Background and aims The mechanism of cholestatic liver injury (CLI) is unclear, and effective therapies are lacking. While peroxisome proliferator-activated receptor alpha (PPARα) agonists show potential hepatoprotective effect and pyroptosis is implicated in hepatocellular damage, how PPARα activation mitigates lithocholic acid (LCA)-induced pyroptosis remains unknown. Methods The hepatoprotective effect of PPARα agonists was evaluated in a mouse model of intrahepatic cholestasis induced by LCA. Liver injury was assessed via serum biochemistry, hematoxylin and eosin and TUNEL staining, and electron microscopy. Pyroptosis pathways were analyzed using real-time quantitative polymerase chain reaction, Western blot, and co-immunoprecipitation. Results Combined morphological, histopathological, and biochemical analyses confirmed that PPARα activation protects against CLI. Compared with LCA treatment alone, PPARα activation significantly attenuated the elevation of serum lactate dehydrogenase (LDH), the increased TUNEL-positive cells, and the formation of hepatocyte membrane pores. Mechanistically, PPARα activation suppressed both NOD-like receptor protein 3 (NLRP3) inflammasome-mediated pyroptosis and apoptosis protease-activating factor-1 (APAF-1)/CASPASE-3/GSDME-mediated pyroptosis. Furthermore, PPARα agonist pretreatment inhibited activation of the nuclear factor-kappa B (NF-κB) and forkhead box O1 (FOXO1) signaling pathways. Conclusions PPARα protects against LCA-induced CLI by inhibiting both NLRP3 inflammasome-mediated pyroptosis associated with NF-κB and APAF-1/CASPASE-3/GSDME-mediated pyroptosis associated with the FOXO1 signaling pathway.

Objective To establish the fingerprint of Xuemai Shutong granules by UPLC-ELSD and determine the contents of 9 components in the preparation simultaneously.Methods The UPLC-ELSD was used to establish the fingerprint of Xuemai Shutong granules,and determine the content of its 9 components.The similarity evaluation system,systematic,cluster analysis,principal component analysis and orthogonal partial least squares discriminant analysis were used to evaluate the quality of different batches of preparation.Results The similarity degrees of UPLC-ELSD fingerprints of 11 batches of Xuemai Shutong granules were from 0.929 to 0.978,17 common peaks were calibrated,of which 11 peaks were identified:peak 3(notoginsenoside R1),peak 4[ginsenoside Rg,(Re)],peak 5(notoginsenoside R2),peak 6(ginsenoside Rb,),peak 9(astragaloside Ⅳ),peak 10(ginsenoside Rk3),peak 11(ginsenoside Rh4),peak 12[20(S)-ginsenoside Rg3],peak 13[20(R)-ginsenoside Rg3],peak 14(ginsenoside Rk1),peak 15(ginsenoside Rg5).The stoichiometric analysis divided 11 batches of samples into 2 classes,and the 2 principal components in PCA analysis reflected the information of 17common peaks,10 peaks which affected the quality difference are screened out.The linear relationship of the 9 components was good in their respective quality ranges in the content analysis(r＞0.999 2),the average recovery rate were between 95.02％-97.78％and the RSD were 0.69％-1.70％(n=6).The contents of notoginsenoside R1,notoginsenoside R2,ginsenoside Rb1,astragaloside Ⅳ,ginsenoside Rh4,20(S)-ginsenoside Rg3,20(R)-ginsenoside Rg3,ginsenoside Rk1 ginsenoside Rg5 in the 11 batches of Xuemai Shutong granules were 0.087 5-0.187 6,0.494 3-0.688 6,0.448 1-0.705 5,0.192 2-0.270 8,1.492 5-2.077 6,0.316 0-0.463 8,0.254 5-0.382 0,0.117 6-0.163 9,3.407 7-4.706 4 mg·g-1,respectively.Conclusions The established fingerprint and content determination method was accurate and reliable,which can improve the quality standard of Xuemai Shutong granules,and provide reference for its overall quality evaluation.

AIM To observe the effects of Yiqi Jiedu Tongluo Formula(YQJDTL)on renal microvascular endothelial function and prevention of renal injury in a rat model of type 2 diabetes mellitus(T2DM).METHODS The SD rats were randomly divided into a normal group and a model group.The model group was administered with high-fat diet combined with a single intraperitoneal injection of STZ to establish the T2DM model.The successfully modeled rats were randomly divided into the model group,the canagliflozin group(9 mg/kg),and the low-dose and high-dose YQJDTL groups(4.77,9.45 g/kg).The corresponding doses of the drug were administered by gavage for a total of 12 weeks,during which the rats underwent observation of their general condition and blood glucose changes.After the end of administration,the rats had their levels of renal index,24-hour UP,serum SCr,BUN,TC,TG,HDL-C,LDL-C,ET-1 and NOS measured;their changes in renal microvasculature and the degree of renal fibrosis observed using HE staining,Masson staining,PAS staining,and PASM staining;their ultrastructure of the glomeruli observed using transmission electron microscopy;their renal protein expressions of TGF-β,SMAD2,SMAD3,Col-1,VEGFA and PKC detected by immunohistochemical staining and Western blot;and their renal mRNA expressions of VEGFA,TGF-β,SMAD2 determined by RT-qPCR.RESULTS Compared with the model group,the high-dose YQJDTL group showed decreased levels of renal index,blood glucose,TG,TC,HDL,24 h UP,BUN,SCr and ET-1(P＜0.05,P＜0.01);increased LDL and NOS levels(P＜0.05,P＜0.01);reduced renal inflammatory infiltration and fibrosis degree,inhibited fusion of foot processes and thickening of basement membrane;decreased renal protein expressions of TGF-β,SMAD2,SMAD3,VEGFA,PKC and Col-1(P＜0.05,P＜0.01);and decreased mRNA expressions of VEGFA,TGF-β and SMAD2(P＜0.01).CONCLUSION In the rat models of T2DM,YQJDTL can reduce their levels of blood glucose and lipids by improving the renal indices levels and the renal microvascular endothelial functions to alleviate renal fibrosis and microangiopathy as well,and the mechanism may be associated with the down-regulated expressions of TGF-β/SMAD and VEGF pathway-related proteins.

Aim To design and synthesize a series of glycyrrhetinic acid derivatives by using glycyrrhetinic acid as the parent nucleus,screen their antitumor activ-ities,and investigate the in vitro and in vivo antitumor effects and mechanisms of the most active compound.Methods MTT assay was used to screen for the com-pound with the most potent antitumor activity.MTT as-say,wound healing assay,colony formation assay and Transwell migration assay were used to evaluate the effects of the compound on tumor cell viability and mi-gration.Flow cytometry was employed to assess the im-pact of the compound on tumor cell cycle progression and apoptosis.Western blot was conducted to verify the effects on the expression of pro-apoptotic proteins Bax,caspase-3 and cleaved caspase-3.A mouse model of hepatocellular carcinoma ascites tumor was estab-lished to examine the antitumor effects of the compound in vivo.Results Compound C22 was identified as having the most significant inhibitory effect on hepato-cellular carcinoma cells.C22 inhibited the viability and migration of hepatocellular carcinoma cells in a time and concentration-dependent manner.C22 upreg-ulated the expression of pro-apoptotic proteins Bax,caspase-3 and cleaved caspase-3 in hepatocellular car-cinoma cells,induced apoptosis,and arrested the cell cycle in the G0/G1 and S phases.C22 significantly re-duced the growth of mouse hepatocellular carcinoma as-cites tumors and prolonged survival.Conclusion Glycyrrhetinic acid derivative C22 significantly inhibits the viability and migration of hepatocellular carcinoma cells in vitro and in vivo,and induces cell cycle arrest and apoptosis.

Objective To establish the fingerprint of Xuemai Shutong granules by UPLC-ELSD and determine the contents of 9 components in the preparation simultaneously.Methods The UPLC-ELSD was used to establish the fingerprint of Xuemai Shutong granules,and determine the content of its 9 components.The similarity evaluation system,systematic,cluster analysis,principal component analysis and orthogonal partial least squares discriminant analysis were used to evaluate the quality of different batches of preparation.Results The similarity degrees of UPLC-ELSD fingerprints of 11 batches of Xuemai Shutong granules were from 0.929 to 0.978,17 common peaks were calibrated,of which 11 peaks were identified:peak 3(notoginsenoside R1),peak 4[ginsenoside Rg,(Re)],peak 5(notoginsenoside R2),peak 6(ginsenoside Rb,),peak 9(astragaloside Ⅳ),peak 10(ginsenoside Rk3),peak 11(ginsenoside Rh4),peak 12[20(S)-ginsenoside Rg3],peak 13[20(R)-ginsenoside Rg3],peak 14(ginsenoside Rk1),peak 15(ginsenoside Rg5).The stoichiometric analysis divided 11 batches of samples into 2 classes,and the 2 principal components in PCA analysis reflected the information of 17common peaks,10 peaks which affected the quality difference are screened out.The linear relationship of the 9 components was good in their respective quality ranges in the content analysis(r＞0.999 2),the average recovery rate were between 95.02％-97.78％and the RSD were 0.69％-1.70％(n=6).The contents of notoginsenoside R1,notoginsenoside R2,ginsenoside Rb1,astragaloside Ⅳ,ginsenoside Rh4,20(S)-ginsenoside Rg3,20(R)-ginsenoside Rg3,ginsenoside Rk1 ginsenoside Rg5 in the 11 batches of Xuemai Shutong granules were 0.087 5-0.187 6,0.494 3-0.688 6,0.448 1-0.705 5,0.192 2-0.270 8,1.492 5-2.077 6,0.316 0-0.463 8,0.254 5-0.382 0,0.117 6-0.163 9,3.407 7-4.706 4 mg·g-1,respectively.Conclusions The established fingerprint and content determination method was accurate and reliable,which can improve the quality standard of Xuemai Shutong granules,and provide reference for its overall quality evaluation.

In order to establish a method for the detection of serum antibodies to donkey-derived e-quine coronavirus(ECoV),recombinant ECoV N protein was expressed in E.coli system,purified by nickel column affinity chromatography and identified by Western blot.After optimizing the re-action conditions,the indirect ELISA(iELISA)detection method was established using the puri-fied recombinant protein as coating antigen and used to detect 143 clinical serum samples.The re-sults showed that the recombinant N protein,which has good reaction activity with serum antibod-y,was successfully expressed.The optimum conditions of the established iELISA method were as follows:the amount of antigen coated was 0.2 μg/well and overnight at 4 ℃,10％skimmed milk powder solution was sealed at 37℃ for 1.5 h,the dilution concentration of serum was 1∶200,and the enzyme-labeled secondary antibody diluted at 1∶10 000.The sensitivity test results showed that the positive serum could be diluted to 1∶6 400.The specificity test results showed that all an-tibodies to several donkey pathogens were negative.The repetitive test results showed that the in-tra-and inter-batch coefficients of variation were 2.90％-6.12％and 2.29％-7.88％respectively.The positive rate of clinical donkey serum was 57.3％.The iELISA established in this study pro-vides a technical support for epidemiological investigation and antibody surveillance.

Aim To design and synthesize a series of glycyrrhetinic acid derivatives by using glycyrrhetinic acid as the parent nucleus,screen their antitumor activ-ities,and investigate the in vitro and in vivo antitumor effects and mechanisms of the most active compound.Methods MTT assay was used to screen for the com-pound with the most potent antitumor activity.MTT as-say,wound healing assay,colony formation assay and Transwell migration assay were used to evaluate the effects of the compound on tumor cell viability and mi-gration.Flow cytometry was employed to assess the im-pact of the compound on tumor cell cycle progression and apoptosis.Western blot was conducted to verify the effects on the expression of pro-apoptotic proteins Bax,caspase-3 and cleaved caspase-3.A mouse model of hepatocellular carcinoma ascites tumor was estab-lished to examine the antitumor effects of the compound in vivo.Results Compound C22 was identified as having the most significant inhibitory effect on hepato-cellular carcinoma cells.C22 inhibited the viability and migration of hepatocellular carcinoma cells in a time and concentration-dependent manner.C22 upreg-ulated the expression of pro-apoptotic proteins Bax,caspase-3 and cleaved caspase-3 in hepatocellular car-cinoma cells,induced apoptosis,and arrested the cell cycle in the G0/G1 and S phases.C22 significantly re-duced the growth of mouse hepatocellular carcinoma as-cites tumors and prolonged survival.Conclusion Glycyrrhetinic acid derivative C22 significantly inhibits the viability and migration of hepatocellular carcinoma cells in vitro and in vivo,and induces cell cycle arrest and apoptosis.

AIM To investigate the variations in mercury dissolution from HgS-containing traditional medicines in three kinds of simulated gastrointestinal dissolution media.METHODS 39 batches of 15 types of HgS-containing traditional medicines were collected,total mercury content and dissolved mercury concentrations in simulated gastric fluid,simulated intestinal fluid,and L-cysteine-containing simulated intestinal fluid were measured.The maximum daily intake of total mercury and soluble mercury was calculated based on the maximum daily clinical dosage.RESULTS Among the 15 types of medicines,the maximum daily intake of total mercury varied by 156 times,the daily intake of soluble mercury varied by 3 502 times in simulated gastric fluid,313 times in simulated intestinal fluid,and 10 663 times in L-cysteine-containing simulated intestinal fluid,approximately.CONCLUSION For the 15 types of HgS-containing traditional medicines,the daily maximum intake of soluble mercury showed greater variations than that of total mercury.Soluble mercury concentration is more closely correlated with intestinal absorption of mercury and thus represents a more rational quality control indicator for HgS-containing traditional medicines.

AIM To observe the effects of Yiqi Jiedu Tongluo Formula(YQJDTL)on renal microvascular endothelial function and prevention of renal injury in a rat model of type 2 diabetes mellitus(T2DM).METHODS The SD rats were randomly divided into a normal group and a model group.The model group was administered with high-fat diet combined with a single intraperitoneal injection of STZ to establish the T2DM model.The successfully modeled rats were randomly divided into the model group,the canagliflozin group(9 mg/kg),and the low-dose and high-dose YQJDTL groups(4.77,9.45 g/kg).The corresponding doses of the drug were administered by gavage for a total of 12 weeks,during which the rats underwent observation of their general condition and blood glucose changes.After the end of administration,the rats had their levels of renal index,24-hour UP,serum SCr,BUN,TC,TG,HDL-C,LDL-C,ET-1 and NOS measured;their changes in renal microvasculature and the degree of renal fibrosis observed using HE staining,Masson staining,PAS staining,and PASM staining;their ultrastructure of the glomeruli observed using transmission electron microscopy;their renal protein expressions of TGF-β,SMAD2,SMAD3,Col-1,VEGFA and PKC detected by immunohistochemical staining and Western blot;and their renal mRNA expressions of VEGFA,TGF-β,SMAD2 determined by RT-qPCR.RESULTS Compared with the model group,the high-dose YQJDTL group showed decreased levels of renal index,blood glucose,TG,TC,HDL,24 h UP,BUN,SCr and ET-1(P＜0.05,P＜0.01);increased LDL and NOS levels(P＜0.05,P＜0.01);reduced renal inflammatory infiltration and fibrosis degree,inhibited fusion of foot processes and thickening of basement membrane;decreased renal protein expressions of TGF-β,SMAD2,SMAD3,VEGFA,PKC and Col-1(P＜0.05,P＜0.01);and decreased mRNA expressions of VEGFA,TGF-β and SMAD2(P＜0.01).CONCLUSION In the rat models of T2DM,YQJDTL can reduce their levels of blood glucose and lipids by improving the renal indices levels and the renal microvascular endothelial functions to alleviate renal fibrosis and microangiopathy as well,and the mechanism may be associated with the down-regulated expressions of TGF-β/SMAD and VEGF pathway-related proteins.