We seek to clarify how these genes are involved in kidney sodium absorption and influence blood pressure, especially emphasizing the underlying mechanisms by which genetic variants alter protein functions and interaction in blood pressure regulation. The present review aims to enhance our understanding of the important role of kidney ion handling genes/channels in blood pressure control.Hypertension affects 30% of adults and is the leading risk factor for cardiovascular disease. Kidney sodium reabsorption plays a vital role in the initial stage and development of essential hypertension. It has been extensively reported that the variants of kidney ion handling genes are associated to blood pressure, and clinical features of hypertension. However, the underlying mechanisms by which these variants alter protein function are rarely summarized. In addition, the variation of one single gene is often limited to induce a significant effect on blood pressure. In the past few decades, the influence by genes × genes (G × G) and/or genotype × environment (G × E) interactions on a given trait, for example, blood pressure, have been widely considered, especially in studies on polygenic genetic traits. In the present review, we discuss the progress in genetics studies on kidney ion handling genes, encoding Na More than 1.1 billion people worldwide have hypertension , which iYellow Emperor’s Classic of Internal Medicine has suggested that ‘the kidneys pass on the diseases to the heart’ [Earlier, people have realized that abnormal renal function is closely related to one’s blood pressure ,6. Over e heart’ . Howevere heart’ , until te heart’ . Increase heart’ ,12. The e heart’ .https://www.ebi.ac.uk/gwas/home) records that about 733 genetic genes/variants in 90 studies associate to persistently high systemic arterial blood pressure. Up to April 2022, in PubMed database (https://pubmed.ncbi.nlm.nih.gov/) more than 56 studies discuss the relationship between genetic variants of genes related to renal sodium reabsorption and blood pressure regulation. These identified renal sodium reabsorption-related genes involved in blood pressure regulation included: (i) Kidney ion handling genes, encoding Na+-Cl− cotransporter (NCC), Na-K-2Cl cotransporter (NKCC2), sodium channels (ENaC), renal outer medullary potassium channel (ROMK), Pendrin, chloride channels (CLC-Kb), respectively, and their upstream kinases, WNKs and SGK1, etc.; and (ii) RAAS genes, including AGT, REN, Ang I, Ang II, ACE, CYP11B2, and aldosterone etc., which participate in the kidney sodium reabsorption via the aldosterone/ENaC pathway , K+ channel (ROMK), and Cl− channels , respectively, and their upstream kinases, WNKs and SGK1, and clarified how they were involved in kidney sodium absorption and influence blood pressure, and elaborated the underlying mechanisms of these genes variations altering protein functions and their interaction in blood pressure regulation, which may provide an opportunity to understand the relationship between genetic background of renal physiology and blood pressure regulation, and individual susceptibility to hypertension [The kidney handles a wide variety of ions including sodium, potassium, chloride, magnesium, calcium, and others to influence blood pressure. However, in the presnt review, we focused on the latest genetic studies of kidney sodium, potassium, and chloride handling genes, especially in the thick ascending limb of Henle’s loop (TALHL), distal convoluted tubule (DCT), and cortical collecting duct (CCD), encoding Nartension , and furThe process of reabsorption in the kidney is, through its specialized ion channels , the nephron removing water and solutes from the tubular fluid and returning them to the circulating blood, and ensuring appropriate electrolyte homeostasis. Given the blood volume closely influenced by renal sodium reabsorption, genetic variants, and mutations of ion channels involving the process of this reabsorption are being paid attention to by researchers and clinical doctors. Two decades of genetics studies are also indicated that these genetic backgrounds may be responsible for individuals’ blood pressure through altering the process of reabsorption on ion channel(s).+ channels in the TAL, DCT, and CCD of kidney are mainly composed of ENaCs, NCC, and NKCC2. Human ENaCs, as the end effector of adrenocortical hormones and excitable epithelial Na+ channel, has four subunits: α, β, γ, and δ encoded by SCNN1A, SCNN1B, SCNN1G, and SCNN1D, respectively [+ in the kidney, and plays an important role in maintaining homeostasis of extracellular fluid volume, blood pressure, and sodium. Gain-of-function mutations in ENaC subunits β and γ cause a rare hereditary hypertension, Liddle syndrome. Conversely, loss-of-function mutations in ENaC result in a kind of hereditary hypotension, pseudohypoaldosteronism type 1 (PHA 1) [SCNN1A [SCNN1B [SCNN1G [Naectively , which a (PHA 1) . Associa [SCNN1A , p.T594M [SCNN1B , and c.G [SCNN1G ) are cloSCNN1A, SCNN1B, and SCNN1G genes mainly change the function of Na+ channels through four mechanisms: (i) Influence the surface expression of ENaC channel A [SCNN1G. For instance, Tong et al. [(ii) Modulate the open probability of channels (Po). The open probability of ion channels is positively correlated with channel activity. With the enhancement of channel open probability, the channel activity increases. Some variants  Affect the proteolytic cleavage of ENaC protein. The activity of ENaC is also affected by the state of proteolytic cleavage. The channel product after proteolytic cleavage has a high-channel open probability due to the release of an inhibitory tract in ENaC protein, while the uncleavage channel is in an inactive state [+ self-inhibition on ENaC channels(iv) Alter the Na, e.g., p.A334T [SCNN1A; p.γH239R [SCNN1G. ENaC selectively allows extracellular sodium ions to enter endothelial cells, and at the same time, elevated extracellular sodium ions have a certain inhibitory effect on ENaC channels, which is referred to as Na+ self-inhibition [+ self-inhibition enables the distal nephron to control Na+ reabsorption based on the urinary Na+ concentrations and maintains sodium homeostasis in body fluid [+ self-inhibition [Functional studies showed that variants of the  p.C618F , p.A334T p.C618F , p.V481M p.C618F , p.C479R p.C618F , and p.A p.C618F ,24 of SCG(-173)A  of SCNN1g et al.  found thf SCNN1A , p.R564  [SCNN1B , and p.L [SCNN1G ) have be p.L511Q  increaseve state . Knight ve state  found ENve state  demonstr p.A334T , p.R476W p.A334T , p.V481M p.A334T , p.H239Rp.γH239R  of SCNN1hibition ,33–35. Ndy fluid . Many sthibition ,36–38. Fhibition  reportedSLC12A3 is specifically expressed in the apical membrane of the DCT cells of kidney, whose genetic variants and mutations , localivariants ), and acrg919Cys ) in membrg919Cys  defined rg919Cys  found th mutants .SLC12A1, it is specifically mainly expressed in the apical membrane of the TALHL and responsible for nearly 24% of kidney sodium reabsorption [SLC12A1 were observed having strong association to blood pressure and hypertension in the general population [SLC12A1 change the protein function through effecting the surface expression , locati p.L505V ), and ac p.L505V ) of NKCC p.L505V .+ channel, encoded by the KCNJ1 gene. ROMK is mainly expressed in two areas of kidney: CCD and TALHL. ROMK strictly regulates the secretion of potassium in CCD and at the same time controls the potassium cycle in TALHL. Potassium plays an important role in the regulation of blood pressure. Diet potassium intake, serum potassium, and urinary potassium are all negatively associated with blood pressure [+ (that is a decrease in intracellular cation concentration). Therefore, maintenance of intracellular charge homeostasis requires ENaC and NKCC2 reabsorb more cation  into the cells [The ROMK (Kir1.1) is a kind of Kpressure ,53, and pressure . Potassipressure . Specifipressure . The acthe cells , ultimathe cells .KCNJ1 gene was previously reported to associate with blood pressure. For instance, loss-of-function mutations of the KCNJ1 gene cause Barter syndrome type II characterized by kidney salt wasting, hypotension, and mild hypokalemia. And Ji et al. [KCNJ1 gene caused a decrease in blood pressure in 3125 individuals and 292 Gitelman or Bartter syndrome patients in the Framingham Heart Study in the United States. Additionally, p.E151K [KCNJ1 gene are observed to associate with blood pressure or hypertension , and confering a gain in regulated-inhibitory gating (p.P166S and p.R169H) of ROMK [The i et al.  determin p.E151K , p.Y314C p.E151K , p.T191N p.E151K , p.E284Q p.E151K , rs67575 p.E151K , rs67538 p.E151K , rs28466 p.E151K , rs28558 p.E151K , and rs2 p.E151K  in the K of ROMK .+ only; therefore, Cl− transporter/channels, such as Pendrin and CLC-Kb, also play important roles in blood pressure control [SLC26A4 gene  is a transmembrane chloride/anion transporter and mediates the secretion of bicarbonate and the reabsorption of chloride, and mainly expressed in the thyroid [− but also totally abolished Cl− reabsorption, and pointed out that the Cl− transport regulated by pendrin is the most important Cl− reabsorption pathway in the collection system. Pendrin gene ablation (SLC26A4−/−) has been reported to associate with reduced renal sodium reabsorption and blood pressure and resistant to hypertension caused by aldosterone in vivo [Extracellular fluid volume is determined by NaCl, but not by Na control . Pendrin thyroid , inner e thyroid ,64, and  thyroid . The exp thyroid . Wall et thyroid  reported in vivo ,69.SLC26A4 gene variants are related to SBP and DBP [Association analyses have also confirmed that  and DBP . In func and DBP , E29Q, V and DBP , V239D,  and DBP , and V51 and DBP  are foun and DBP . To date and DBP , which pCLCNKB gene. The ClC-Kb channel and its accessory subunit barttin (encoded by the BSND gene) are expressed in Henle’s loop, DCT and CCD of the kidney, and their function is to help the reabsorption of chloride, and the maintenance of urine concentration [CLCNKB cause Barter syndrome type III, while gain-of-function mutations in CLCNKA and CLCNKB can cause rare salt-sensitive hypertension [CLCNKB gene variants, such as p.S12A, p.E192Ter [CLCNKB gene mutations affect the function of CLC-Kb channel through increasing the activity , or  p.T481S , p.A242E p.T481S , p.R351W p.T481S , p.R30X  p.T481S , and p.A p.T481S ).With-no-lysine (WNK) and SGK1 are two important kinases regulating kidney ion channels, and hence involved in kidney sodium reabsorption and hypertension. WNK is a serine/threonine protein kinase, and four members of the WNKs family (WNK1–4) have been identified in mammals (Supplementary Table S2). WNKs are widely expressed in human organs and tissues. Except for WNK2, all the other WNKs are expressed in kidney . Alterna(i) WNK1 and WNK3 exist compensation effects. In mice lacking WNK3, no obvious salt consumption phenotype was observed for compensatory up-regulation of WNK1/SPAK axis [(ii) WNK1/WNK3 and WNK4 inhibit each other. In addition to act alone, WNK1 can regulate renal sodium handling genes by inhibiting WNK4 [(iii) KS-WNK1 inhibits the activity of L-WNK1/WNK3. Based on observations in genetically inactivated mice of two WNK1 isoforms, the effect of activating L-WNK1 exceeds that of KS-WNK1 [There are interactions between WNKs as follows. PAK axis ; (ii) WNing WNK4 . WNK3 caing WNK4 . Meanwhiing WNK4 ; (iii) K KS-WNK1 .in vivo and generated Wnk4D561A/+ knockin mice. They found this knockin mice increased apical expression of phosphorylated NCC protein in the DCTs through activation of the OSR1/SPAK-NCC phosphorylation cascade, and showed the phenotypes of hyperkalemia and hypertension.WNKs are extremely important kinases that connect angiotensin II, aldosterone, and renal sodium and potassium transporters. Mutations of WNK1 and WNK4 cause Gordon syndrome  accompanied by hypertension, elevated serum potassium, and acidosis . LaliotiWNK1 gene , which has at least three isoforms: SGK1 (iso-1), and its two N-terminal variant subtypes Sgk1_i2 (iso-2) and Sgk1_i3 (iso-3). Iso-2 and iso-3 are more stable than iso-1; Therefore, they are considered to be the main players for the role of SGK1 in hypertension . SGK1 isrtension . In contrtension .SGK1 gene have been demonstrated to relate to blood pressure or hypertension, including rs1057293, rs1743966 [SGK1 gene was associated with risk of hypertension (P=7.4 × 10−3) in the Chinese Han population [Some SNPs in the s1743966 , rs27581s1743966 ,119, rs9s1743966 , rs93760s1743966 , rs17634s1743966   and/or genotype × environment (G × E) on a given trait   have bee−/HCO3− transporter, and NCC is a Na+-Cl− cotransporter, both of which play an important role in renal sodium reabsorption. When pendrin and NCC were mutated separately, the renal function of mice including sodium chloride excretion, urine output, and blood urea nitrogen were comparable with wild-type mice. However, when pendrin and NCC were double knocked out in mice, and they showed volume depletion or hypotension under salt restriction conditions [Pendrin is a Clnditions . Therefonditions . Similarnditions . In futuSimilar to NCC and Pendrin, compensation also exists between NCC and ENaCs, and their interaction maintains the homeostasis of systemic blood pressure. For example, it is generally believed that NCC activation causes hypertension. However, the result observed in KS-WNK1-KO mice is that NCC activity is significantly enhanced, but systemic blood pressure only showed a slight increase, and failed to cause hypertension, which is related to decreased expression of ENaC . ConversTaken together, there are strong compensatory effects between NCC and Pendrin/ENaCs. When NCC or Pendrin/ENaC is knocked out or malfunction, the expression or activity of another protein will compensatively increase to maintain the homeostasis of individual’s salt wasting, urine output maintains, and blood pressure. However, NCC and ENaCs, or NCC and pendrin were both knocked out or inactivited, exceeding the compensatory ability between genes, which will lead to serious salt wasting or blood pressure variation.− restriction alone [Pendrin and ENaCs are expressed in aldosterone-sensitive kidney areas, mainly including DCT, CNT, and CCD. Previous studies showed that Pendrin works in tandem with ENaC, while NCC works alone . In animon alone . Meanwhion alone .Pech et al.  showed tWNKs protein and isoforms are composed of WNK1–4, and KS-WNK1 etc. Except for WNK2, all of the others are expressed in the kidney. As the difference of WNKs in structure, their regulatory functions for renal ion channels also vary.pathways 1 and 2) [There are many regulatory mechanisms whereby WNKs regulate the function of NCC and NKCC2 A,B. Firs1 and 2) ,145. Som1 and 2) .pathway 1). WNK3 is expressed in DCT of the kidney, and its carboxyl-terminal can activate NCC and is one of the important members of up-regulating NCC [in vitro, the overall effect of this kinase on NKCC2 in vivo seems to be limited.Second, WNK3 increases NCC and NKCC2 through SPAK pathway . WNK4 has been reported to directly inhibit NCC and is a strong inhibitor of NCC, which deletion causes overactivity of NCC and thus hypertension [− plays a vital role in the conversion of positive and negative regulations of WNK4 on NCC. They confirmed that WNK4 is a chloride-sensitive kinase, and its effect on the SPAK-NCC pathway is regulated by Cl− concentration. Under low-chlorine conditions, WNK4 is automatically phosphorylated through a SPAK-dependent mechanism to activate NCC and play a positive regulatory role; Under high-chlorine conditions, WNK4 cannot be automatically phosphorylated due to its significant negative effects on WNK1 and WNK3, and inhibits NCC, plays as a negative regulator. NKCC2 is regulated by WNK4 via SPAK/OSR1 in a kinase activity-dependent manner [Third, the regulation of WNK4 on NCC is complicated, and besides the above positive regulation, WNK4 also plays an inhibitory role on NCC  [pathway 5) [Fourth, previous studies showed KS-WNK1 antagonizes the ability of L-WNK1 to inhibit NCC and NKCC2 (thway 4) ,149. KS-thway 5) .pathway 1); and (ii) WNK1-SGK1-ENaC pathway (pathway 2) [pathway 2) [pathway 3) [pathway 4) [WNK1 activates ENaC via two pathways: (i) WNK1-Nedd4-ENaC pathway (thway 2) ,152. WNKthway 2) ; and (iithway 3) . Howeverthway 4) .pathway 1) [pathway 2). Third, KS-WNK1 has no kinase activity and cannot block ROMK directly. It indirectly activates ROMK by antagonizing the inhibition of L-WNK1 on ROMK [in vivo.First, WNK1 and WNK4 inhibit ROMK via enhancing clathrin-dependent endocytosis, and the C terminus of ROMK plays an important role in the inhibitory effect (thway 1) . Second, on ROMK . Cheng e on ROMK  found th+, K+, and Cl− channels in the kidney. Among them, the regulation of SGK1 on ENaC, ROMK, and CLC-Kb has been well summarized in the review of Valinsky et al. [SGK1 exerts a vital role in regulating Nay et al.  In addity et al.  and NCC y et al. , and enhHypertension affects 30% of adults and is the leading risk factor for major cardiovascular events such as heart attack and stroke, chronic kidney disease, and heart failure . Kidney + T cells [Over the past decades, there has been a rapid increase in our understanding that the immune system plays a vital role in the regulation of renal ion channels . For exa T cells , IL-17A  T cells , TNF-α [ T cells , and int T cells  have bee T cells , which pIn the present review, we discuss the progress of genetics studies on kidney ion handling genes and their interaction in blood pressure regulation, and provide a panel of genes effecting kidney sodium reabsorption. Since hypertension is a polygenic disease and there are G × G interactions between kidney sodium reabsorption-related genes, it may be more effective to detect a gene panel than to detect a single susceptibility gene for patients with hypertension. Taking into account the key role of kidney sodium reabsorption in blood pressure control, in future studies, combining all the mutations of renal sodium reabsorption-related genes for a comprehensive analysis is more helpful and powerful for clarifying the mechanisms of essential hypertension.Click here for additional data file.


Multiple myeloma (MM) is a cancer of white blood cells known as plasma cells. It is hard to treat cancer, thus requires new treatments. Herein, a plant extracted compound, guggulsterone (GS), has been investigated for its anticancer activity in MM cells. The results from this study revealed that GS could be used for the effective treatment of MM due to its ability to cause cell death in MM cells. It exhibits anticancer activity itself and also increases the effectiveness of other drugs when combined. Therefore, it could be further investigated for its possible utilization in clinics to treat MM patients.Multiple myeloma (MM) is a hematological disorder characterized by the abnormal expansion of plasma cells in the bone marrow. Despite great advances over the past three decades in discovering the efficacious therapies for MM, the disease remains incurable for most patients owing to emergence of drug-resistant cancerous cells. Guggulsterone (GS), a phytosteroid, extracted from the gum resin of guggul plant, has displayed various anticancer activities in vitro and in vivo; however, the molecular mechanisms of its anticancer activity have not been evaluated in MM cells. Therefore, in this study, we investigated the anticancer activity of GS in various MM cell lines  and the mechanisms involved. GS treatment of MM cells caused inhibition of cell proliferation and induction of apoptotic cell death as indicated by increased Bax protein expression, activation of caspases, and cleavage of poly (ADP-ribose) polymerase. This was associated with the downregulation of various proliferative and antiapoptotic gene products, including cyclin D, Bcl-2, Bcl-xL, and X-linked inhibitor of apoptosis protein. GS also suppressed the constitutive and interleukin 6-induced activation of STAT3. Interestingly, the inhibition of Janus activated kinase or STAT3 activity by the specific inhibitors or by siRNA knockdown of STAT3 resulted in the downregulation of HMGB1, suggesting an association between GS, STAT3, and HMGB1. Finally, GS potentiated the anticancer effects of bortezomib (BTZ) in MM cells. Herein, we demonstrated that GS could be a potential therapeutic agent for the treatment of MM, possibly alone or in combination with BTZ. Multiple Myeloma (MM) is a rare form of hematologic malignancy that is characterized by excessive proliferation and accumulation of malignant clonal plasma cells in the bone marrow microenvironment, resulting in anemia, hypercalcemia, extensive skeletal destruction, abnormal bleeding, and renal failure ,2. Over Various studies have revealed the involvement of Janus-activated kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway in the pathogenesis of MM. The JAK/STAT pathway is a pleiotropic signaling cascade and a key player in various physiological processes, including immune function, hematopoiesis, cell growth, and apoptosis . Accumulnuclear factor-κB pathway, evidenced by the increased sensitivity to chemotherapy and induction of DNA damage and cellular apoptosis upon downregulation of HMGB1 [Compelling evidence has indicated the association of HMGB1 with various hallmarks of cancer, such as angiogenesis, apoptosis, tissue invasion, metastasis, inflammation, and insensitivity to growth inhibitors ,11. In Mof HMGB1 ,13. In pof HMGB1 . CollectCommiphora mukul (Hook. Ex Stocks) Engl., also known as Commiphora wightii (Arn.) Bhandari [Guggulsterone -pregnadiene-3,16-dione, Bhandari . Owing tBhandari ,16,17,18Bhandari . However-pregnadiBhandari ,22. HoweBhandari . In addiBhandari ,25,26. OBhandari ,28. Ahn Bhandari . HoweverThe human MM cell lines MM.1S, RPMI 8266, and U266-B1 were procured from the American-type culture collection . Fetal bovine serum (FBS), penicillin-streptomycin (PenStrep), and RPMI-1640 media were obtained from Thermo Fisher Scientific . Z-GS and JAK inhibitor AG-490 were purchased from Tocris Bioscience  and were prepared in dimethyl sulfoxide (DMSO). Z-VAD-FMK was purchased from Calbiochem . Cell counting kit-8 (CCK-8) and N-acetylcysteine were purchased from Sigma Aldrich . Live/dead cell viability/cytotoxicity kit was purchased from Molecular Probes . Annexin-V FITC apoptosis detection kit, fixation/permeabilization solution kit, JC-1, BV421 mouse anti-gamma H2AX (pS139), PE rabbit anti-active caspase-3 and Alexa Fluor 700 mouse anti-cleaved PARP (Asp214) were obtained from BD Biosciences . The HMGB1 ELISA kit was purchased from Biomatik . Caspase 3/7 green detection reagent was purchased from Invitrogen . STAT3 siRNA was purchased from Life Technologies , and control siRNA was purchased from Qiagen . Antibodies such as p-H2AX, cytochrome c, caspase-3, cleaved caspase-3, caspase-9, PARP, Bcl-2, Bax, Bcl-xL, cyclin D1, LC3, Atg7, Atg5, p62, STAT3, p-STAT3, SHP-1, and HMGB1 were purchased from Cell Signaling Technology . Other antibodies, such as BID, XIAP, p21, and p27, were purchased from Abcam . Loading control antibodies, such as GAPDH, β-actin, and HSP60, were obtained from Santa Cruz Biotechnology .2 at 37 °C. Peripheral blood mononuclear cells (PBMCs) were obtained from healthy volunteers and were maintained under the same conditions as MM cells.MM cells were cultured in RPMI 1640 medium supplemented with 20% FBS, 1% L-glutamine, 1% penicillin-streptomycin and incubated with 5% COHuman MM.1S, U266-B1, and RPMI 8266  were seeded in 96-well plates and treated with increasing concentrations of GS . The cells were then incubated for 24, 48, 72, and 96 h. After the completion of the time point, 10 µL of CCK-8 reagent was added to the cells and the plate was incubated for 1 h at 37 °C. The absorbance was measured at 450 nm, and the number of viable cells was estimated as described previously .3 cells/well in a 6-well plate and treated with GS for 48 h. Live/dead stain was prepared by adding 5 μL of ethidium homodimer-1 (EthD-1) and 5 μL of calcein AM to 10 mL PBS . The cells were stained with the prepared dye for 30 min and then images were captured using the EVOS FLoid cell imaging system .U266 cells were seeded at a density of 5 × 103) were treated with GS for 48 h in a 12-well plate. After the treatment time point was completed, caspase-3/caspase-7 green detection reagent was added to the cells, and the cells were incubated for 30 min at 37 °C. The cells were then visualized, and images were captured using the EVOS FLoid cell imaging system .U266 cells , and β-mercaptoethanol was added. SDS-PAGE technique was used to separate protein cell lysates (30–60 µg), which were transferred onto the polyvinylidene difluoride (PVDF) membrane. The membranes were immunoblotted with antibodies and developed on the ChemiDoc imaging system .6) were incubated with various concentrations of GS for 48 h. The cells were collected in 1× annexin binding buffer after washing with PBS and stained with Annexin V-FITC and PI for 20 min. The cells were analyzed and quantified as live , early apoptotic , late apoptotic , and necrotic  using BD LSRFortessa cell analyzer . Total apoptosis percentages were measured by adding early and late apoptosis percentages [U266 cells (at a seeding density of 1 × 10centages .5) were fixed and permeabilized using a permeabilization and fixation kit and stained with 5 µL H2AX (pS139)-Alexa Fluor 647 antibody. After staining, the cells were then analyzed using the BD LSRFortessa cell analyzer .To measure DNA double-strand breaks, U266 cells  were fixed, permeabilized, and stained with AF700 tagged cleaved PARP, and BV605 tagged active caspase-3 for 30 min. The analysis was carried out via flow cytometry, as previously described [U266 cells .TM System (Lonza) was used for the transfection of U266 cells with control siRNA  and STAT3 siRNA  according to the manufacturer’s protocol. After transfection, the cells were incubated for 48 h at 37 °C and then lysed and immunoblotted with different antibodies.4D-NucleofactorTM Cytometric Bead Array kit (BD Biosciences). The supernatant of U266 was collected, and IL-6 levels were measured. In tubes containing cytokine standards, detection antibodies were added and after 2 h incubation, Streptavidin-PE was added to the mix and subsequently washed with Tween 20 and PBS (PBS/T). After an additional 1 h incubation, the samples were washed with PBS/T, and the beads were resuspended in PBS with 10% FBS. The samples were analyzed by a BD LSRFortessa cell analyzer .Cytokine measurement was performed using a multiplexing array BDp ≤ 0.05.The statistical analysis was performed using GraphPad Prism software version 7.0 . One-way analysis of variance (ANOVA) was utilized to evaluate the statistical differences between control and treatment groups, followed by Sidak’s post hoc test. Results are presented as mean ± standard deviation (SD). The differences between groups were considered statistically significant at 50) was not achieved for all three cell lines. However, at 48 h, IC50 for U266 and RPMI 8226 cells was observed at 11.9 µM and 35.6 µM  and early apoptotic cell population (lower-right quadrant). In comparison, the upper quadrants represent the late apoptotic cell population (upper-right quadrant) and the necrotic cell population (upper-left quadrant) A. A concThe Bcl-2 family of proteins controls the intrinsic apoptotic pathway, primarily by affecting the mitochondrial membrane integrity. Under stress conditions, pro-apoptotic proteins of the Bcl-2 family promote the permeabilization of the mitochondrial outer membrane and the subsequent release of cytochrome c into the cytoplasm . Our resNext, to explore the effect of GS on the activation of caspase-cascades, U266 and RPMI 8226 cells were treated with increasing concentrations of GS and immunoblotted against caspase-3, cleaved-caspase-3, and PARP antibodies. To assess the role of HMGB1 in GS-treated U266 cells, HMGB1 ELISA was performed. Our results indicated a significant reduction in the secretion of HMGB1 in a concentration-dependent manner after 48 h of GS treatment A. In addTo further explore the effect of GS on the STAT3 signaling pathway, U266 cells were treated with GS and immunoblotted with antibodies against p-STAT3, STAT3, Bcl-xL, XIAP, cyclin D1, p21, and p27. GS treatment was found to inhibit the phosphorylation of STAT3 at Tyr705, in a concentration-dependent manner A withoutIL-6 plays a vital role in sustaining the growth and proliferation of MM  via the Next, we sought to investigate if subtoxic concentrations of GS and BTZ could potentiate the anticancer effects in U266 cells. BTZ is a food and drug administration (FDA)-approved proteasome inhibitor used to treat refractory MM patients . HoweverWe further sought to determine whether GS can sensitize MM cells to BTZ when used in combination. We found that the combination of subtoxic concentrations of GS (10 µM) and BTZ (10 nM) significantly inhibited cell proliferation A and indC. mukul, possesses anticancer, anti-inflammatory, and antioxidant properties [Recent advances in the treatment of MM have led to an unprecedented response rate and patient survival. Despite these improvements, the cure for MM remains elusive, highlighting the importance of discovering novel therapeutic targets and potent anticancer agents against MM . GS, a poperties ,44. Hencoperties ,45. MoreHMGB1 is actively involved in tumor growth, cell proliferation, metastasis, and invasion . This maThe activation of STAT3 regulates the transcription of various antiapoptotic genes, such as Bcl-xL. Our results demonstrated that GS suppressed the phosphorylation of STAT3, thereby downregulating the downstream targets of STAT3, including Bcl-xL, cyclin D, and XIAP. In addition to these molecular targets, the expression of a PTP, SHP-1, was upregulated upon GS treatment which was associated with the downregulation of STAT3 phosphorylation. PTPs are negative regulators of the JAK/STAT signaling pathway , and SHPThe involvement of IL-6 in MM pathogenesis and inhibition of IL-6-mediated STAT3 activation by other anticancer agents in MM cells has been described earlier ,51. Our In recent years, combination therapy involving two or more therapeutic agents to enhance the efficacy of anticancer drugs has gained widespread attention. Although the conventional monotherapy approach is still a standard treatment modality for different types of cancer, it is less effective than the combination therapy approach as it is more vulnerable to drug resistance. Usually, it targets proliferating cells non-selectively, leading to the death of both healthy and tumor cells . CombinaIn the current study, we explored the anticancer effect of GS treatment on MM cells by evaluating the intrinsic and extrinsic apoptotic pathways. The findings of our study are consistent with other studies, where GS induced anticancer effects in gastric cancer cells and cholangiocarcinoma and promoted apoptosis via the intrinsic pathway ,55. Our Despite the fact that the currently available drugs have, to some extent, significantly improved the quality of life of MM patients, the clinical outcome is still unsatisfactory due to toxicity, relapse, and acquired drug resistance, which has emerged as the primary barrier to chemotherapy’s failure in the clinical treatment of MM. Therefore, further exploration of the potential alternatives to existing therapeutic options and their molecular mechanisms is highly urgent for seeking effective treatment strategies for MM. The expression of HMGB1 is upregulated in various tumor tissues compared to normal tissues. This upregulation is associated with tumor invasion, metastasis, progression, and other aspects of tumor growth. Additionally, HMGB1 can be produced from a variety of tumor cells in response to chemo or radiation therapy, and as a result, it plays a role in the regulation of cancer cells’ chemoresistance and sensitivity. The proteasome inhibitor BTZ, has marked efficacy against MM, but the major limitations of this drug are intrinsic and acquired resistance. Considering all these factors, we thought of investigating the potential of GS as a natural anticancer drug alone and in combination with BTZ in MM cells. The results of this study, which show that GS can target STAT3 and HMGB1 and improve the effectiveness of BTZ, suggest the therapeutic relevance of this safe and affordable nutraceutical as a viable supplemental medicine for the treatment of MM. Understanding the molecular underpinnings of HMGB1′s role as a protumor protein in cancer requires additional research. Additionally, even though numerous xenografts or transplantable tumor models have been employed, generating HMGB1-associated spontaneous tumor models is crucial.In conclusion, our results elucidated that GS, as a single agent, is active against MM. However, the toxicity and bioavailability studies of GS have not been performed yet in cancer clinical studies. Therefore, this is an area of future investigation. Our study offers proof of concept for more research into the function of HMGB1 and the possible clinical use of GS in treating MM. It also provides a preclinical framework for integrating GS and BTZ in clinical settings. Our study, along with other previously published studies, provides synthetic and structural biologists with some justification to modulate the chemistry of this alkaloid, such as active moiety, including its phenolic and alcoholic groups, the unsaturation pattern, and the presence of an amine group, to make this compound more therapeutically feasible with increased bioavailability and less toxicity.


Vitis vinifera) bZIP gene from group K, VvbZIP36, acts as a negative regulator of anthocyanin biosynthesis. Knocking out one allele of VvbZIP36 in grapevine with CRISPR/Cas9 promoted anthocyanin accumulation. Correlation analysis of transcriptome and metabolome data showed that a range of anthocyanin biosynthesis genes were activated in VvbZIP36 mutant plants relative to the wild type, resulting in the accumulation of related metabolites, including naringenin chalcone, naringenin, dihydroflavonols, and cyanidin-3-O-glucoside. The synthesis of stilbenes (α-viniferin), lignans, and some flavonols  was significantly inhibited, and several genes linked to their metabolism were downregulated in the mutant plants. In summary, our results demonstrate that VvbZIP36 is a negative regulator of anthocyanin biosynthesis that plays a role in balancing the synthesis of stilbenes (α-viniferin), lignans, flavonols, and anthocyanins.Anthocyanins are plant secondary metabolites that have a variety of biological functions, including pigmentation. The accumulation of anthocyanins is regulated by both transcriptional activators and repressors. Studies have shown that bZIP family members act primarily as positive regulators of anthocyanin biosynthesis, but there are few reports of negative regulation. Here, we report that a grapevine ( Flavonoids are a family of specialized plant metabolites that includes flavonols, flavones, proanthocyanidins , and anthocyanins . They acArabidopsis thaliana [Solanum lycopersicum) [Fragaria × ananassa) [Prunus persica) [Pyrus communis) [O-glucosyltransferase (UFGT), glutathione S-transferase (GST), and anthocyanin 3-O-glucoside-6′′-O-acyltransferase (3AT) to synthesize anthocyanins [Flavonols, Pas, and anthocyanins are all end-products of the flavonoid pathway, which has been well characterized in many species, such as thaliana , tomato ersicum) , strawbenanassa) , peach (persica) , pear (Pommunis) , and graommunis) . The flaommunis) . Subsequommunis) . Anthocyommunis) , which aocyanins . PAs areocyanins .AtMYB113 and AtMYB114  [MdBBX22 and MdHY5  [FvbHLH9 (strawberry) [PpMYB10 and PpbHLH64 (pear) [PpNAC1 and PpMYB10.1 (peach) [VvMYBA1, VvMYBA2, and VvMYBPA1 (grapevine) [FaMYB1 in Lotus corniculatus inhibits the expression of LAR and ANR and significantly reduces the content of PAs in leaves [Populus) MYB182 protein significantly reduces the content of anthocyanins and PAs in poplar by reducing the expression of DFR1, ANS1, and ANR1 [PpMYB18 acts as a negative regulator, balancing the accumulation of anthocyanins and PAs during peach fruit ripening and at juvenile stages of development [The biosynthesis of flavonoids is controlled by a complex network of structural and regulatory genes. Many transcriptional activators involved in regulating flavonoid biosynthesis, especially anthocyanins and PAs, have been identified in various species. These include mestica) , FvbHLH9awberry) , PpMYB104 (pear) , PpNAC1  (peach) , and VvMapevine) . It has n leaves ; the popand ANR1 ; and PpMVitis vinifera) are economically important fruit-producing plants rich in flavonoids [VvMYBPA1/VvMYBPA2, VvMYBA1/VvMYBA2, VviMYB86, VvBBX44, and VvWRKY26 [VvMYBPA1 or VvMYBPA2 activate the expression of VvANR and VvLAR and promote PA accumulation [VvMYBA1 activates the transcription of Vv3AT in the white grape variety Chardonnay, causing the fruit to accumulate anthocyanins and turn red, whereas silencing of VvMYBA1 or VvMYBA2 in the red grape variety Shiraz results in a significant decrease in fruit anthocyanin content [HY5, have also been shown to play key roles in the regulation of flavonoid biosynthesis in many species, including Arabidopsis [Grapevines , wheat (Triticum aestivum), and maize (Zea mays) [CsLOB1 increased resistance to citrus canker [clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 ribonucleoproteins (RNPs) were successfully delivered to apple protoplasts to edit DIPM-1, DIPM-2, and DIPM-4, thereby increasing fire blight resistance [VvWRKY52, VvMLO3, and VvPR4b were successfully mutated using CRISPR/Cas9, increasing resistance to Botrytis cinerea, downy mildew, and powdery mildew, respectively [Genome editing technology has proven effective for loss-of-function research in many horticulturally important crops, such as tomato, tobacco, rice (ea mays) . There hea mays) , citrus ea mays) , and appea mays) ; this iss canker , and tars canker . In applsistance . In grapectively –39.VvbZIP36 and found that it contributes to the regulation of anthocyanin levels by balancing the synthesis of stilbenes (α-viniferin), lignans, flavonols, and anthocyanins.In the current study, we used CRISPR/Cas9 to target the grapevine bZIP family gene V. vinifera) [VvbZIP36 has high expression in leaves, stems, flowers, tendrils, and especially fruits [VvbZIP36 is expressed in the roots, tendrils, stems, leaves, and fruit of grapevine, with the highest expression in mature stems, leaves at fruit set, and senescent leaves, as well as ripening fruit  [AtbZIP60 and OsbZIP74 play important roles in the endoplasmic reticulum (ER) stress response in Arabidopsis and rice, respectively [http://blast.ncbi.nlm.nih.gov/Blast.cgi) database for VvbZIP36 homologs and found 25 sequences from other species, all of which contained the bZIP domain and a putative TMD  . Tissue-y fruits . In thisng fruit , similarin (TMD) . Previouectively , 42. To tive TMD . When anVvbZIP36 in grapevine (Thompson Seedless) using the CRISPR/Cas9 genome editing system and obtained one mutant grapevine plant (KO45) [VvbZIP36 [In our previous study, we successfully knocked out one allele of t (KO45) . No off-t (KO45) . SubsequVvbZIP36 , but onlVvbZIP36 plays a role in regulating anthocyanin biosynthesis. We therefore collected leaves from the mutant lines (KO21 and KO45) and the wild type (WT) to determine the total anthocyanin contents. As shown in We observed that the leaves of KO21 and KO45 turned red  and hypoVvbZIP36 and anthocyanin biosynthesis, we performed an RNA-seq analysis of KO45  and WT CHS; chalcone isomerase, CHI; flavanone 3-hydroxylase, F3H; flavonoid 3′-hydroxylase, F3′H; dihydroflavonol 4-reductase, DFR; flavonol synthase, FLS; and anthocyanidin synthase, ANS) related to flavonoid biosynthesis based on the KEGG and MapMan enrichment analysis results  and WT plants. The results of normalized cluster analysis and Pearson’s correlation analysis show that the biological replicates of each group are highly correlated glucoside, cyanidin-3-O-(6′′-O-p-coumaroyl)glucoside, and peonidin-3,5-O-diglucoside were higher in KO45 than in the WT (O-(6′′-O-caffeoyl) glucoside was lower in KO45  was generally higher in KO45 than in WT, and the corresponding metabolites, such as naringenin chalcone, naringenin, dihydroflavonols, and anthocyanins, were more abundant to varying degrees  and two Type-I flavone synthase genes  was higher. The PAs and most of their precursor metabolites (flavanols) also accumulated to higher levels in KO45 than in the WT, accompanied by higher expression levels of VvLAR1 (leucoanthocyanidin reductase 1: VIT_01s0011g02960) and VvANR (anthocyanidin reductase: VIT_00s0361g00040). The content of some flavonols was higher in KO45 than in the WT, whereas the abundance of others was lower  and lower expression of two FLS genes  and two FLR genes .In other branches of the flavonoid biosynthesis pathway, such as flavanones and flavones, the levels of most metabolites were significantly higher in KO45 than in the WT. Correspondingly, the expression of as lower , accompaPAL genes from the early general phenylpropanoid pathway were expressed at higher levels in KO45  were identified, one of which showed higher expression in KO45 than in the WT, whereas the other showed lower expression.Interestingly, all the differentially abundant lignan metabolites were lower in KO45 than in the WT, whereas five  in KO45 . NotablyVvF3H2 and VvLAR1, their expression patterns were consistent with the transcriptome data.Subsequently, we randomly selected 15 of the 30 DEGs involved in the phenylpropanoid pathway from VvbZIP36 was highly expressed in leaves, stems, flowers, tendrils, and especially fruits [VvbZIP36 might be involved in the regulation of anthocyanin biosynthesis. Knocking out one allele of VvbZIP36 did not affect expression  have been related to endoplasmic reticulum stress [VvbZIP36) participate in plant anthocyanin biosynthesis.The basic region/leucine zipper (bZIP) TFs are important regulators in plants and are involved in regulating many metabolic processes, such as energy metabolism, development, senescence, light signaling, anthocyanin biosynthesis, and response to biotic and abiotic stimuli , 44. In y fruits . Similary fruits –31, we spression , Fig. S9pression . This repression , suggestin apple , 45, SlHn tomato , 28, and in pear , 29. Sim and FLS , and VvHm stress , 42. ThiVvbZIP36 influences anthocyanin biosynthesis, we performed transcriptome analysis of the leaves of a CRISPR/Cas9-mediated VvbZIP36 mutant line (KO45) and WT plants. A total of 1965 and 2138 genes were expressed at higher and lower levels, respectively, in KO45 compared with the WT  fruit ripening [VvbZIP36 with photosynthesis and the central role that photosynthesis plays in plant growth and development [VvbZIP36 knock-out grapevine lines with single allele mutations glucoside, cyanidin-3-O-(6′′-O-p-coumaroyl)glucoside and peonidin-3,5-O-diglucoside) were more abundant in KO45  found that knocking out SlHY5 (a bZIP gene) in tomato can reduce anthocyanin levels and expression of these anthocyanin biosynthesis genes [O-(6′′-O-caffeoyl)glucoside was lower in KO45 than in the WT glucoside synthesis will be an interesting area of future study.Stilbenes, lignans, and flavonoids are all synthesized from the same precursor metabolite, royl-CoA . Subsequ in KO45 . Naringeis genes . Similaris genes , 31. Thin the WT , indicatO-rhamnoside, kaempferol-3-O-rhamnoside, and kaempferol-7-O-rhamnoside)  was lower in the transgenic plants. These results suggest that VvbZIP36 is involved in balancing the synthesis of flavonols and anthocyanins. Similar results have also been found in the stilbene (α-viniferin) and lignan biosynthetic pathways, which share the same precursor metabolite, p-coumaroyl-CoA, as the precursor of flavonoids, naringenin chalcone. We infer from our results that the action of VvbZIP36 causes more p-coumaroyl-CoA to be used to synthesize stilbenes (α-viniferin) and lignans, and more dihydroflavonols to be synthesized into flavonols , thereby restricting the synthesis of naringenin chalcone and anthocyanins, ultimately leading to relatively low levels of anthocyanin accumulation. By contrast, more anthocyanin accumulation was seen in mutant plants because of the inhibition of flavonol, stilbene (α-viniferin), and lignan synthesis  increased significantly in the mutant lines . Howevermnoside) . Correspynthesis . That knleaf age .VvbZIP36 contributes to the regulation of anthocyanin biosynthesis by balancing the synthesis of stilbenes (α-viniferin), lignans, flavonols, and anthocyanins in grapevine. This study provides new insights into the regulation of anthocyanin biosynthesis that will be valuable for grapevine breeding programs.In conclusion, the results reported here indicate that To investigate the relationship between VvbZIP36 and other bZIP TFs, multiple amino acid sequence alignments were generated using DNAMAN software  with default parameters, and a phylogenetic analysis was carried out using the neighbor-joining (NJ) method and MEGA software (version 11.0.8), as previously described .VvbZIP36 was analyzed using the Grape eFP Browser (http://bar.utoronto.ca/efp_grape/cgi-bin/efpWeb.cgi).The tissue-specific expression of V. vinifera L. cv. Thompson Seedless) was performed as described in a previous study [−2 s−1; #G2-1000D3, Youke, China) for four months.Vegetative propagation of mutant lines 21 and 45 (KO21 and KO45) and WT grapevine . The PrimeSTAR Max DNA Polymerase kit  was used to amplify potential mutation regions in the first exon of VvbZIP36, and the PCR products were inserted into the pClone007 Simple Vector  to select single clones for sequencing.Identification of mutant lines was performed as previously described , 43. SpeThe anthocyanin contents of leaves from three seven-month-old transgenic (KO21 and KO45) and WT plants, grown in a growth chamber , were measured after sampling the third to fourth leaf from the top of the plants. The total anthocyanin content was calculated according to Li et al. (2012) . In brieV. vinifera reference genome (PN40024) (http://plants.ensembl.org/index.html) using HISAT (v2.1.0). FPKM was calculated using featureCounts (v1.6.2), and differential gene expression was determined using DESeq2 (v1.22.1) with a threshold of corrected P-value < 0.05 and absolute log2FC (fold change) > 1.The third to fourth leaves from the tops of seven-month-old transgenic (KO45) and WT plants were collected, and their transcriptomes were characterized by RNA-seq analysis. Samples from three plants per line were pooled and used as one independent sample, and three independent samples were analyzed per genotype. Total RNA was extracted from the transgenic and WT leaves using TRIzol reagent . The RNA library preparation and sequencing were performed by Metware Biotechnology Co., Ltd. . In brief, RNA-seq libraries were generated using the NEBNext Ultra RNA Library Prep Kit for Illumina  following the manufacturer’s instructions before sequencing on the Illumina HiSeq platform. The original data were filtered using fastp (v0.19.3) to remove reads with adapter sequences. The resulting cleaned reads were mapped to the https://mapman.gabipd.org/home) for pathway analysis. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database (https://www.genome.jp/kegg) was used for gene functional annotation, and KEGG enrichment analysis was performed using TBtools software [https://cloud.metware.cn/#/tools/tool-list).All differentially expressed genes (DEGs) were imported into MapMan software  system  was used to analyze sample extracts as described 2FC (fold change) ≥ 1 were considered to be differentially regulated between groups.The third to fourth leaves from the tops of three seven-month-old transgenic (KO45) and WT plants were collected for a widely targeted metabolome analysis. Approximately 5 g of leaf tissue per line was used as one independent sample, and three independent samples were analyzed. Sample extraction, metabolite identification, and quantification were performed by Metware Biotechnology Co., Ltd. . An ultra-performance liquid chromatography-electrospray tandem mass spectrometry  and WT plants grown in a growth chamber using the E.Z.N.A. Plant RNA Kit . First-strand cDNA was synthesized using the HiScript II Q RT SuperMix for qPCR(+g DNA wiper) Kit  according to the manufacturer’s protocol. qRT-PCR was performed using the SYBR qPCR Master Mix  on a StepOnePlus RT-PCR instrument (Thermo Fisher Scientific), and the gene-specific primers used are listed in Supplementary Data analysis was performed using Excel 2016 software . Data were plotted using SigmaPlot . Tests for significant differences were performed using SPSS statistics software .Web_Material_uhac022Click here for additional data file.


The overall erythrocyte count, packed cell volume, prostaglandin, and sperm counts were all considerably lower in the experimental animals. Histological changes were found in the liver, spleen, and testes of rats administered lornoxicam alone. In rats, pretreatment with ginger extract reduced the majority of the negative effects of conventional and high dosages of lornoxicam.Lornoxicam is a potent oxicam-class nonsteroidal anti-inflammatory drug (NSAID) with analgesic, anti-inflammatory, and antipyretic effects. Its impacts on many biological functions are not fully understood. We measured various biomarkers in male albino rats provided an oral aqueous ginger extract before IM administration of therapeutic and 2× the therapeutic doses of lornoxicam. The aqueous ginger plant extract was characterized by mass spectroscopy, and its effects were determined by examining free radical scavenging activity, blood parameters, renal and hepatic function, semen quality, proinflammatory cytokines, antioxidant markers, and histopathology. Rats administered lornoxicam had significantly higher liver and kidney function biomarker values, TNF- Prevention and treatment of musculoskeletal illnesses are public health priorities in humans and other animals . To treaα and IL-1β activity.The NSAID lornoxicam (chlorotenoxicam) is an oxicam derivative, suppressing polymorphonuclear (PMN) leukocyte motility, PMN leukocyte superoxide release, and macrophage nitric oxide release . Used inZingiber officinale).There are many variables that might lead to an increased risk of NSAID hepatotoxicity, including excessive intake, prescription overdose, drug interactions, and unique patient susceptibilities. . In partMedicinal herbs have been utilized to heal various infections since antiquity. According to the World Health Organization, 80 percent of the world's population uses diverse plant fractions and their dynamic ingredients as traditional remedies , 15. TheThe effectiveness of ginger extracts in mitigating the harmful effects of lornoxicam administration has yet to be demonstrated. In addition, the effects of high dosages of lornoxicam (two times the therapeutic dose) on biochemistry, hematological, histology, oxidative stress, cytokine production, and sperm characteristics remain unknown. Therefore, we aimed to investigate the effect of an aqueous ginger extract on these parameters in male albino rats treated with therapeutic and 2× the therapeutic doses of lornoxicam.Lornoxicam -1,2-thiazine-3-carboxamide-1,1-dioxide) was obtained from Eva Pharma Pharmaceuticals Inc.  in powder form. The powder was diluted in 2 mL of distilled water before to injection, giving a final dosage of 8 mg/mL lornoxicam. Botanists from the Faculty of Agriculture, Benha University, obtained ginger rhizomes from a local market. The rhizomes were crushed in a blender and air-dried. A total of 125 g of air-dried powder was macerated in 200 mL of distilled water for 12 hours at room temperature before filtration to obtain a final aqueous extract concentration of 120 mg/mL for use in the experiment.μm (film thickness) capillary column . The components were determined by comparing their relative mass spectra fractions to those of the WILEY 09 and NIST 14 mass spectral databases [In the current study, the chemical composition of the aqueous ginger extract was determined using a Trace GC–TSQ mass spectrometer with a direct TG − 5MS 30 m × 0.25 mm × 0.25 atabases .The radical-scavenging ability of aqueous ginger extract was evaluated using 2,2-diphenyl-1-picrylhydrazyl (DPPH) as a reagent according to previous studies . To geneFifty male Wister rats  were housed in separate cages. The laboratory environment was conventional , with a 12-hour light/dark cycle. Throughout the experiment, rats were fed a typical diet of bread, barley and milk, and water ad libitum. For adaption and to prevent stress during the experimental period, the rats were acclimatized to the laboratory conditions for two weeks prior to treatment. The Institutional Animal Care and Use Committee for Research Ethics of Benha University (# BUFVTM3921) approved all study procedures, which followed the principles of the Declaration of Helsinki.The 50 rats were divided into five groups of 10 rats. The rats in the first group were given no therapy and served as a control. The rats in the second group were administered IM lornoxicam at a therapeutic dose  once a After the end of the experiment, hematological assays were performed on whole blood samples taken from the retroorbital venous plexus. Total erythrocyte count (TEC), packed cell volume % (PCV%), hemoglobin concentration (Hb), mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV), and mean corpuscular hemoglobin concentration (MCHC) were detected using an automated cell counter .α and IL-6 concentrations were determined by a commercial ELISA kit [Serum samples were used to estimate the activity of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) , catalasLISA kit .Liver, kidney, testes, and spleen tissue samples were taken from each group's slaughtered animals and processed for histopathological procedures that were performed using standard techniques . Masson'P value ≤0.05.Data were statistically analyzed by one-way ANOVA with a post hoc Duncan multiple comparison test using a statistical software program . Differences were considered significant at a α-curcumene, α-zingiberene, β-sesquiphellandrene, β-bisabolene, zingiberene, shogaol, α-sitosterol, 6-gingerol, and 2,6,10-dodecatrien-1-ol, 3,7,11-trimethyl  of kidney function parameters  and 2× therapeutic doses (group 4) resulted in significantly higher levels  resulted in significantly lower sperm counts  resulted in significantly elevated TNF-s of MDA  in the sZingiber officinale) aqueous extract on the pathological grading of lornoxicam in renal, hepatic, splenic, and testicular tissues of different treated groups.Tables 6Histological examination of kidney tissues by H&E staining revealed that control rats  showed lH&E staining of the liver tissues revealed that, unlike the control , lornoxiCompared to the control group , hemorrhH&E staining of the testicular tissues revealed that, compared to the control , adminisIn the current study, the antioxidant capacity (42.79% ± 3.1%) of the aqueous ginger extract was higher than that detected in a similar, previous study  in whichUrea is a waste product of protein catabolism produced in the liver and discharged by the kidneys , which rThe liver is the major target organ for drug metabolism. Hepatic biotransformation reactions induce hepatocyte apoptosis –60. TherIn Swiss albino mice, hepatotoxicity, hepatitis, and elevated levels of the liver enzymes AST, ALT, and ALP had previously been documented  and ratsThe protective effect of ginger has been attributed to its relatively high concentration of either vitamin C (35–38 mg per 100 g)  or shogaThe significant changes in hematological parameters  observed in rats treated with lornoxicam at 2× the therapeutic dose may provide evidence of the drug's toxicity. The reduction of red blood cells with subsequent anemia was dose-dependent. This anemia results in reduced oxygen-carrying capacity in the blood and the amount of oxygen delivered to the tissues , 75. SimH&E staining of the spleen showed hemorrhage with trabecular thickening and hemosiderosis in splenic tissue of rats injected with 2× the therapeutic dose of lornoxicam without ginger pretreatment. Similarly, N-acetyl-p-aminophenol (140 mg/kg) was found to induce overall splenic congestion, congested sinusoids, widened red pulp, and atrophy of lymphoid follicles . Rats prA significant fall in PG levels in rats injected with lornoxicam without ginger pretreatment was previously recorded . LornoxiPreviously, it was discovered that aspirin causes hypercholesterolemia in rats via blocking PG production. This inhibition could result in altered cholesterol metabolism and androgen biosynthesis . AndrogeIn the current study, lornoxicam injection induced more sperm abnormalities and significantly fewer total sperm than the control. These findings are also reflected in testis histopathology. Desquamation in the spermatogonia and interstitial edema and apoptosis of some cells were the main features identified in rats injected with lornoxicam. A previous study  reportedα are proinflammatory cytokines produced by activated macrophages. They help regulate various biological processes, including cell proliferation, differentiation, apoptosis, immune response, and activation of cell signaling proteins involved in systemic inflammation. The overproduction of TNF-α has been implicated in various diseases [α levels, probably by inhibiting prostaglandin production [α and IL-6 are widely known to induce hepatorenal toxicity and tissue damage. In previous studies, intoxication with the NSAID paracetamol was found to induce oxidative stress, triggering a secondary inflammatory cascade associated with cytokine release from Kupffer cells [α and significantly decreased CAT and GPX. Furthermore, hepatic GSH, antioxidant enzymes, and lipid peroxidation increased after lornoxicam injection in rats [α) and significant elevation of CAT and GPX. This observation may be attributed to the effect of the antioxidant components, gingerol and shogaols, of ginger extract [α, IL-6, IL-8, IL-2 and IL-1β, PLA2, iNOS, COX-2, and PGE2) [IL-6 and TNF-diseases , 88. Cytoduction . TNF-α aer cells –92. Moreer cells  and aceter cells  were pre in rats . We prop extract , 95, 96. extract . The keynd PGE2) . High lend PGE2) , 100.α, and IL-6, and sperm abnormalities. However, RBC, PCV, prostaglandin, and sperm counts were significantly decreased in rats administered lornoxicam without ginger extract pretreatment. Congestion, hydropic degeneration, desquamation, and focal necrosis were observed in the kidney, liver, spleen, and testes of rats administered either lornoxicam dose. All observed detrimental effects were more pronounced in rats receiving 2× the therapeutic dose than those administered the therapeutic dose of lornoxicam. These detrimental effects in rats administered 2× the therapeutic dose of lornoxicam were improved significantly by pretreatment with ginger. We recommend providing a ginger extract pretreatment in animals to avoid any possible hepatic, renal, splenic, and testicular injuries induced by administering a high dose of lornoxicam.Administration of the therapeutic dose and 2× the therapeutic dose of the NSAID lornoxicam to rats without ginger extract pretreatment resulted in significant increases in the serum activities of ALT, AST, and ALP, concentrations of urea, creatinine, TNF-


Shewanella species have been identified as progenitors of several clinically important antibiotic resistance genes. The aim of our study was to analyze Shewanella baltica strains isolated from the gut contents of wild Atlantic mackerel (Scomber scombrus) for the presence of both known and novel variants of antibiotic resistance genes (ARGs), using Illumina-based whole-genome sequencing (WGS). Thirty-three S. baltica strains were isolated from Atlantic mackerel collected in the northern North Sea. WGS revealed the presence of several new variants of class C and class D beta-lactamases. Nearly 42% (14/33) of the strains carried the mobile colistin resistance gene mcr-4.3. To understand the genetic context of mcr-4.3, we determined the complete genome sequence of strain 11FHM2, using a combination of Oxford Nanopore- and Illumina-based sequencing. The complete genome sequence is 5,406,724 bp long, with one contig representing a chromosome of 5,068,880 bp and three contigs representing novel plasmids . Plasmid pSBP2_mcr4 contains the mobile colistin resistance gene mcr-4.3, as well as the mercury resistance operon merRPAT. Plasmid pSBP1 carries genes encoding resistance against copper, zinc, chromium, and arsenic. Plasmid pSBP3 does not carry any antibiotic or heavy metal resistance genes. Analysis of the flanking region of mcr-4.3 suggests that a phage integrase may be involved in the mobilization of mcr-4.3 in Shewanella spp. Our results provide insights into the mobile mcr-4.3 present in Shewanella spp. and highlight the importance of the marine environment in the emergence and dissemination of clinically important resistance genes.IMPORTANCE We identified two new plasmids in Shewanella baltica isolated from wild Atlantic mackerel (Scomber scombrus) collected from the northern North Sea, one plasmid carrying the mcr-4.3 gene for colistin resistance and the operon merRPAT for mercury resistance and the other carrying multiple heavy metal resistance genes. The marine environment has been recognized as a source of new resistance genes that are found in human pathogens. Selection pressure from heavy metals is seen in the marine environment, especially associated with human activities, such as waste discharge, mining, and in aquaculture settings. This would help maintain and disseminate these plasmids in the environment. Our study provides insights into the mobilization of colistin resistance genes in Shewanella spp. and highlights the importance of the marine environment in the emergence and dissemination of clinically important antibiotic resistance genes. Here, we describe a novel plasmid in S. baltica strain 11FHM2 that carries the mcr-4.3 gene and a mercury resistance operon. This is the first report of plasmid-borne mcr-4.3 in Shewanella spp. and the first report of mcr-4.3 in S. baltica isolated from wild fish. Furthermore, we show the presence of several new variants of class C and class D β-lactamases, as well as a novel catB-related chloramphenicol O-acetyltransferase in S. baltica isolates.The aim of our study was to analyze n = 20) were collected from the northern North Sea in October 2018 in sterile plastic bags. The fish were dissected, and the gut contents were collected aseptically in sterile 15-mL tubes. Serial dilutions of the individual gut content samples were inoculated onto Mueller-Hinton agar with ampicillin (100 μg/mL) and onto MacConkey agar with ampicillin (100 μg/mL) and incubated at 30 °C for 24 to 48 h. A total of 73 bacterial isolates were obtained and identified, using matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) . A total of 33 isolates were identified as S. baltica. We sequenced the genomes of these 33 strains using the Nextera DNA Flex library prep kit  for sequencing library preparation, followed by sequencing on the Illumina MiSeq platform, using 2 × 300-bp chemistry and analysis as described previously   strains carried the mcr-4.3 gene. A phylogenetic tree including the MCR variant detected in this study (MCR-4.3), along with other known variants of MCR, is presented in Fig. S1.Species identification of the 33 strains as r (TYGS) . Analysimcr-4.3 gene, we determined the complete, closed genome sequence of S. baltica strain 11FHM2 (carrying mcr-4.3 and a novel catB variant), using an Oxford Nanopore MinION instrument, as described previously  shows that it shares 99.9% nucleotide sequence identity to fragments of several plasmids carrying mcr-4.3, as well as 98.2% nucleotide sequence identity to the DNA fragment carrying the mcr-4.3 gene from the chromosome of the proposed progenitor species, Shewanella frigidimarina   and Salmonella sp. plasmid pMCR_R3445 (MF543359), respectively (mcr-4.3 gene is flanked by an integrase that contains the protein domains Integrase_1 (Pfam PF12835) and Phage_int_SAM_2 (Pfam PF12834). Both domains belong to a family of DNA-binding prophage integrases found in Proteobacteria, suggesting a role of phage integrases in the mobilization of mcr-4.3 in Shewanella spp. Our study suggests that the mobilization event of the mcr-4.3 gene discovered in S. baltica may be independent of the mobilization event for the gene variant found on plasmids in Enterobacteriaceae. Having observed that, extensive follow-up studies are needed to ascertain this point (Plasmid pSBP2_mcr4 contains 86 open reading frames (ORFs), with an average GC content of 43.9%. The ntegrase . AlignmeP000447)  13). Th. Thmcr-4ectively , 15. In is point , 15.Antibiotic selection pressure is required for maintenance of resistance plasmids. In the absence of selection pressure, i.e., by exposure to antibiotics, plasmids may be lost . Other mcopABCD, copper-translocating P-type ATPase, copper oxidase, copper resistance protein NlpE, copper chaperone PCu(A) chromate efflux transporter, CusA/CzcA family heavy metal efflux RND, and arsenic resistance protein. Both plasmids, pSBP1 and pSBP2_mcr4, carry multiple conjugal transfer genes, indicating that these plasmids are potentially conjugative. Marine environments, especially around Norway, have heavy metal selection pressure from the presence of zinc and copper due to mining, as well as the use of heavy metals in aquaculture and antifouling paints , CP051530 (plasmid pSBP1), CP051531 (plasmid pSBP2_mcr4), CP051532 (plasmid pSBP3), respectively. The draft genome sequences of the other strains are deposited in GenBank under the following accession numbers JAAHCG000000000, JAAHCI000000000, JAAHCQ000000000, JAAHCS000000000, JAAHCT000000000, JAAHCU000000000, JAAHCV000000000, JAAHCY000000000, JAAHDA000000000, JAAHDB000000000, JAAHDC000000000, JAAHDD000000000, JAAHDK000000000, JAAHDT000000000, JAAHDZ000000000, JAAHEE000000000, JAAHEI000000000, JAAHEJ000000000, JAAHEK000000000, JAAHEL000000000, JAAHEM000000000, JAAHER000000000, JAAHEU000000000, JAAHEV000000000, JAAHEW000000000, JAAHEX000000000, JAAHEY000000000, JAAHEZ000000000, JAAHFA000000000, JAAHFB000000000, JAAHFC000000000, JAAHCM000000000.The complete genome sequence of


Staphylococcus aureus colony-counting method. Furthermore, we evaluated the biocompatibility of the ABS@NPs in vitro. This study suggests that ABS@NPs with improved antioxidant and antibacterial properties can be used to treat diseases related to various oxidative stresses and can be applied in many fields, such as pharmaceuticals, cosmetics, and foods.Bioactive compounds are widely used in the bio-industry because of their antioxidant and antibacterial activities. Because of excessive oxidative stress, which causes various diseases in humans, and because preservatives used in bioproducts cause allergies and contact dermatitis, it is important to use natural bioactive compounds in bioproducts to minimize oxidative stress. α-bisabolol (ABS) is a natural compound with both antioxidant and antibacterial properties. However, its water-insolubility makes its utilization in bioproducts difficult. In this study, ABS-loaded polyglyceryl-4 caprate nanoparticles (ABS@NPs) with improved aqueous stability and ABS loading were fabricated using an encapsulation method. The long-term stability of the ABS@NPs was analyzed with dynamic light scattering and methylene blue-staining to determine the optimized ABS concentration in ABS@NPs (10 wt%). The ABS@NPs exhibited excellent antioxidant activity, according to the 2,2-diphenyl-1-picrylhydrazyl assay and in vitro reactive oxygen species generation in NIH-3T3 fibroblast cells, and an outstanding antibacterial effect, as determined using the  They cause extreme damage to biomolecules in cells and mitochondria, thereby inducing inflammation and excessive oxidative stress in the body resulting in various diseases, such as cancer and Alzheimer’s disease (AD), in humans [Bioactive compounds have various properties, such as antioxidant and antibacterial activities, and they are widely used in the pharmaceutical, cosmetic, and food industries ,2,3. Tern humans ,10,11,12n humans ,14,15.Antibacterial activity is an important property for bioproduct development . CompareMatricaria chamomilla, and several studies have indicated that it has useful pharmacological and cosmetic activities, including anti-inflammatory, anti-irritant, anti-tumor, antioxidant, antibacterial, and anti-allergic properties [In this study, we focused on α-bisabolol (ABS), a natural bioactive compound with antioxidant and antibacterial properties. ABS is a naturally occurring sesquiterpene alcohol that is widely present in plant sources, such as operties ,31,32,33operties ,35,36,37operties ,30,36,37operties ,30,31,37In this study, we developed a nanoprecipitation technique using amphiphilic polyglyceryl-4 caprate, which contains a hydrophilic carboxyl group segment and a hydrophobic hydrocarbon chain segment, to fabricate ABS-loaded nanoparticles (ABS@NPs) with higher loading and stability of ABS than previously achieved. The objective of this study was to improve the long-term stability and effectiveness of ABS as an antioxidant and antibacterial material in an aqueous environment . To eval® , and acetonitrile (HPLC grade) and phosphoric acid were obtained from Merck® . For the dye solubility method, methylene blue was obtained from Sigma-Aldrich . For the in situ antioxidants experiment, the reagents 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ascorbic acid (AA) were obtained from Sigma-Aldrich . For in vitro cell incubation, penicillin–streptomycin, trypsin (0.25%), fetal bovine serum (FBS), and dulbecco’s modified Eagle’s medium (DMEM) were purchased from Gibco . For in vitro experiments, reagents such as 2,7-dichlorodihydrofluorescein diacetate (H2DCFDA) and 3--2,5-diphenyltetrazolium bromide (MTT) were obtained from Invitrogen , hydrogen peroxide  was purchased from Junsei Chemical Co. , dimethyl sulfoxide-d6  was obtained from Sigma-Aldrich , and phosphate-buffered saline  was purchased from HyClone . For the Gram-positive bacterium, Staphylococcus aureus ATCC 6538, culture, Luria–Bertani (LB) agar and Luria–Bertani (LB) broth were obtained from BD Difco . For the antibacterial assay, the Gram-positive bacterium, Staphylococcus aureus ATCC 6538 was obtained from the American Type Culture Collection . All solvents were used as received without additional purification.ABS and tetrahydrofuran  were purchased from Sigma-Aldrich , polyglyceryl-4 caprate was purchased from Special Chemical Co. , and deionized water (DIW) were obtained from HyClone  for fabricating ABS nanoparticles. For high-performance liquid chromatography (HPLC), deionized water (DIW) was purchased from Milli-QABS@NPs were fabricated through self-assembly in the aqueous phase after mixing hydrophobic ABS with amphiphilic polyglyceryl-4 caprate, which contains a hydrophilic carboxyl group segment and a hydrophobic hydrocarbon chain segment. First, ABS  and polyglyceryl-4 caprate (20 mg) were dissolved in THF (1 mL) in 4 mL vial under magnetic stirring at room temperature for 2 h. Then, utilizing a syringe pump , the THF solution with ABS and polyglyceryl-4 caprate was added dropwise to 5 mL of DIW with stirring at 530 rpm. The ABS@NPs  were stabilized by mild stirring (530 rpm) for 1 h. Finally, the solvent was completely evaporated via vacuum evaporation for 2 h and the remaining was diluted with DIW to 5 mL. The resulting products are denoted as ABS@NP × wt%, where × wt% represents the ABS loading respect to polyglyceryl-4 caprate. The same method was used to prepare empty nanoparticles (NPs) of polyglyceryl-4 caprate without the addition of ABS (denoted as ABS@NP 0 wt%) . The zetTo visually confirm the encapsulation, stability, and transparency of the dispersed phase of the fabricated ABS@NPs with various ABS concentrations , a dye solubility method was carried out utilizing methylene blue, a hydrophilic dye. First, methylene blue was dissolved in DIW at 120 nM concentration. Next, 2 mL of the dye solution was added to 2 mL of the ABS@NPs suspension and mixed in a 4 mL vial. Finally, the solution was visually examined to confirm the precipitation or dispersion of ABS@NPs ,38.The stability of ABS@NPs was analyzed over 112 days via DLS. The PDI and particle size of ABS@NPs were estimated at ABS concentrations of 0, 5, and 10 wt% in DIW on days 0, 1, 3, 7, 14, 28, 56, and 112.The antioxidant activity of ABS@NPs was evaluated utilizing a DPPH assay . As cont2O2, as an oxidative stress agent. After sample treatment, changes in the ROS levels were estimated using the following steps: first, suspensions of ABS@NP 10 wt% with various ABS concentrations (1–1000 nM) and 5 μM hydrogen peroxide (H2O2) were added to fibroblast cells and cultured for 8 h. For comparison, a negative control group without H2O2 and a positive control group with H2O2 were used. Then, after washing with PBS (200 µL to each 96-well plate) to eliminate the remaining sample solution, the NIH 3T3 cells were treated with a 10 μM H2DCFDA solution, which is an ROS fluorescence indicator, and seeded for 90 min in the dark. Finally, the in vitro antioxidant activity of the samples was estimated by measuring the fluorescence intensity of dichlorofluorescein (DCF), which was oxidized by ROS, at the emission wavelength of 535 nm (excitation wavelength: 485 nm) and using a microplate reader [To estimate the intracellular antioxidant effect of the ABS@NPs with ABS concentrations of 5 and 10 wt%, mouse embryonic fibroblast cells (NIH 3T3) were cultured in 96-well plates at a density of 10,000 cells/well and incubated for 24 h. ROS were induced in NIH 3T3 fibroblast cells via stimulation with He reader ,31.Staphylococcus aureus ATCC 6538 (S. aureus ATCC 6538) was used. The bacteria were incubated in Luria–Bertani (LB) agar plates  containing 1.5% agar at 37 °C. Before utilization, a single colony was inoculated in the LB broth and incubated overnight at 37 °C. This culture was subsequently diluted in the LB broth until the optical density (OD600) reached 0.1. Next, ABS@NPs (with 0 and 10 wt% ABS) were added to each culture to estimate the inhibition of bacterial growth over 24 h in an incubator. PBS and the ABS solution in DIW were used as negative control groups. Finally, after incubation, the suspensions were diluted to a factor of 10⁷, and 100 μL of each suspension was spread over an agar plate using glass beads. After overnight incubation, the number of colonies was counted to determine the antibacterial activity against S. aureus ATCC 6538 [To estimate the antibacterial activity of ABS@NPs, a colony-counting method using TCC 6538 ,43.The biocompatibility of ABS@NPs was evaluated utilizing NIH 3T3 cells, which were incubated in the DMEM solution including 1% penicillin–streptomycin and 10% FBS. Cell viability analysis was performed using the 3--2,5-diphenyltetrazolium bromide (MTT) assay 24 h after the addition of ABS@NPs. First, the cells were cultured at a density of 10,000 cells/well in 96-well plates. After 24 h of culture, the cells were incubated with different concentrations of ABS@NPs (1–10 µM) and cultured at 37 °C for 24 h. Thereafter, a 1 mg/mL MTT solution was added to the medium, and DMEM solution (containing 1% penicillin–streptomycin and 10% FBS) was added to each well of the plate and incubated for 3 h. Finally, the medium was washed with PBS and DMSO-d6 was used to dissolve the purple formazan dye crystals. Using a microplate reader , the absorbance of the formazan generated by live cells was estimated at 570 nm. The percentage of viable cells was estimated using the following equation: ,36,44, and a hydroxyl radical (OH−). They cause extreme damage to biomolecules in cells and mitochondria, thereby inducing inflammation and excessive oxidative stress in the body, resulting in various diseases, such as cancer and AD in humans. Therefore, compounds with antioxidant properties have become of great interest for the development of various bioproducts in modern urbanized society, where many factors, such as ultraviolet rays, fine dust, and blue light, induce the formation of active oxygen species. The antibacterial activity of natural substances is an important property for bioproduct development as it minimizes preservatives, such as formaldehyde, iodopropynyl butylcarbamate, and triclosan, which induce issues such as allergies and contact dermatitis in users. ABS, a sesquiterpene alcohol, is a natural hydrophobic bioactive compound with various effects, including antioxidant and antibacterial activities. However, its application in the bio-industry is difficult owing to its low solubility and stability in the aqueous phase. In this study, we demonstrated the successful and facile fabrication of ABS@NPs, which have potential antioxidant and antibacterial effects, using an emulsification method. The optimized ABS@NPs were thermodynamically stable and exhibited excellent stability for up to 112 days. Moreover, the improved aqueous solubility of ABS@NPs led to a remarkable increase in the bioavailability of ABS and induced improved effects of ABS compared to those of non-encapsulated ABS. We confirmed that ABS@NPs successfully scavenge ROS and kill bacteria without toxicity. The ABS@NPs are expected to be used as drug carriers with outstanding stability in various fields, including foods, cosmetics, and pharmaceuticals.Bioactive compounds have various properties, such as antioxidant and antibacterial activities, and are widely used in the pharmaceutical, cosmetic, and food industries. ROS, which are highly reactive forms of intermediates, include H


The comprehensive parent-metabolite model covers biotransformation via carboxylesterase (CES) 1, CES2, CYP2C19, CYP3A4, and uridine 5′-diphospho-glucuronosyltransferase 2B7. Moreover, CYP2C19 was incorporated for normal, intermediate, and poor metabolizer phenotypes. Good predictive performance of the model was demonstrated for the DGI involving CYP2C19, with 17/19 predicted DGI AUClast and 19/19 predicted DGI Cmax ratios within 2-fold of their observed values. Furthermore, DDIs involving bupropion, omeprazole, montelukast, pioglitazone, repaglinide, and rifampicin showed 13/13 predicted DDI AUClast and 13/13 predicted DDI Cmax ratios within 2-fold of their observed ratios. After publication, the model will be made publicly accessible in the Open Systems Pharmacology repository.The antiplatelet agent clopidogrel is listed by the FDA as a strong clinical index inhibitor of cytochrome P450 (CYP) 2C8 and weak clinical inhibitor of CYP2B6. Moreover, clopidogrel is a substrate of—among others—CYP2C19 and CYP3A4. This work presents the development of a whole-body physiologically based pharmacokinetic (PBPK) model of clopidogrel including the relevant metabolites, clopidogrel carboxylic acid, clopidogrel acyl glucuronide, 2-oxo-clopidogrel, and the active thiol metabolite, with subsequent application for drug–gene interaction (DGI) and drug–drug interaction (DDI) predictions. Model building was performed in PK-Sim The antiplatelet agent clopidogrel is widely used in the prevention of atherothrombotic events, such as secondary prophylaxis after a myocardial infarction or in the event of an acute coronary syndrome . In 2019Clopidogrel is classified as a BCS class II drug . Oral upCYP2C19 gene, resulting in rapid (RM), normal (NM), intermediate (IM), or poor metabolizer (PM) phenotypes [A high interindividual variability in plasma levels of clopidogrel and the effect on platelet aggregation can be observed, resulting mainly from the so-called “clopidogrel resistance”. Not well defined, this term is generally associated with a considerable attenuation of the inhibitory effect on platelet aggregation, with the specific mechanisms not yet conclusively investigated . Geneticenotypes ,17,18. Aenotypes .Furthermore, drug–drug interactions (DDIs) are suspected to play a role with clopidogrel as the victim, especially in association with CYP3A4 and CYP2C19 interacting perpetrators ,21. WhilTo ensure effective therapy of clopidogrel and concurrent drugs while maintaining adequate control of adverse events, the complex pharmacokinetics (PK) of clopidogrel and its metabolites, characterized by their high potential for DDIs, DGIs, and DDGIs, should be thoroughly investigated. Physiologically based pharmacokinetic (PBPK) modeling has been a capable tool for such approaches for many years, as it allows the study of complex metabolic processes and pharmacokinetic aspects. Where relevant, different genotypes as well as interactions involving multiple drugs can be considered ,38. AddiThe objectives of the present work were (a) the development of a whole-body PBPK model of clopidogrel including all relevant metabolites  as well as the application of the model to predict (b) the DGI involving CYP2C19, along with (c) DDIs involving CYP2B6, CYP2C19, CYP3A4, and specifically CYP2C8. Once published, the model will be made publicly accessible in the Open Systems Pharmacology (OSP) repository. The ® and MoBi® . Digitization of published clinical study data was carried out via Engauge Digitizer 12.1 , and local sensitivity analysis were accomplished using PK-SimMitchell , 2019) aMitchell . CalculaPlasma concentration-time profiles of clopidogrel and its four metabolites  were digitized from published literature. All studies were conducted in healthy participants covering a wide dosing range, SD and MD, intravenous and peroral administration. Considering the digitized studies, training ,50,51,52Development of the clopidogrel parent-metabolite model was initiated with an extensive literature search for physicochemical properties as well as information regarding absorption, distribution, metabolism, and excretion (ADME) processes of clopidogrel and its metabolites Clo-COOH, Clo-AG, 2-Oxo-Clo, and Clo-AM.®. Relative expression for enzymes and transporters of interest in the different organs were specified according to the PK-Sim® expression database [A virtual individual was created for each included study. If available, mean and mode data on age, sex, weight, height, body mass index, and ethnicity from the respective study were incorporated. If demographic data were missing, a default individual was generated based on data for different ethnicities provided in PK-Simdatabase . Further® were fitted using the training dataset. Due to the complexity of the model, a stepwise approach was applied. First, only clopidogrel parameters were optimized to establish the accurate ratio between the two major pathways. Next, separate optimizations were performed for parameters related to the pathways leading to the formation of Clo-AG  and Clo-AM , respectively. Since many enzymes inhibited by clopidogrel are responsible for its own biotransformation, relevant inhibition parameters of clopidogrel and its metabolites were incorporated during model development. Studies quantifying clopidogrel and Clo-AM in CYP2C19 PMs were used to define CYP2C19 independent metabolism [®, can be found in During model development, model input parameters missing from the literature or involved in calculation methods of PK-Simtabolism ,51. An olast) and maximum plasma concentration (Cmax) values for all predicted versus observed profiles. Predictions within the 2-fold deviation of observed values were considered successful. Quantitative evaluations were performed by calculating mean relative deviations (MRDs) for all plasma concentration predictions along with geometric mean fold errors (GMFEs) for all predicted AUClast and Cmax values according to Equations (1) and (2), respectively.last or Cmax value of study i, last or Cmax value of study i, and m = number of studies.The clopidogrel parent-metabolite model was evaluated both graphically and quantitatively. Predicted concentration-time profiles were plotted alongside their respective observed data points for visual comparison. Furthermore, goodness-of-fit (GOF) plots were used to display the agreement between predicted and observed concentration values. GOF plots were additionally generated to compare the calculated area under the concentration-time curve between first and last concentration measurements  unchanged while varying the catalytic rate constant (kcat) depending on the phenotype. For CYP2C19 NMs, activity was assumed to be 100%, for IMs 50%, and for PMs 0%, based on reported activity scores [Metabolism via CYP2C19 was implemented according to Michaelis–Menten kinetics. Different levels of CYP2C19 activity between phenotypes were modeled by maintaining the Michaelis–Menten constant ), with subsequent comparison to corresponding observed ratios computed analogously.last or Cmax, PK parameterDGI = AUClast or Cmax of IM or PM phenotype, and PK parameterReference = AUClast or Cmax of NM phenotype.DGI model evaluation was performed by plotting and comparing predicted to observed plasma concentration-time profiles of each phenotype. Predicted DGI AUClast and Cmax ratios according to Equation (2).The limits proposed by Guest et al.  were appDDI performance of the clopidogrel model was assessed by building a CYP2B6/CYP2C8/CYP2C19/CYP3A4 DDI network centered around clopidogrel, coupling the clopidogrel model with publicly accessible models of bupropion, montelukast, omeprazole, pioglitazone, repaglinide, and rifampicin ,78,79,80last and Cmax ratios were calculated for every predicted and observed profile according to Equation (4), with subsequent comparison, applying the limits proposed by Guest et al. [last or Cmax, PK parameterDDI = AUClast or Cmax of victim drug during perpetrator co-administration, and PK parameterControl = AUClast or Cmax of victim drug control.Evaluating the DDI network, corresponding predicted as well as observed plasma concentration-time profiles of the victim drug administered with and without the respective perpetrator drug were plotted and compared graphically. Moreover, DDI AUCt et al.  to deterlast and Cmax ratios according to Equation (2).Quantitative assessment was performed by calculating GMFE values for all predicted DDI AUCBuilding and evaluation of the clopidogrel parent-metabolite model was performed using one clinical study involving intravenous application of 0.1–300 mg clopidogrel (SD) as well as 31 studies administering clopidogrel perorally at common doses of 75 to 600 mg (SD and MD), providing a total of 23 clopidogrel, 21 Clo-COOH, 3 Clo-AG, 1 2-Oxo-Clo, and 18 Clo-AM plasma concentration-time profiles. Information on all profiles utilized is listed in M values were adopted from the literature, while kcat and nonspecific clearance parameters were fitted. Moreover, all (auto)inhibition parameters of clopidogrel and its metabolites were implemented as literature values. A full list of model parameters of clopidogrel and its metabolites is provided in For each biotransformation process, KThe final clopidogrel parent-metabolite model allows good description (training dataset) and prediction (test dataset) of clopidogrel, Clo-COOH, Clo-AG, 2-Oxo-Clo, and Clo-AM plasma concentration-time profiles following intravenous and peroral clopidogrel administration. last and Cmax values, divided into training and test datasets. Considering both datasets for all five compounds, 76% of the predicted concentration measurements, 63/66 predicted AUClast, and 55/58 predicted Cmax values lie within the 2-fold range of their respective observed counterparts. In total, the model shows a mean MRD of 1.91 as well as mean GMFEAUClast and GMFECmax values of 1.38 and 1.35, respectively, thus, confirming its adequate descriptive and predictive performance. Individual MRD and GMFE values for all profiles are listed in last of clopidogrel, 2-Oxo-Clo, and Clo-AM to be most sensitive to perturbation of the respective optimized lipophilicity, while the AUClast of Clo-COOH and Clo-AG showed the highest sensitivity to perturbation of the optimized kcat of UGT2B7 and fraction unbound adopted from the literature, respectively. The complete quantitative assessment of the sensitivity analyses, along with a list of all parameters evaluated for their influence on AUClast are provided in Sensitivity analyses of an MD simulation over ten days with the administration of 75 mg clopidogrel daily revealed the AUCCYP2C19 genotyping was conducted for alleles *1, *2, and *3 with subsequent assignment of genotypes to phenotypes NM, IM, and PM according to The DGI model was developed using five DGI studies, with one study quantifying clopidogrel, three studies measuring Clo-AM, and one study investigating both compounds in relation to different phenotypes ,71,81,82last and Cmax ratios for each profile. In total, 15/19 predicted DGI AUClast and 16/19 predicted DGI Cmax ratios fall within the limits proposed by Guest et al. [t et al. , with loThe DDI network centered around clopidogrel was built and evaluated using nine DDI studies. Regarding clopidogrel as the victim, two studies examined the impact of the CYP2C19 mechanism-based inactivator omeprazole on the PK of Clo-AM when co-administered with clopidogrel for several days ,26, whillast and Cmax ratios for each profile. Overall, 11/13 predicted DDI AUClast and 13/13 predicted DDI Cmax ratios lie within the limits proposed by Guest et al. [last ratios (1.39) and for predicted DDI Cmax ratios (1.15) demonstrate a good DDI prediction performance. Individual GMFE values for all DDI profiles are available in Classified according to whether clopidogrel represents the victim or perpetrator, t et al. , while aIn this work, a clopidogrel parent-metabolite whole-body PBPK model was successfully built and evaluated, capable of adequately describing and predicting plasma concentrations of clopidogrel and its metabolites Clo-COOH, Clo-AG, 2-Oxo-Clo, and Clo-AM over a wide dosing range of intravenously  and perorally  administered clopidogrel. Moreover, the application of the model allowed the successful prediction of the DGI involving CYP2C19, along with DDIs involving CYP2B6, CYP2C8, CYP2C19, and CYP3A4.Predicted intestinal absorption ranges from 97–100% across the dosing range investigated, which is in line with the literature data on clopidogrel excretion, showing that more than 50% of administered clopidogrel is absorbed . While clast following intravenous application [Comparison of the pharmacokinetic profiles of dose-matched intravenously and perorally administered clopidogrel revealed a 170-fold higher AUClication ,85. A nolication .According to the literature, various CYP enzymes are responsible for the formation of Clo-AM from clopidogrel via 2-Oxo-Clo as part of the second main metabolic pathway, e.g., CYP1A2, CYP2B6, CYP2C9, CYP2C19, and CYP3A4 ,15,87,88*2/*2 and *3/*3 genotypes were both classified as PM, and therefore, assigned the same activity score, thus, not allowing differentiation of genotypes by the model. Here, sufficient data on individual genotypes might be required to enable differentiated modeling. Additionally, Clo-AM in particular is subject to a high interindividual variability, e.g., Cmax values of Clo-AM after peroral administration of 75 mg clopidogrel vary between 9.6 and 27.9 ng/mL in different individuals [CYP2C19 genotype information.The final clopidogrel model was applied for successful prediction of the DGI involving CYP2C19, which is especially noteworthy considering that CYP2C19 is incorporated at two metabolic steps and no phenotype data were available for the intermediate metabolite 2-Oxo-Clo. However, incomplete published data caused limitations of the modeled DGI. For example, due to the exclusive availability of data obtained from Asian individuals, the predictive performance might vary for other ethnicities. Moreover, activity scores were used for DGI modeling, with the classification of genotypes according to phenotypes and assignment of the phenotypes to activity scores adopted from the literature . Consequividuals ,71. HereFurthermore, a CYP2B6/CYP2C8/CYP2C19/CYP3A4 DDI network was successfully built by coupling the final clopidogrel parent-metabolite model with models of bupropion, montelukast, omeprazole, pioglitazone, repaglinide, and rifampicin ,78,79,80CYP2C19 variant allele carriers as well as the comprehensive DDI network involving several perpetrator and victim drugs.Previously published PBPK models of clopidogrel can be found in the literature ,95,96,97The developed clopidogrel parent-metabolite whole-body PBPK model shows a good descriptive and predictive performance for all modeled compounds, especially considering model complexity and the partly sparse data availability, e.g., regarding 2-Oxo-Clo. In addition, the model has been successfully applied not only for the prediction and study of the DGI involving CYP2C19 but also of DDIs centered around clopidogrel as CYP2C19 and CYP3A4 substrate as well as CYP2B6, CYP2C8, and CYP2C19 inhibitor. Potential applications of the model include the support of MID3 or the provision of dose recommendations. Further expansion of the model to other DDI partners may be considered in the future once appropriate DDI studies and corresponding perpetrator/victim PBPK models become available. For instance, simulation of DDIs involving potent CYP3A4 inhibitors such as ketoconazole would be beneficial to further examine the role of CYP3A4 in the metabolism of clopidogrel . Althoug