Abstract:Objective To investigate the effect of silybin in inhibiting fibrosis after glaucoma filtration surgery and the underlying mechanism.Methods Twenty-five healthy rabbits undergoing unilateral trabeculectomy on the left eye were randomly assigned into 5 groups for treatment with subconjunctival injections of sterilized water(control)or 50,100,200,and 250 μmol/L silybin for 7 consecutive days.Postoperative intraocular pressure(IOP)and filtration bleb morphology(Krofeld classification)were monitored.On day 28,tissues samples were harvested from the operated eyes for HE staining,Masson's trichrome staining,and immunofluorescence detection of fibronectin and collagen I.In cultured rabbit Tenon's capsule fibroblasts with TGF-β1-induced fibrosis,the effects of silybin on autophagy and apoptosis were analyzed using Western blotting(LC3II/LC3I ratio and p62 expression)and flow cytometry.Results From postoperative day 7 to day 21,the rabbits with silybin treatment showed significantly lower IOP than those in the control group,and in the 200 and 250 μmol/L silybin groups,IOP reduction was maintained up to postoperative day 28.From postoperative day 14 to day 21,silybin dose-dependently increased the formation rate of functional filtering blebs,the 200 and 250 μmol/L silybin groups maintained more than 40%functional filtering blebs from postoperative day 14 to day 28.HE staining revealed obviously lessened inflammatory cell infiltration in silybin treatment groups compared with that in the control group.Masson's trichrome staining showed progressively reduced fibroblast numbers and collagen deposition in the silybin groups.Immunofluorescence staining confirmed that silybin dose-dependently reduced fibronectin and collagen I-positive cells.In cultured rabbit Tenon's capsule fibroblasts,silybin treatment effectively reversed TGF-β1-induced fibroblast fibrotic phenotype,increased the LC3II/LC3I ratio,decreased p62 expression,and promoted apoptosis of the fibroblasts.Conclusion Silybin significantly inhibits postoperative fibrosis in rabbit models of glaucoma likely by activating cellular autophagy and inducing apoptosis to reduce fibroblast activation.

Abstract:Objective To investigate the protective effect of 2-week dietary folic acid(FA)supplementation against renal fibrosis induced by high-salt(HS)diet in mice.Methods Twenty-eight mice were randomly divided into control,HS(8%NaCl)diet group,HS+low-dose FA(FA:0.56 mg/kg)group and HS+high-dose FA(5.6 mg/kg)group.The changes in body weight,food intake,water consumption,tail artery systolic pressure,and heart rate of the mice were monitored,and serum levels of creatinine(SCr)and inflammatory cytokines(IL-1β,IL-6,and TNF-α)were measured.Renal histopathology of the mice was assessed using HE,periodic acid-Schiff(PAS),Masson,and Sirius red staining.Renal expressions of Kim1,vimentin,α-Sma,and E-cadherin mRNAs and E-cadherin,vimentin,and α-SMA proteins were detected using RT-qPCR and immunohistochemistry.Results In mice feeding a HS diet,FA supplementation significantly reduced their water intake,lowered tail artery systolic pressure,heart rate,renal index and serum SCr levels,reducing also serum levels of TNF-α,IL-1β,and IL-6.Masson and Sirius red staining revealed markedly reduced glomerular and interstitial fibrosis indices in HS+low-dose FA group,while the mice in HS+high-dose FA group showed mixed fibrotic improvements.Immunohistochemical results demonstrated that FA significantly upregulated E-cadherin protein expression and suppressed vimentin expression in renal tissues.Both FA doses inhibited HS-induced mRNA and protein expressions of fibrosis markers(Vimentin,Kim1 and α-Sma),attenuated interstitial collagen deposition,and alleviated renal fibrosis.FA also upregulated renal E-cadherin mRNA expression,contributing to fibrosis mitigation.Conclusion FA intervention ameliorates renal inflammation and delays fibrosis progression in HS-fed mice.Low-dose FA supplementation produces better protective effect than high-dose FA,likely mediated by regulation of renal E-cadherin,Kim1,vimentin,and α-Sma expressions.

Abstract:Objective To investigate the effect of niranthin(Nir)in mice with Crohn's disease(CD)-like colitis and its therapeutic mechanism.Methods In a mouse model of CD-like colitis induced using 2,4,6-trinitrobenzene sulfonic acid,the effects of niranthin on colitis symptoms were evaluated by measuring changes in disease activity index(DAI)score,body weight,colon length,and colonic pathologies.Intestinal barrier function and cell apoptosis were evaluated using AB-PAS staining,immunofluorescence staining,Western blotting,and TUNEL staining.The changes in Th1 and Th2 cells in the mesenteric lymph nodes and colonic TNF-α and IL-10 expression levels were determined with flow cytometry and ELISA.In lipopolysaccharide(LPS)-induced mouse colon organoids,the effects of niranthin on organoid budding number and barrier protein expressions were observed.Network pharmacology and in vivo experiments were employed to explore and verify the therapeutic mechanism of niranthin on colitis.Results In the mouse models of CD-like colitis,niranthin treatment obviously improved weight loss,DAI scores,and colorectal shortening and significantly reduced tissue inflammation scores,goblet cell loss,and intestinal epithelial cell apoptosis.Niranthin significantly increased the budding number in LPS-induced mouse colon organoids.In both mouse colon tissues and LPS-induced mouse colon organoids,niranthin obviously increased the expressions of ZO-1 and claudin-1,downregulated the expressions of cleaved caspase-3 and BAX,and upregulated Bcl-2 expression.The niranthin-treated mouse models showed significantly ameliorated Th1/Th2 imbalance in the mesenteric lymph nodes,downregulated TNF-α and upregulated IL-10 levels in the colon tissues.Network pharmacology predicted that the therapeutic mechanism of niranthin for CD-like colitis involved the PI3K/AKT pathway,which was validated in the mouse models treated with niranthin and the PI3K/AKT pathway activator Recilisib.Conclusion Niranthin ameliorates colitis in mice by antagonizing epithelial apoptosis and regulating Th1/Th2 balance via inhibiting the PI3K/AKT pathway.

Abstract:Objective To explore the regulatory effects of 3 Astragalus herb pairs(Astragalus-Salvia miltiorrhiza,Astragalus-Rehmannia glutinosa,and Astragalus-Dioscorea opposita)in Qidan Dihuang Granule on PTGS2-mediated lipid peroxidation in mice with diabetic kidney disease(DKD).Methods Network pharmacology was used to screen active components and targets of Astragalus membranaceus,Salvia miltiorrhiza,Rehmannia glutinosa,and Dioscorea opposita in Qidan Dihuang Granule to construct herb pair-active component-target networks,followed by intersection analysis with DKD-related targets for PPI network construction and enrichment analysis.Molecular docking was used to verify the binding of the key active components to PTGS2.In 25 C57BL/6J mouse models of streptozotocin-induced DKD,the therapeutic effects of treatments with saline,irbesartan,and the 3 Astragalus herb pairs(n=5)for 8 weeks were tested,with 5 normal mice serving as the control group.Results Network pharmacology showed extensive intersections between the active components of each herb pair and DKD-related targets,with PTGS2 as the key target.The major active components exhibited good binding affinity to PTGS2.The DKD mouse models in Astragalus-Salvia miltiorrhiza and Astragalus-Rehmannia glutinosa groups,particularly those in the former group,showed significant improvements in body weight,fasting blood glucose,serum creatinine,urea nitrogen,and 24-h urinary albumin.The Astragalus-Dioscorea opposita pair only slightly improved blood glucose and creatinine without improving urea nitrogen or urinary albumin in the mouse models.The Astragalus-Salvia miltiorrhiza pair,but not the other two pairs,markedly reduced the elevation of PTGS2 expression,significantly enhanced SOD activity,reduced MDA content,and upregulated GPX4 expression in the mouse models,and the therapeutic effect was only moderate in Astragalus-Rehmannia glutinosa group and the poorest in Astragalus-Dioscorea opposita group.Conclusion The 3 Astragalus herb pairs from Qidan Dihuang Granule can improve DKD in mice by reducing PTGS2-mediated lipid peroxidation,and the Astragalus-Salvia miltiorrhiza pair shows the strongest efficacy.

Abstract:Objective To investigate the neuroprotective effects of formononetin(FMN)against hypoxic-ischemic brain damage(HIBD)in neonatal mice and the underlying mechanism.Methods Twenty-four neonatal C57BL/6J mice were randomly divided(n=6)into sham-operated group,HIBD model group,HIBD+FMN-L(50 mg/kg)group,and HIBD+FMN-H(100 mg/kg)group.Mouse models of HIBD were established by left common carotid artery ligation followed by hypoxia(92%N₂,8%O₂)for 40 min.FMN at the two doses was administered by intraperitoneal injection,and 3 days later,brain tissues from the cortical ischemic penumbra were collected for assessing expressions of ferroptosis-related proteins(P53,SAT1,and ACSL4)using Western blotting and immunofluorescence staining and for detecting the levels of Fe²⁺,superoxide,malondialdehyde(MDA),and glutathione(GSH).In cultured HT22 neurons with oxygen-glucose deprivation(OGD),the effects of 100 μmol/L FMN,10 μmol/L Nutlin-3(a P53 agonist),or their combination on expressions of ferroptosis proteins,intracellular Fe²⁺,reactive oxygen species(ROS),lipid peroxidation,GSH,mitochondrial membrane potential,and cell viability were evaluated.Results In the neonatal mouse models of HIBD,FMN treatment significantly suppressed the protein expression of P53,SAT1,and ACSL4,reduced Fe²⁺,ROS,and MDA levels and increased GSH content in the cortical ischemic penumbra.In HT22 neurons with OGD,FMN obviously alleviated OGD-induced ferroptosis as shown by lowered expressions of the key ferroptosis proteins,reduced Fe²⁺ accumulation and lipid peroxidation,and significant increases of GSH levels,mitochondrial membrane potential,and cell viability.Mechanistic experiments showed that activation of P53 signaling by Nutlin-3 markedly reversed the protective effects of FMN.Conclusion FMN produces neuro-protective effects against HIBD in neonatal mice by mitigating neuronal ferroptosis,primarily through downregulation of the P53/SAT1/ACSL4 signaling pathway.

Abstract:Objective To investigate the protective effect of Huoxue Qingjie Ling(HXQJL)agaisnt bile duct ligation(BDL)-induced liver fibrosis in rats and the underlying mechanism.Methods SD rats were randomized into sham-operated group,BDL model group,low-and high-dose HXQJL treatment groups,Ex527(a Sirt1 inhibitor)group(EX527),and HXQJL+Ex527 group.Liver histopathological changes and collagen deposition were observed using HE and Sirius Red staining,and serum levels of ALT,AST,ALP,and GGT of the rats were measured using biochemical assays.The expression levels of α-SMA,FN,COL I,Atg5,Beclin1,p62,LC3B,and Sirt1 were determined by RT-PCR and Western blotting.Immunofluorescence staining was used to detect the expression of α-SMA,LC3B,and Sirt1.Results Compared with the sham-operated rats,the rats in BDL model group exhibited increased hepatocyte injury,inflammatory cell infiltration,and collagen deposition area with elevated serum levels of ALT,AST,ALP,and GGT,enhanced haptic expressions of α-SMA,FN,COL I,Atg5,and Beclin1,an increased LC3B-II/I ratio,and lowered hepatic expressions of p62 and Sirt1.All these changes were significantly alleviated in the two HXQJL treatment groups but aggravated in EX527 treatment group.Compared with those in high-dose HXQJL group,the protective effects of HXQJL on liver tissue injury,liver function,liver fibrosis,and autophagy were obviously mitigated by treatment with Ex527.Conclusion HXQJL has protective effect against BDL-induced liver fibrosis in rats by modulating the Sirt1-autophagy signaling pathway.

Abstract:Objective To explore the molecular mechanism by which selenocystine(SeC)inhibits colon cancer cell growth in vitro.Methods Colon cancer cells(RKO,HCT-116,and LoVo)were cultured and treated with 5,10,or 20 μmol/L SeC for 24 h and 48 h.MTT assay was used to detect the cell viability,and wound healing assay was used to examine changes in cell migration.Flow cytometry with PI staining was used to analyze cell cycle arrest and apoptosis.Fluorescence probes were employed to monitor reactive oxygen species(ROS)generation,mitochondrial morphology and membrane potential,and the changes in ferroptosis were evaluated by detecting malondialdehyde(MDA),glutathione(GSH)and ferrous ion(Fe2+)levels;Western blotting was used to detect the changes in protein expressions.Results SeC at all the 3 doses significantly inhibited proliferation and migration of colon cancer cells,down-regulated the expression of cell cycle-related proteins CDK2 and CDK4 and activated the apoptotic proteins PARP and caspase-9.Western blotting showed that SeC decreased the expression of ferroptosis proteins FTH1 and xCT and increased the expression of DMT1.The levels of MDA and Fe2+were increased and GSH level was decreased in SeC-treated cells.Fluorescence staining results showed that SeC treatment induced mitochondrial structure damages and promoted cellular ROS production.SeC treatment also increased phosphorylation of oxidative damage proteins and lowered the expression levels of NRF2 and HO-1 proteins.ROS scavenger significantly reversed the up-regulation of DMT1,PARP and p-H2A.X protein induced by SeC in colon cancer cells.Conclusion SeC induces apoptosis and ferroptosis of colon cancer cells by promoting ROS generation and initiating oxidative damage,suggesting the potential of SeC as a potential chemotherapeutic agent for colon cancer.

Abstract:Objective To investigate the therapeutic effect of modified Chaihu Shugan San(CHSGS)in mice with cholesterol gallstones(CS)and liver depression syndrome and its potential mechanism.Methods Sixty male C57BL/6 mice were randomly divided into blank group(n=9)and model group(n=51)with CS induced by high-lithogenic diet feeding.After verification of successful modeling in 3 mice,the remaining 48 mouse models were divided into CS group,CS-liver depression(CS-LD)group,CHSGS group,and ursodeoxycholic acid(UDCA)group,and the latter 3 groups were subjected to CS-LD modeling by chronic unpredictable mild stress(CUMS)and solitary raising and treated with saline,CHSGS or UDCA gavage for 3 weeks.The mice were assessed for general state,body weight,depressive behavior,ileal pathologies,biliary total cholesterol(TC)and total bile acid(TBA)levels,and ileal mRNA expressions of TGR5,GLP-1,and GLP-2 using q-PCR.16S rDNA sequencing and metabolomics studies were used to analyze fecal microbiota and bile acids of the mice.Results Compared to the blank control and CS mice,the CS-LD mouse models showed decreased body weight,depressive behaviors,enlarged gallbladder with crystals,intestinal villi damage,increased biliary TC,decreased TBA,and increased ileal GLP-1/2 and TGR5 mRNA expressions,which were obviously improved by CHSGS treatment.Microbiota analysis revealed decreased alpha diversity in CS and CS-LD groups,with altered abundances of Verrucomicrobiota,Akkermansia,Bacteroidota,and Muribaculaceae.Bile acid profiling revealed significant upregulation of TDCA,DCA,and GDCA in CS group and increased CA and 3-ketodeoxycholic acid levels in CS-LD group.Correlation analysis suggested positive correlations between pro-lithogenic bile acids and the harmful gut bacteria.Conclusion Modified CHSGS alleviates liver depression and cholesterol gallstones in mice likely by ameliorating gut microbiota dysbiosis and bile acid metabolism disorders.

Abstract:Objective To investigate the value of serum gasdermin D(GSDMD)level for predicting 30-day major adverse cardiovascular events(MACE)in patients following primary percutaneous coronary intervention(PPCI)for acute ST-segment elevation myocardial infarction(STEMI)and construct a predictive nomogram.Methods A total of 100 STEMI patients undergoing PPCI were prospectively enrolled.Serum GSDMD levels of the patients were measured by ELISA before and on the second morning after PPCI and compared using Wilcoxon signed-rank test.The patients were divided into high-GSDMD and low-GSDMD groups based on the optimal cut-off value of postoperative GSDMD levels determined by the Youden index.During the 30-day follow-up,the patients were categorized into MACE group(n=26)and non-MACE group(n=74).The key predictors were selected using univariable and LASSO regression,followed by multivariable logistic regression to identify the independent risk factors.A nomogram was constructed and evaluated by ROC curve analysis,Bootstrap internal validation,Hosmer-Lemeshow test,calibration plots and decision curve analysis(DCA).Results Post-PPCI serum GSDMD levels were significantly elevated relative to the pre-PPCI levels(Z=-4.848,P<0.001).The incidence of 30-day MACE was significantly higher in the high GSDMD group(P=0.01).Post-PPCI GSDMD level was identified as an independent risk factor for short-term MACE.The final nomogram incorporated Killip classification,number of stents,albumin,and post-PPCI GSDMD and demonstrated good predictive performance with an AUC of 0.847(P<0.001,95%CI:0.759-0.936),a sensitivity of 84.6%and a specificity of 79.7%.All the validation analyses confirmed good predictive efficacy and clinical utility of the model.Conclusion Elevated post-PPCI serum GSDMD level is an independent risk factor for 30-day MACE in STEMI patients.The nomogram model based on this biomarker provides a reliable tool for short-term risk stratification of these patients.

Abstract:Objective To investigate the regulatory mechanism of Yiqi Jiedu Formula on immune microenvironment of hepatocellular carcinoma(HCC)in mice.Methods Forty male BALB/c mice bearing subcutaneous Hep3B cell xenografts were randomized equally into model group(with normal saline treatment),Huachansu Tablet group,and low-(0.5 g/kg),medium-(1 g/kg),and high-dose(2 g/kg)Yiqi Jiedu Formula groups.All the mice received daily gavage of the corresponding treatments for 28 consecutive days,and the changes in tumor weight and volume were recorded every 7 days,and tumor inhibition rate was calculated after the final administration.Histopathological changes in the tumor tissues were observed with HE staining,and polarization of M1/M2 macrophages was analyzed with flow cytometry.The mRNA and protein expressions of iNOS,Arg-1,p65,and p50 in the tumor tissues were detected using q-PCR and Western blotting,and CD86 and CD206 protein expressions were observed with immunofluorescence staining.Serum levels of IL-4,IL-6,IL-10,IFN-γ,TNF-α,and TGF-β1 of the mice were measured using ELISA.Results The mice in the 3 Yiqi Jiedu Formula groups showed significant reductions in tumor weight and volume with decreased tumor cell count,increased tumor cell apoptosis,increased percentage of M1 macrophages,and decreased percentage of M2 macrophages.The treatment obviously upregulated CD86 and downregulated CD206 expression in the tumor tissue,lowered the protein and mRNA expressions of iNOS,Arg-1,p65,and p50,reduced serum levels of IL-4,IL-10,TNF-α,and TGF-β1,and increased serum levels of IL-6 and IFN-γ in the tumor-bearing mice.Conclusion Yiqi Jiedu Formula effectively inhibits Hep3B cell xenograft growth in mice and induces a shift of M1/M2 ratio by promoting M1 polarization,thereby ameliorating the immunosuppressive tumor microenvironment and enhancing anti-tumor immunity possibly in association with inhibition of NF-κB signaling pathway overactivation.

Abstract:Objective To investigate the mechanism of eurycomanone(EN)for ameliorating ischemia/reperfusion(IR)-induced acute kidney injury(AKI)in mice.Methods Twenty-four male C57BL/6J mice were randomly divided into 4 groups(n=6)for sham operation,IR modeling by bilateral renal pedicle clamping,or intraperitoneal injections of EN at 0.25 or 1.0 mg/kg for two days prior to surgery.Network pharmacology and molecular docking were used to identify the potential molecular targets and signaling pathways and evaluate the binding affinities.Renal function,histopathological changes,expression of tubular injury markers(KIM-1 and NGAL),phosphorylation levels of STAT3,PI3K,and JAK2,renal inflammation,mitochondrial biogenesis,and mitochondrial function of the mice were assessed.The interaction between EN and STAT3 was examined using surface plasmon resonance(SPR).Another 24 male C57BL/6J mice receiving sham operation,IR modeling,or EN or EN+ML115(a STAT3 agonist)treatment prior to modeling(n=6)were used to validate the role of STAT3 in EN-mediated renal protection.Results Both low-and high-dose EN significantly alleviated renal injury and improved renal function in mice with IR-induced AKI.The core targets of EN were associated with inflammation-related signaling pathways.EN treatment markedly reduced renal levels of IL-6,MCP-1,and TNF-αand decreased macrophage infiltration(F4/80-positive cells)in renal interstitial tissue of the mice.EN also significantly increased renal ATP content and mitochondrial DNA copy number(P<0.05),and upregulated the expressions of PGC-1α,TFAM,and Nrf2 mRNAs and protein levels of PGC-1α and TOM20.Molecular docking identified STAT3 and PI3K as key molecular targets of EN.In mice with IR-induced AKI,EN significantly suppressed phosphorylation of STAT3 and PI3K in the renal tissues without affecting p-JAK2 levels.SPR analysis confirmed a direct and specific interaction between EN and STAT3.Notably,activation of STAT3 by ML115 significantly reversed the renoprotective effects of EN.Conclusion EN mitigates IR-induced AKI in mice by directly binding to STAT3 to inhibit its phosphorylation,suppressing inflammation,and enhancing mitochondrial biogenesis and function,suggesting the potential of EN as a promising therapeutic candidate for IR-induced AKI.

Abstract:Objective To investigate the protective effect of mefloquine HCl(MQ)against X-ray irradiation-induced DNA damage in lung epithelial cells.Methods Human lung epithelial cells(BEAS-2B)were divided into blank control group,irradiation group,and irradiation+MQ treatment group.The effects of MQ on cell proliferation and radiosensitivity after X-ray irradiation were assessed using CCK-8 assay,EdU-488 assay,and colony formation assay.Apoptosis and cell cycle distribution of BEAS-2B cells with different treatments were detected by flow cytometry.The effect of MQ in promoting DNA double-strand break(DSB)repair was observed using immunofluorescence staining and comet assay,and the molecule ar mechanism was explored using Western blotting,qPCR,and luciferase reporter assays.Results MQ at 0-10 μmol/L did not significantly affect BEAS-2B cell viability.Compared to the irradiated cells,treatment with 0-10 μmol/L MQ enhanced the cell viability,and the effect was the most conspicuous at 10 μmol/L.MQ treatment obviously promoted proliferation and increased clonogenic survival rate of irradiated BEAS-2B cells while reducing their radiosensitivity,and significantly lowered cell apoptosis rate following the irradiation.Cell cycle analysis revealed that MQ alleviated G2/M phase arrest induced by irradiation,and comet assay and immunofluorescence staining showed reduced comet tail moment and γH2AX foci formation in irradiation+MQ group.Western blotting and qPCR demonstrated that MQ treatment for 48 h significantly increased CtIP promoter activity and upregulated CtIP expressions at both the mRNA and protein levels.Conclusion MQ promotes DSB repair in BEAS-2B cells by upregulating CtIP expression and may thus alleviate radiation-induced lung epithelial cell injury.

Abstract:Objective To investigate the regulatory effects of Streptococcus salivarius-derived extracellular vesicles(SS-EVs)on natural killer(NK)cell activation and cytotoxic function and their role in progression of systemic lupus erythematosus(SLE).Methods Peripheral blood and fecal samples were collected from 35 SLE patients and 38 healthy individuals to assess the number and proportion of peripheral T cells,B cells,and NK cells.Fecal DNA was extracted for PCR amplification of S.salivarius DNA fragments and quantitative analysis using agarose gel electrophoresis.SS-EVs isolated by ultracentrifugation and characterized using transmission electron microscopy,nanoparticle tracking analysis,and Western blotting were co-cultured with NK cells,whose activation status and cytotoxicity were evaluated in vitro using qPCR and flow cytometry.Twenty-four MRL/lpr mice were randomized into 3 groups(n=8)for treatment with gavage of 100 μL PBS or 20 μg E.coli-EVs or SS-EVs in 100 μL PBS.Kidney pathology,immune complex deposition,and peripheral cytokine levels were assessed with HE staining,immunofluorescence staining,and ELISA.Results The number and proportion of peripheral NK cells were significantly reduced(P<0.0001)and the fecal abundance of S.salivarius was markedly increased in positive correlation with SLEDAI scores(R²=0.8369)in SLE patients.In vitro studies showed that SS-EVs significantly upregulated NK cell-activating receptors and enhanced NK-mediated cytotoxicity.In MRL/lpr mice,SS-EV treatment markedly exacerbated renal inflammation,promoted C3 and IgG immune complex deposition,and significantly increased serum levels of IL-6,TNF-α,IL-8,and CCL20.Conclusion S.salivarius is enriched in the gut of SLE patients and promotes NK-cell activation by releasing SS-EVs,which induce renal inflammation and immune complex deposition and contributes to SLE progression.S.salivarius and its EVs may represent novel therapeutic targets for treatment of SLE.

Abstract:Objective To investigate the protective effects of carbon quantum dots(CQDs)derived from Allium macrostemon(Xiebai)administered orally or via intraperitoneal injection in a mouse model of cisplatin-induced acute kidney injury(AKI)and explore the underlying mechanisms.Methods Male C57BL/6 mice were randomly divided into control group,AKI model group,intraperitoneal injection group,and oral administration group(n=6).In all but the control group,the mice received a single intraperitoneal injection of cisplatin(20 mg/kg)on day 3 to induce AKI;intraperitoneal injections of Xiebai-derived CQDs(0.25 mL/day)were administered on a daily basis for 5 consecutive days,and oral CQD solution was given at the dose of 1 mL/day.On day 6,blood samples were collected to measure serum creatinine(CRE)and blood urea nitrogen(BUN).HE staining and transmission electron microscopy(TEM)were used to evaluate kidney tissue structure,mitochondrial morphology,and podocyte injury.Expression levels of renal injury markers(KIM-1 and NGAL)and inflammatory cytokines(IL-6,TNF-α,and IL-1β)were determined with with RT-qPCR and Western blotting.Results Compared with those in the control group,AKI mice exhibited significant weight loss,renal enlargement,increased kidney-to-body weight ratio,and elevated serum CRE and BUN levels.In both Xiebai CQDs treatment groups,kidney/body weight ratios and serum CRE and BUN levels were reduced and the expression levels of KIM-1,NGAL,and inflammatory cytokines were lowered significantly.Histological and ultrastructural analyses revealed more intact renal architecture,reduced inflammatory infiltration,restored mitochondrial morphology,and alleviated podocyte foot process fusion and basement membrane thickening in the two treatment groups,particularly in the intraperitoneal injection group.Conclusion Intraperitoneal administration of Xiebai-derived CQDs effectively attenuates cisplatin-induced AKI in mice,improves renal function,suppresses inflammatory responses,and repairs mitochondrial damage,thus offering better renal targeting and protective effects for AKI prevention and treatment.

Abstract:Objective To screen key genes related to efferocytosis in osteoarthritis(OA)based on bioinformatics and machine learning methods,and explore their diagnostic value,immune microenvironment characteristics,and potential therapeutic targets of traditional Chinese medicines(TCM).Methods OA-related datasets GSE55235,GSE55457,and GSE117999 were obtained from the GEO database.An efferocytosis-related gene set was retrieved from GeneCards.Differential expression analysis was performed to identify OA-related differentially expressed genes(DEGs)and their intersection with efferocytosis-related genes,followed by GO and KEGG enrichment analyses.Three machine learning algorithms(Random Forest,LASSO regression,and SVM)were used to screen feature genes,and their diagnostic efficacy was evaluated using ROC curves.qRT-PCR was used to validate the feature gene expressions in a rat OA model.Immune cell infiltration was analyzed using CIBERSORT,GSEA was used to explore the related pathways,and the Coremine database was utilized to predict TCMs associated with the feature genes.Results A total of 959 OA-related DEGs were identified,including 15 efferocytosis-related genes,which were enriched in leukocyte migration,extracellular matrix,and inflammatory pathways.Machine learning identified 3 feature genes,namely UCP2,EGLN3,and IL1B,which showed good diagnostic performance in both the training(GSE55235)and validation sets(GSE55457 and GSE117999)and varying expression patterns in the mouse models.Immune infiltration analysis showed significant differences in resting mast cells,resting memory CD4⁺ T cells,and activated mast cells between OA patients and healthy controls.The feature genes were closely associated with the adipocytokine signaling pathway,sulfur metabolism,and spliceosome pathway.A total of 100 TCMs were predicted,which were primarily herbs for tonifying deficiency,clearing heat,and promoting blood circulation,such as Lycium barbarum,Epimedium brevicornu,Rehmannia glutinosa,Sophora flavescens,Ligusticum chuanxiong,and Achyranthes bidentata.Conclusion Efferocytosis-related genes play important roles in OA pathogenesis.UCP2,EGLN3,and IL1B have diagnostic value for OA.The predicted TCMs may serve as potential agents for OA prevention and treatment.

Abstract:Objective To determine whether dentin sialophosphoprotein(DSPP)modulates oxaliplatin efficacy for colorectal cancer(CRC)and explore the underlying integrin αvβ3-dependent mechanism.Methods Immunohistochemistry was used to compare the expression levels of DSPP between oxaliplatin-sensitive and oxaliplatin-resistant CRC tissues.The changes in oxaliplatin sensitivity in parental and oxaliplatin-resistant CRC cell lines after DSPP knockdown or overexpression were assessed using CCK-8 assay,and Western blotting was used to evaluate the efficacy of DSPP modulation and MAPK pathway activity.The interaction between DSPP and integrin αvβ3 was examined by immunofluorescence staining,co-immuno-precipitation,and immunohistochemistry.HE and immunofluorescence staining were used to confirm the establishment of CRC organoid models.Patient-derived xenograft(PDX)and nude mouse subcutaneous xenografts were used to evaluate the in vivo effect of targeting DSPP on oxaliplatin response.Results DSPP expression was significantly elevated in oxaliplatin-resistant patients and in oxaliplatin-resistant HCT8 cells.DSPP knockout significantly increased oxaliplatin sensitivity in oxaliplatin-resistant HCT8 cells and in HCT116 and SW620 cells.Co-immunoprecipitation revealed binding between DSPP and integrin αvβ3 in tumor cells,and immunofluorescence staining demonstrated their co-localization.Immunohistochemistry showed a positive correlation between DSPP expression and integrin αvβ3 expression in CRC tissues.Western blotting indicated that DSPP upregulated the phosphorylation levels of ERK and P53 in the MAPK signaling pathway,whereas the integrin αvβ3-targeted inhibitor(Cyclo)effectively abrogated this regulatory effect.In the xenograft and PDX models,targeted inhibition of DSPP or integrin αvβ3 suppressed tumor growth and improved the efficacy of oxaliplatin,for which the anti-DSPP monoclonal antibody was more effective than integrin αvβ3-targeted inhibitor.Conclusion We identified a DSPP-integrin αvβ3 axis that mediates oxaliplatin resistance,and DSPP may serve as a therapeutic target to restore chemosensitivity in advanced CRC.

Abstract:Objective To investigate the mechanism by which Acorus tatarinowii Schott volatile oil(VOA)alleviates tic disorder(TD)in rats.Methods Forty-eight 3-week-old SD rats were randomly divided into blank group(n=8)and TD model group with intraperitoneal injection of iminodipropionitrile(n=40).The rat models were further randomized into 5 groups(n=8)for treatment with daily gavage of saline(model group),tiapride(47.91 mg/kg)or VOA(51.12 mg/kg),intraperitoneal injection of TAK-242(a TLR4 inhibitor;3 mg/kg),or both VOA gavage and TAK242 injection for 28 days.Behavioral changes of the rats were assessed,and Nissl staining was used to observe neuronal morphology in the striatum.The levels of TNF-α,C1q,TGF-β,and VEGF in the serum and striatum were measured using ELISA.The mRNA and protein expressions of TLR4,MyD88,and NF-κB p65 in the striatum were detected using RT-PCR and Western blotting,and NF-κB p65 nuclear translocation and expressions of Iba1(a MG-specific marker),CD86 and CD206 were analyzed using immunofluorescence staining.Results VOA treatment significantly reduced tic-like behavior scores and ameliorated striatal neuronal damage in the rat models,resulting also in lowered levels of TNF-α and C1q and increased levels of TGF-β and VEGF in both the serum and striatum,decreased mRNA and protein expression levels of TLR4,MyD88,and NF-κB p65 in the striatum,and reduced nuclear translocation of NF-κB p65.Immunofluorescence staining revealed significantly weakened expressions of Iba1 and CD86 and enhanced CD206 expression in the striatum of VOA-treated rats.All these ameliorative effects of VOA were significantly attenuated by treatment with TAK-242.Conclusion VOA alleviates tic symptoms in rats by inhibiting microglia activation and polarization to reduce neuroinflammation possibly through suppression of the TLR4/MyD88/NF-κB signaling pathway.

Abstract:Objective To construct and validate a diagnostic model for osteonecrosis of the femoral head(ONFH)based on alcohol exposure-related genes using machine learning methods.Methods The transcriptomic data related to alcohol exposure and ONFH were obtained from the GEO database for construction of a diagnostic model for ONFH.The differentially expressed genes(DEGs)of alcohol exposure and ONFH were identified,and functional enrichment analysis of the intersecting genes was performed.Single sample gene set enrichment analysis(ssGSEA)was used to quantify immune infiltration.A total of 113 combinations of 12 machine learning algorithms were tested on the training set,and 10-fold cross-validation was used to construct the diagnostic model of ONFH,which was validated on the test set.The expressions of the DEGs in the model were detected by qRT-PCR in alcohol-treated MC3T3-E1 cells to verify the reliability of the constructed model.The Enrichr platform was used to identify potential drugs for ONFH.Results Twenty-one intersecting DEGs closely related to alcohol exposure and ONFH were identified,which were also involved in immune processes.Immune infiltration analysis showed that the patients with alcohol exposure and ONFH had significant differences in immune cell infiltration compared with the healthy controls.The diagnostic model was constructed based on 8 genes(SOAT1,GMCL1,GMPR,CISD2,ST3GAL6,AHSP,UBL3,and PTPN12),which showed significant differential expressions in alcohol-treated MC3T3-E1 cells.Ten potential drugs for ONFH treatment were predicted.Conclusion By integrating bioinformatics analysis and machine learning methods,a reliable model for diagnosing ONFH has been successfully constructed based on the DEGs shared by alcohol exposure and ONFH.

Abstract:Objective To compare the biomechanical properties of a novel Ti-3Zr-2Sn-3Mo-25Nb β-titanium alloy and a traditional Ti-6Al-4V titanium alloy pedicle screw-rod fixation system in posterior lumbar interbody fusion(PLIF).Methods In vitro mechanical tests were conducted to evaluate the bending,tensile,compressive,and torsional performance of the β-titanium alloy screws,connecting rods,and the assembled screw-rod fixation system.Two PLIF finite element models were constructed to compare the effects of Ti-6Al-4V versus β-titanium screw-rod systems on range of motion(ROM)and stresses in the endplate and implants.Results In vitro tests showed that the β-titanium alloy screw exhibited good resistance to bending(455.95±18.66 N)and torsion(9.03±0.20 N·m).The maximum tensile load of the β-titanium rod was 17 647.06±101.89 N,and the β-titanium screw-rod system showed a maximum compressive load of 417.65±5.09 N and a maximum torque of 25.00±0.70 N·m.Compared with Ti-6Al-4V,the β-titanium model showed a 2.6%-8.3%increase in ROM,and the peak stresses in the interbody bone graft,cage,and endplate increased by 1.4%-8.5%,2.2%-9.4%,and 2.2%-10.1%,respectively,whereas the peak stresses at the bone-screw interface and within the screw-rod system decreased by 8.8%-23.7%and 19.0%-33.1%,respectively.Conclusion The β-titanium pedicle screw-rod fixation system exhibits good in vitro mechanical performance to provide stability comparable to the conventional Ti-6Al-4V system while markedly reducing stress concentration within the screw-rod construct,suggesting its great potential for clinical application.

Abstract:Objective To explore the effect of Shengmai San(SMS)for improving osimertinib resistance in non-small cell lung cancer cells and the underlying mechanism.Methods Cultured A549 cells were treated with osimertinib alone or in combination with SMS-medicated rat serum,and the changes in cell viability and glucose and lactate levels were determined.The effect of SMS combined with osimertinib for improving osimertinib resistance of A549 cells was assessed in a mouse model bearing subcutaneous A549 cell xenograft.Western blotting,RT-qPCR,and immunofluorescence staining were used to analyze the effects of SMS and lactate on the Wnt/β-catenin/LDHA signaling pathway.Results SMS significantly increased osimertinib sensitivity of A549 cells in a concentration-dependent manner.Compared with osimertinib alone,the combined treatment with SMS and osimertinib significantly inhibited cell viability,colony formation ability,and tumor growth in nude mice.SMS concentration-dependently decreased glucose and lactate levels in A549 cells.The results of Western blotting showed that SM inhibited the protein expression of LDHA,total β-catenin,and cytoplasmic and nuclear β-catenin,while lactate obviously activated the expressions of total β-catenin protein,nuclear β-catenin,and LDHA in A549 cells.Immunofluorescence concentration-dependent staining showed that lactic acid activated nuclear accumulation of β-catenin protein,while SMS significantly inhibited the expression of nuclear β-catenin protein;RT-qPCR demonstrated that lactate significantly increased mRNA expressions of the Wnt/β-catenin downstream target genes(c-myc,CD44,Axin2,Oct3/4,survivin,and CCND1),while SMS inhibited their expressions.Conclusion SMS improves osimertinib resistance in non-small cell lung cancer cells by inhibiting lactate/Wnt/β-catenin/LDHA pathway-mediated glycolysis.