Aim To compare the cytotoxicity of geni-poside (GS)and its metabolite genipin (GP)on hu-man hepatocelluar HepG2 cells and explore the sub-stance and mechanism of hepatotoxicity induced by Fructus Gardeniae.Methods The cytotoxic effect of GS and GP was analyzed by MTT method;the antioxi-dant enzyme activities of manganese superoxide dis-mutase (Mn-SOD),catalase (CAT)and levels of glu-tathione (GSH)were detected by respective kits;the change of intracellular reactive oxygen species (ROS ) was measured by 2′,7′-dichlorofluorescin diacetate (DCFH-DA)staining method;multiparameter cytotox-icity analysis (cell loss,nuclear size and morphologi-cal changes,DNA content,cell membrane permeabili-ty,mitochondrial membrane potential changes,cyto-chrome C release ) were measured simultaneously by high content screening (HCS)assays.Results No cytotoxicity was found in GS (20~1 000 μmol·L-1 ) groups (P>0.05 ),but GP was found to exert obvi-ous cytotoxic effect,and 50μmol·L-1 GP could obvi-ously inhibit HepG2 cell proliferation (P<0.05 ),and the IC50 value was (450.00 ±26.15)μmol·L-1;GS showed no obvious effects on Mn-SOD, CAT, GSH ,ROS (P >0. 0 5 ),GP could significantly decrease the activity of Mn-SOD,CAT and the level of GSH,and obviously increase the content of ROS (P<0.05 or P <0.01 );treatment with 50,500,1 000μmol·L-1 GP resulted in loss of mitochondrial mem-brane potential,cytochrome C release and increased cell permeability (P<0.05 or P<0.01),500,1 000μmol· L-1 GP could also show abvious nuclear con-densation,increased total nuclear intensity and cell loss (P<0.0 1 ).Opposed with GP,GS had no signif-icant effect on the cytotoxic parameters (P>0.05 ). Conclusion GP is the direct substance of hepatotox-icity induced by Fructus Gardeniae,and the mecha-nism might be associated with oxidative stress,mito-chondria injury and apoptosis.

Objective To investigate the effect and machanism of Scutellaria baicalensis formula granule (SBFG)on proliferation and apoptosis of HepG2 human hepatoma cells.Methods HepG2 cells were di-vided into normal control group and six SBFG groups at concentration of 0.05,0.1,0.2,0.4,0.8 and 1.6 g/L.After HepG2 cells were treated by SBFG for 24 hours,the proliferation was analyzed by using MTT method,apoptosis was observed by Hoechst 33342 staining method,and the changes of cell count, nuclear size,nuclear DNA content,permeability,mitochondria membrane potential(MMP)and cyto-chrome C were tested by high content screening assays(HCS).Results The inhibitory effect of SBFG on proliferation of HepG2 cells was significant (P <0.01)with concentration-dependence,and IC50 of (0.38 ±0.016)g/L.By Hoechst 33342 staining,the morphological changes were observed including hyperchromatic nuclei,concentrated karyoplasms and karyorrhexis.Furthermore,HCS indicated that 0.1 g/L SBFG decreased MMP and increased cytochrome C release significantly (P <0.01);0.4 g/L SBFG further decreased MMP and increased cytochrome C release,and increased the membrane permeability significantly,pyknosis and nuclear fluorescence intensity (P <0.01 );in addition to the effects above, 1.6 g/L SBFG also reduced the number of cells (P <0.01).Conclusion SBFG can significantly in-hibit the proliferation of HepG2 cells,and induce cell apoptosis.The mechanisms may be related to de-creasing the MMP,promoting the release of cytochrome C and increasing membrane permeability.

BACKGROUND:Piezo1,a mechanosensitive protein,is tightly connected to osteogenic differentiation,and it has been demonstrated that TAZ has a role in regulating osteogenic differentiation.It is unclear whether TAZ participates in the regulation of osteogenic differentiation of human bone marrow mesenchymal stem cells by Piezo1,so it is crucial to investigate its unique mechanism to prevent osteonecrosis of the femoral head. OBJECTIVE:To elucidate what function Piezo1 plays in osteogenic differentiation and TAZ expression in human bone marrow mesenchymal stem cells. METHODS:The siRNA targeting Piezo1 was constructed and transfected into 293T cells.The silencing efficiency was detected by RT-qPCR.The selected Piezo1-Home-2337 was packaged according to the silencing efficiency,and its optimal multiplicity of infection value was assayed by immunofluorescence staining.The packaged Piezo1 silencing recombinant lentivirus was transfected into human bone marrow mesenchymal stem cells,and its silencing effect was detected by RT-qPCR and western blot assay.Alizarin red staining,alkaline phosphatase activity analysis,immunofluorescence staining,RT-qPCR and western blot assay were utilized to analyze the effect of silencing Piezo1 on the osteogenic differentiation of human bone marrow mesenchymal stem cells. RESULTS AND CONCLUSION:(1)The mRNA and protein levels of Piezo1 in human bone marrow mesenchymal stem cells transfected by si-Piezo1 were decreased significantly,with a statistically significant difference compared with normal and negative control groups.(2)The alkaline phosphatase activity in the si-Piezo1 group was much lower and the calcium deposition in the si-Piezo1 group was significantly reduced compared with the negative control group.(3)The mRNA levels of osteogenesis-related genes including Runt-related transcription factor 2(Runx2),osteopontin(OPN),distal-less homeobox 5(DLX5),osteocalcin,β-catenin and Tafazzin(TAZ)in the si-Piezo1 group were significantly decreased compared with the negative control group.Afterward,the expression levels of TAZ and β-catenin protein in the si-Piezo1 group were down-regulated significantly compared with the negative control group,whereas the expression levels of p-TAZ and p-β-catenin protein in the si-Piezo1 group had the opposite condition.(4)The results of immunofluorescence staining showed that the expression of TAZ and β-catenin in human bone marrow mesenchymal stem cells in the si-Piezo1 group was less compared with the negative control group.(5)These findings indicate that Piezo1 can promote the osteogenic differentiation of human bone marrow mesenchymal stem cells.The osteogenic ability of human bone marrow mesenchymal stem cells is significantly reduced after silencing Piezo1,and the expression of TAZ is also reduced.

Objective To explore the anti-inflammatory effect of Qingre Jiedu(QRJD)formula on mice with gout and its effect on gut microbiota.Methods Forty C57BL/6 mice weighing 20～22 g were divided into control(CON),model(MOD),allopurinol(ALLO),and QRJD formula(QRJD)groups,and i.g.10 g/0.1 mL carboxymethyl cellulose was administered to the CON every morning from 1 to 35 days.A hyperuricemia mouse model was prepared by intragastric injection of a potassium oxalic acid(500 mg/kg)and yeast extract(10 g/kg)suspension.On day 29,50 μL sterile carboxymethyl cellulose was injected into the right ankle of mice in the CON group under isoflurane-induced anesthesia,and a gouty arthritis model was prepared by injecting the same volume of sodium urate(50 mg/mL)into the right ankle of mice in the other groups.Each group was treated with corresponding drugs every day.On day 35,samples were collected from mice that had been fasted for 6 hours without water.Blood indexes,such as uric acid,creatinine,and urea nitrogen,were assessed.Hematoxylin-eosin and saffranine O-fast green staining was performed on ankle joints.Anti-inflammatory indexes of interleukin-10(IL-10)and transforming growth factor-β1(TGF-β1)were detected by ankle joints immunohistochemical assay.The cecum contents of mice were collected,and changes in gut microbiota were analyzed by high-throughput sequencing of 16S rDNA.Results(1)After 7 days of treatment,compared with the MOD group,QRJD formula effectively reduced the blood concentrations of uric acid(P＜0.001),creatinine(P＜0.01),and urea nitrogen(P＜0.05),and effectively protected renal functions.(2)Compared with the MOD group,HE staining showed that synovial hyperplasia and inflammatory cell infiltration were reduced in the QRJD formula group after treatment.The cartilage arrangement of the compound was more orderly than before,cartilage destruction was less than that in the MOD group,and no matrix loss was observed.(3)Immunohistochemical analysis of the ankle joint indicated that IL-10 and TGF-β1 were not significantly increased in CON and MOD groups.Compared with the MOD group,IL-10 and TGF-β1 expression in the QRJD formula group were increased(P＜0.05).(4)In terms of biodiversity,the number of MOD-specific OTUs increased by 75 compared to the CON group,while the QRJD was able to reduce the number of MOD-specific OTUs to more closely resemble the CON group;no significant difference was found in α-diversity among the four groups(P＜0.05),whereas β-diversity was more similar to the CON group(P=0.001).(5)Compared with the CON group,the MOD group exhibited increased abundances(P＜0.05)of Ruminococcaceae spp.,Dubosiella sp.,Tyzzerella sp.,Ileibacterium sp.,and Bacteroidales spp..In contrast to the MOD group,the QRJD formula group showed elevated abundances(P＜0.05)of Lactobacillus sp.,Ligilactobacillus sp.,and Bacteroides sp..Furthermore,an interaction network of gut microbiota indicated mutual interactions among these microorganisms.(6)In the correlation analysis between gut microbiota and renal functions as well as anti-inflammatory factors,the relative abundances of Dubosiella sp.,Tyzzerella sp.,and Bacteroidales spp.were significantly positively correlated to SUA and SCR(P＜0.05).However,Lactobacillus sp.,Ligilactobacillus sp.,and Mitochondria spp.exhibited a positive correlation to anti-inflammatory factors IL-10 and TGF-β1 with a more significant association observed for TGF-β1(P＜0.05).(7)COG function prediction suggested that the functions of the QRJD formula group were concentrated on inorganic ion transport and metabolism,and carbohydrate transport and metabolism.Conclusions QRJD effectively modulates immune inflammation and gut microbiota dysbiosis,thereby treating gout.Its mechanism of gout prevention and treatment may involve regulation of gut microbiota diversity and abundance,as well as the control of the abundance of differential bacterial species,such as Ruminococcaceae spp.,Dubosiella sp.,and Lactobacillus sp.,to achieve gout therapy.