AIM To investigate the effects of Yunpi Tongchang Formula on intestinal mucosal barrier damage in rats with opioid-induced constipation(OIC)of Spleen-Kidney Yang Deficiency Syndrome.METHODS In contrast to the 10 rats of the blank group,the 50 rats of the modeling group were induced into models of OIC of Spleen-Kidney Yang Deficiency Pattern by 7 days consecutive administration of both subcutaneous loperamide injection and alternating gavage of activated carbon ice water and vinegar.Following successful modeling,rats were randomly allocated into the model group,the mosapride citrate tablet group(1.35 mg/kg),and the high-dose,medium-dose,and low-dose Yunpi Tongchang Formula groups(15.12,7.56,3.78 g/kg),with 8 mice in each group.Upon the completion of the 14 days treatment,the rats had their TCM Syndrome scores assessed;their fecal water content,initial black stool excretion time,and small intestine propulsion rate measured;their colon tissue morphology observed by HE staining;their serum levels of IL-6,TNF-α,and IL-1β detected by ELISA;their expressions of occludin and zonula occludens-1(ZO-1)in colon tissues detected by immunohistochemistry;their mRNA expressions of MyD88,TLR4 and NF-κB p65 in the colon tissues detected by RT-qPCR;and their protein expressions of MyD88,TLR4 and NF-κB p65 in the colon tissues detected by Western blot.RESULTS Compared to the blank group,the model group had higher TCM Syndrome scores(P＜0.01);lower fecal water content and small intestine propulsion rate(P＜0.05,P＜0.01);longer initial black stool excretion time(P＜0.01);more mucosal edema in colon tissue,obvious inflammatory infiltration,and glandular disorder;increased serum levels of IL-6,TNF-α and IL-1 β(P＜0.05);decreased colon expressions of ZO-1 and occludin(P＜0.01);and increased mRNA and protein expressions of TLR4,MyD88 and NF-κB p65(P＜0.01).Compared to the model group,both the medium-dose Yunpi Tongchang Formula group and the mosapride citrate tablet group demonstrated effectively reduced TCM syndrome scores(P＜0.01);increased fecal water content and small intestine propulsion rate(P＜0.05,P＜0.01);and shorter initial black stool excretion time(P＜0.01);improved colon mucosal edema and inflammatory infiltration;decreased serum levels of IL-6,TNF-α and IL-1β(P＜0.01);upregulated protein expressions of ZO-1 and occludin(P＜0.01);and downregulated mRNA and protein expressions of TLR4,MyD88 and NF-κB p65(P＜0.05,P＜0.01).CONCLUSION Yunpi Tongchang Formula significantly ameliorates constipation symptoms in OIC rat models of Spleen-Kidney Yang Deficiency Syndrome because of its efficacy in attenuating intestinal inflammation and preserving the integrity of intestinal epithelial barrier structure,with its mechanistic action in downregulating TLR4/MyD88/NF-κB signaling pathway activation.

Forensic medicine has been designated as a first-level discipline,presenting new opportunities and challenges for the development of forensic medicine.Since the 1980s,the establishment of foren-sic medicine discipline and the cultivation of high-level forensic talents have become hot topics in the development of forensic medicine in China.Since the 13th Five-Year Plan,the forensic team of Shanxi Medical University has been aiming at the forefront,proposing the development goals of"Five First-class"and the discipline development path"Six Major Achievements".It has selected benchmark disci-plines,identified gaps in disciplinary development,unified thoughts,formulated completion timelines,concentrated superior resources,assigned tasks to individuals,and created an"Organized Fill-in-the-Blank Format"forensic medicine discipline construction model with the characteristics of the new era.The construction model of forensic medicine has achieved good results in the goals,discipline frame-work,scientific research,talent cultivation,discipline team and platform construction,forming a rela-tively complete discipline construction and management system,and accumulating valuable experience for the construction of first-level discipline and high-level talent cultivation of forensic medicine.

Objective To establish a chromatography-tandem mass spectrometry method for detecting chlorfenapyr and its metabolite tralopyril in blood,and to investigate the toxicokinetics in rats.Methods Chlorfenapyr(8 mg/kg)was administered orally to rats,and blood samples were collected from rats'canthus vein at 5 min,15 min,30 min,1 h,3 h,6 h,12 h,24 h and 48 h after administration.The blood samples were extracted using 100 μL of 5%formic acid solution and 400 μL of acetonitrile.Chlorfena-pyr was qualitatively and quantitatively detected by triple quadrupole gas chromatography-tandem mass spectrometry(GC-MS/MS)and tralopyril was detected by triple quadrupole liquid chromatography-tandem mass spectrometry(LC-MS/MS).The DAS 3.0 software was used to fit the toxicokinetic equa-tions and calculate the toxicokinetic parameters.Results Chlorfenapyr was detectable from 5 min to 24 h with a peak time of 1 h.Tralopyril was detectable from 15 min to 48 h with a peak time of 3 h.The toxicokinetic process of chlorfenapyr in rat blood conformed to a first-order absorption one-compartment open model,with the toxicokinetic equation described as C=e-0.265t-e-0.175t.Tralopyril con-formed to the first-order absorption three-compartment model,and the toxicokinetic equation was C=47 361.069e-2.209t-35 404.962e-1.486t+11 956.363e-0.512t.In the equations,C stands for the concentration of the target substance in the blood,e is the natural constant(≈2.718 28),and t stands for time.Conclu-sion This study optimized the detection method for chlorfenapyr and its metabolite tralopyril in blood.The toxicokinetic equations and parameters of chlorfenapyr and tralopyril can provide a reference for the estimation of oral intake time of chlorfenapyr.

OBJECTIVE: Lipid oxidation is involved in the pathogenesis of atherosclerosis and may be contribute to the development of Ischemic stroke (IS). However, the lipid profiles associated with IS have been poorly studied. We conducted a pilot study to identify potential IS-related lipid molecules and pathways using lipidomic profiling. METHODS: Serum lipidomic profiling was performed using LC-MS in 20 patients with IS and 20 age- and sex-matched healthy controls. Univariate and multivariate analyses were simultaneously performed to identify the differential lipids. Multiple testing was controlled for using a false discovery rate (FDR) approach. Enrichment analysis was performed using MetaboAnalyst software. RESULTS: Based on the 294 lipids assayed, principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) models were used to distinguish patients with IS from healthy controls. Fifty-six differential lipids were identified with an FDR-adjusted P less than 0.05 and variable influences in projection (VIP) greater than 1.0. These lipids were significantly enriched in glycerophospholipid metabolism (FDR-adjusted P = 0.009, impact score = 0.216). CONCLUSIONS Serum lipid profiles differed significantly between patients with IS and healthy controls. Thus, glycerophospholipid metabolism may be involved in the development of IS. These results provide initial evidence that lipid molecules and their related metabolites may serve as new biomarkers and potential therapeutic targets for IS.
Humans ; Pilot Projects ; Lipidomics ; Male ; Female ; Biomarkers/blood* ; Middle Aged ; Ischemic Stroke/blood* ; Aged ; China ; Lipids/blood* ; Adult ; Case-Control Studies ; East Asian People

Hepatocellular carcinoma(HCC),which is essentially primary liver cancer,is closely related to CD8+T cell immune infiltration and immune suppression.We constructed a CD8+T cells related risk score model to pre-dict the prognosis of HCC patients and provided therapeutic guidance based on the risk score.Using integrated bulk RNA sequencing(RNA-seq)and single-cell RNA sequencing(scRNA-seq)datasets,we identified stable CD8+T cell signatures.Based on these signatures,a 3-gene risk score model,comprised of KLRB1,RGS2,and TN-FRSF1B was constructed.The risk score model was well validated through an independent external validation co-hort.We divided patients into high-risk and low-risk groups according to the risk score and compared the differ-ences in immune microenvironment between these two groups.Compared with low-risk patients,high-risk patients have higher M2-type macrophage content(P＜0.0001)and lower CD8+T cells infiltration(P＜0.0001).High-risk patients predict worse response to immunotherapy treatment than low-risk patients(P＜0.01).Drug sensitivity a-nalysis shows that PI3K-β inhibitor AZD6482 and TGFβRII inhibitor SB505124 may be suitable therapies for high-risk patients,while the IGF-1R inhibitor BMS-754807 or the novel pyrimidine-based anti-tumor metabolic drug Gemcitabine could be potential therapeutic choices for low-risk patients.Moreover,expression of these 3-gene mod-el was verified by immunohistochemistry.In summary,the establishment and validation of a CD8+T cell-derived risk model can more accurately predict the prognosis of HCC patients and guide the construction of personalized treatment plans.

Objective:To explore the role of nuclear receptor-binding SET-domain protein 1(NSD1)in the pathogenesis of nonobstructive azoospermia(NOA)by regulating the expressions of relevant genes.Methods:We detected the expression of NSD1 in the testis tissue of 7 male patients with obstructive azoospermia(OA)and 18 with NOA by qPCR and immunofluorescence assay,and determined the modification level of H3K36me2 in the testes of two groups of patients by immunofluorescence staining,Western blot and immunoprecipitation(IP).We examined the difference in the enrichment of H3K36me2 in the testis tissue by chromatin IP-based sequencing(ChIP-Seq),analyzed the genomic distribution and target genes using bioinformatics,and verified the expression levels of the target genes in the testes of the two groups of patients by qPCR.Results:Compared with the patients with OA,those with NOA showed dramatically decreased mRNA and protein expressions of NSD1(P=0.000 8).The binding of NSD1 to H3K36me2 was observed in the testis tissue of both the two groups of patients,while the modification level of H3K36me2 was evidently reduced in the NOA males.H3K36me2 was distributed mainly in the intergenic region in the testes of the two groups of patients,but the enrich-ment of H3K36me2 was obviously decreased in the NOA group.The differentially H3K36me2-enriched genes were involved in various biological processes,including tissue development,and cell morphogenesis.Results of ChIP-Seq and qPCR showed significantly down-regulated expressions of the target genes KIT,SPO11 and ACRV1 in the testis tissue of the NOA males compared with those in the OA patients(P＜0.01).Conclusion:The levels of NSD1 and H3K36me2 are decreased in testis tissue of the NOA patient,H3K36me2 is highly enriched in the spermatogenesis-related key genes KIT,SPO11 and ACRV1,and the down-regulated expression of NSD1 impairs spermatogenesis.

AIM To investigate the in vivo pharmacokinetics of chlorogenic acid,isoquercetin,ferulic acid,isorhamnetin and shionone in Asteris Radix et Rhizoma in rats.METHODS Twelve rats were randomly assigned into 2 groups and given intragastric administration of conventional dose(0.63 g/kg)and large dose(3.3 g/kg)of Asteris Radix et Rhizoma extracts,respectively,after which blood collection was made at 0.083,0.167,0.33,0.5,0.75,1,1.5,2,4,6,8,12,24 h,UPLC-MS/MS was adopted in the plasma concentration determination of various effective constituents,and main pharmacokinetic parameters were calculated.RESULTS Various effective constituents in the large dose group demonstrated prolonged t1/2,MRT0-∞ as compared with those in the conventional dose group(P＜0.05).After dose correction,Cmax of chlorogenic acid,isoquercetin,shionone in the large dose group displayed no obvious changes(P＞0.05),while Cmax of ferulic acid,isorhamnetin,and AUC0-t,AUC0-∞ of various effective constituents were higher than those in the conventional dose group(P＜0.05).CONCLUSION Various effective constituents in the high dose of Asteris Radix et Rhizoma can maintain high-concentration and long-time effects on rat bladder tissue.

AIM To investigate the effects of Yunpi Tongchang Formula on intestinal mucosal barrier damage in rats with opioid-induced constipation(OIC)of Spleen-Kidney Yang Deficiency Syndrome.METHODS In contrast to the 10 rats of the blank group,the 50 rats of the modeling group were induced into models of OIC of Spleen-Kidney Yang Deficiency Pattern by 7 days consecutive administration of both subcutaneous loperamide injection and alternating gavage of activated carbon ice water and vinegar.Following successful modeling,rats were randomly allocated into the model group,the mosapride citrate tablet group(1.35 mg/kg),and the high-dose,medium-dose,and low-dose Yunpi Tongchang Formula groups(15.12,7.56,3.78 g/kg),with 8 mice in each group.Upon the completion of the 14 days treatment,the rats had their TCM Syndrome scores assessed;their fecal water content,initial black stool excretion time,and small intestine propulsion rate measured;their colon tissue morphology observed by HE staining;their serum levels of IL-6,TNF-α,and IL-1β detected by ELISA;their expressions of occludin and zonula occludens-1(ZO-1)in colon tissues detected by immunohistochemistry;their mRNA expressions of MyD88,TLR4 and NF-κB p65 in the colon tissues detected by RT-qPCR;and their protein expressions of MyD88,TLR4 and NF-κB p65 in the colon tissues detected by Western blot.RESULTS Compared to the blank group,the model group had higher TCM Syndrome scores(P＜0.01);lower fecal water content and small intestine propulsion rate(P＜0.05,P＜0.01);longer initial black stool excretion time(P＜0.01);more mucosal edema in colon tissue,obvious inflammatory infiltration,and glandular disorder;increased serum levels of IL-6,TNF-α and IL-1 β(P＜0.05);decreased colon expressions of ZO-1 and occludin(P＜0.01);and increased mRNA and protein expressions of TLR4,MyD88 and NF-κB p65(P＜0.01).Compared to the model group,both the medium-dose Yunpi Tongchang Formula group and the mosapride citrate tablet group demonstrated effectively reduced TCM syndrome scores(P＜0.01);increased fecal water content and small intestine propulsion rate(P＜0.05,P＜0.01);and shorter initial black stool excretion time(P＜0.01);improved colon mucosal edema and inflammatory infiltration;decreased serum levels of IL-6,TNF-α and IL-1β(P＜0.01);upregulated protein expressions of ZO-1 and occludin(P＜0.01);and downregulated mRNA and protein expressions of TLR4,MyD88 and NF-κB p65(P＜0.05,P＜0.01).CONCLUSION Yunpi Tongchang Formula significantly ameliorates constipation symptoms in OIC rat models of Spleen-Kidney Yang Deficiency Syndrome because of its efficacy in attenuating intestinal inflammation and preserving the integrity of intestinal epithelial barrier structure,with its mechanistic action in downregulating TLR4/MyD88/NF-κB signaling pathway activation.

This study systematically investigated the molecular mechanisms underlying the involvement of mesoderm development-associated genes in melanoma progression through integrated bioinformatics analy-sis and experimental validation.Utilizing the GSVA(gene set variation analysis)algorithm to perform enrichment analysis of 7 752 biological functions in 406 skin cutaneous melanoma(SKCM)cases,we i-dentified for the first time the significant activation of mesoderm development pathways during SKCM pathogenesis.Four core regulatory genes(SMAD4,NODAL,BMPR1A,and ZFP36L1)were screened using LASSO-COX regression analysis and a prognostic risk-scoring system was established.Gene Ontolo-gy(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analyses revealed predomi-nant enrichment of these genes in mRNA metabolic processes and TGF-β signaling pathways.Experimen-tal validation through Quantitative Polymerase Chain Reaction(qPCR),Western blotting,and immuno-histochemistry(IHC)demonstrated that:(1)Downregulation of SMAD4 and BMPR1A in tumor tissues was significantly correlated with poor prognosis(P＜0.05);(2)NODAL promoted tumor invasion and metastasis by regulating epithelial-mesenchymal transition(EMT);(3)High ZFP36L1 expression was associated with enhanced chemotherapy sensitivity.Further analyses revealed significant correlations be-tween core gene expression levels and tumor immune infiltration characteristics as well as immune check-point molecules.By integrating multi-omics analysis with experimental validation,this study elucidates the critical roles of mesoderm development-associated genes in SKCM progression,particularly clarifying the molecular mechanisms through which SMAD4/NODAL/BMPR1A/ZFP36L1 influence tumor biologi-cal behaviors via immune microenvironment regulation and EMT processes.These findings provide novel theoretical foundations for molecular subtyping and targeted therapy in melanoma.

Aim To investigate the effects of the fangchinoline derivative LYY-32 on the biological prop-erties of the BLM642-1290 DNA helicase,in order to lay a foundation for further research on its antitumor activity.Methods Fluorescence polarization assay,malachite green-phosphate and ammonium molybdate colorime-try,and fluorescein-labeled DNA gel electrophoresis experiments were conducted to study the effects of fangchinoline derivative LYY-32 on the DNA binding activity,ATPase activity,and DNA unwinding activity of BLM642-1290 DNA helicase.The effects of LYY-32 on the DNA unwinding activity of DNA helicase in cells were studied using fluorescent techniques and time-lapse microscopy.Ultraviolet spectral scanning was used to investigate the effects of LYY-32 on the confor-mation of the BLM642-1290 DNA helicase.Results At a concentration of 10 μmol·L-1,the inhibition rate of LYY-32 on BLM642-1290 DNA helicase binding to dsDNA was 53.17％.At a concentration of 5 μmol·L-1,the inhibition rate of LYY-32 on BLM642-1290 DNA helicase binding to ssDNA was 88.49％.The inhibition rate of LYY-32 on the ATPase activity of BLM642-1290 DNA he-licase was 89.3％at a concentration of 50 μmol·L-1.When the concentration of LYY-32 exceeded 5μmol·L-1,its inhibition rate on the DNA unwinding activity of BLM642-1290 DNA helicase was 100％.LYY-32 also significantly inhibited the DNA unwinding ac-tivity of DNA helicase in cells.However,LYY-32 had no effect on the conformation of BLM642-1290 DNA heli-case.Conclusion The DNA binding activity,AT-Pase activity,and DNA unwinding activity of BLM642-1290 DNA helicase could be significantly inhibi-ted by the fangchinoline derivative LYY-32.