radical, which is produced by iNOS  to the proton to generate H2O2 and O2  were observed when compared with the model group, suggesting that RA protects the CCl4-induced liver oxidative damage by elevation of antioxidant enzyme activity.Antioxidant enzymes play an important role in preventing the CCl2 and O2 . CAT conon of HO . GSH conon of HO . Changes4-induced liver injury through activating the Nrf2 signaling pathway and upregulating the downstream antioxidant enzymes. Our result was consistent with the previous study that RA could promote the Nrf2 nucleus transport and then upregulate the expression of HO-1, NQO1, GCLC, and GCLM in lipopolysaccharide/D-galactosamine-induced acute liver injury in mice  initiates TNF-α, which activates nuclear factor kappa B (NF-κB)  . NF-κB ptivation , 31, 32.tivation . TherefoThe correlation analysis between antioxidant makers  and liver damage-related indicators  was performed. The divergences in correlations between different antioxidant makers and various liver damage-related indicators could be attributed to the divergence in antioxidant mechanism of the individual antioxidant maker. For examples, HO-1 catalyzes substrate into ferrous iron, CO, and bilirubin, which play a key role in antioxidant and anti-inflammatory , whereas4 in mice. Caspase-3, an important member of the apoptotic protease family, plays an important role in cell apoptosis, and its level could reflect the degree of apoptosis  with RA predominant possessed significant protective effect on CCl4-induced hepatotoxicity in vivo and in vitro (In our previous study, free phenolics from in vitro , 38. Bas4-induced liver injury through activating Nrf2 signaling pathway, reducing antioxidant damage, suppressing inflammatory response, and inhibiting hepatocyte apoptosis. Furthermore, the results of in vitro experiment also revealed that RA could attenuate BRL hepatocyte ROS production, DNA oxidative damage, inflammatory response, and apoptosis induced by CCl4 exposure. Further research is still imperative to elucidate the activation mechanism of the Nrf2 signaling pathway and anti-inflammatory mechanism of RA in CCl4-induced hepatic injury.In conclusion, the results of in vivo experiment indicated that RA exerted a protective effect against CCl