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Aim To investigate the effects of DNMT3A regulating Drp1 mediated mitochondrial fission on the proliferation and migration of active hepatic stellate cells. Methods HSC-T6 cells were activated by 5 μg·L-1 TGF-β1 for 24 h, and DNMT3A lentivirus infection model was established to silence DNMT3A. The experiment was divided into control group, TGF-β1 group, TGF-β1+LV5-NC group and TGF-β1+ LV5-DNMT3A group. The effects of DNMT3A on related mRNA and protein expression were detected by RT-qPCR and Western blot. The cell proliferation was detected by CCK-8. The effect of DNMT3A on the migration ability of HSCs cells was observed by Wound healing assay and Transwell migration assay. Results Lentivirus infection successfully constructed a DNMT3A silencing model. Compared with the control group, the level of DNMT3A significantly increased, the mRNA and protein levels of the fibrosis markers collagen and α-SMA in the TGF-β1 group significantly increased, and the mRNA and protein levels of the mitochondrial fission marker Drp1 significantly increased; At the same time, the proliferation and migration ability of HSCs cells was significantly improved. Compared with the NC group, the DNMT3A level of the DNMT3A silenced group was significantly reduced, the expressions of collagen I, α-SMA and Drp1 were significantly inhibited, and the proliferation and migration capabilities of HSCs were also significantly inhibited. Conclusions Silencing DNMT3A inhibits the level of Drp1 and inhibits the proliferation and migration of HSCs at the same time. It is suggested suggest that DNMT3A-mediated low level DNA methylation modification may inhibit the occurrence of mitochondrial fission by inhibiting the level of Drp1, thereby inhibiting the activation of HSCs and affecting the occurrence and development of liver fibrosis. ,,,,.
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Objective:To investigate the effect of endothelin-1 (ET-1) on the expression of phosphorylated myosin light chain Ⅱ(p-MLCⅡ)and myosin light chain Ⅱ(MLCⅡ)protein in rat hepatic stellate cells HSC-T6 and explore the intervention effect of Danggui Shaoyao San(DSS)drug-containing serum. Method:After HSC-T6 cells were seeded, DMEM and blank rat serum with final concentrations of 2.5%, 5%, 10%, 15% and 20% were added to each well. The viability of HSC-T6 cells was determined by methyl thiazolyl tetrazolium(MTT) assay to screen the suitable serum concentration range. The cells were divided into blank serum control group (5%, 10%, 15%) and DSS drug-containing serum group (5%, 10%, 15%). ELISA was used to detect the content of ET-1 in cell culture supernatant under basic state. The cells were divided into blank serum control group (10%), DSS drug-containing serum low (5%), medium (10%) and high dose (15%) groups. Real-time fluorescent quantitative polymerase chain reaction (Real-time PCR) was used to detect the level of ET-1 mRNA in cell culture supernatant under basic state. The cells were divided into blank serum control group (10%), model group (10%), DSS drug-containing serum low (5%), medium (10%), high dose (15%) groups and Y-27632 inhibitor group (100 μmol·L-1). Except the blank serum control group, the other groups all received 10 nmol·L-1 ET-1 to induce HSC-T6 cells. Western blot was used to detect the expression of p-MLCⅡ and MLCⅡ in HSC-T6 cells induced by ET-1. Result:Serum concentrations of 5%, 10% and 15% were used as drug-containing serum concentrations. As compared with the blank serum control group, the DSS drug-containing serum group significantly reduced the relative content of ET-1 and ET-1 mRNA in the basic state (PPPPPConclusion:DSS drug-containing serum may down-regulate the expression of p-MLCⅡ and MLCⅡ by down-regulating the content of ET-1 and inhibiting the autocrine of ET-1.
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Aim: To investigate the effect of salvianolic acid B (Sal B) on liver fibrotic cells in vivo and in vitro from die perspective of apoptosis, as well as the effect on cleaved caspase-9. Methods: Diethylnitrosamine (DEN) was used to induce liver fibrosis in mice for 12 weeks. The padiological changes were detected by HE staining, and the fibrotic lesion area was determined. The cell apoptosis in the fibrotic area was observed by Hoechst 33258 fluorescence staining. The expression of cleaved caspase-9 in fibrotic tissues was detected by Western blot. The apoptotic rate of each group was detected by double standard method AnnexinV-FITC/PI, and the expression of apoptotic protein cleaved caspase-9 in HSC-T6 was detected by Western blot. Results: HE staining suggested that 12 weeks were the period of liver fibrosis in mice. No pseudoplobular structure was formed in group with low and high dose of Sal B, and the degree of fibrosis was lower than that in model group. In the fibrotic lesion area, the fluorescence staining of Hoechst 33258 showed that apoptotic cells significantly increased in group with low Sal B and high dose compared with model group. The results of the AnnexinV-FITC/PI method showed that TGF-β1 inhibited the apoptosis of HSC-T6, and Sal B promoted the apoptosis of HSC-T6 after TGF-β1 intervention and showed a concentration dependence (P <0. 01). Western blot results showed that in fibrotic liver tissues, Sal B increased the expression of cleaved caspase-9 in HSC-T6 cells compared with model group. Compared with TGF-β1 group, Sal B increased cleaved caspase-9 protein expression (P < 0. 01). Conclusions: Sal B can significantly promote apoptosis of liver fibrotic cells in vitro and in vivo, and its pro-apoptosis mechanism may be related to the up-regulation of cleaved caspase-9.
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OBJECTIVE： To optimize the proteolytic enzymes for enzymolysis technology of degreasing ointment from Periplaneta americana， and to improve the extraction rate and activity of anti-liver fibrosis active part from P. americana. METHODS： Using degreasing ointment of P. americana as control， ninhydrin method and folin-ciocalteu method were used to investigate the hydrolysis degree of trypsin （TR）， pepsin （PE）， alkaline protease （AL）， papain （PA） and neutral protease （NE） to the degreasing ointment. Macroporous resin isolation and purification method was used to investigate the yield of elution part from hydrolyzate， with 50％， 60％， 70％， 95％ ethanol as eluting solvents. Inhibition test in vitro of rat hepatic stellate cells HSC-T6 was performed， and anti-liver fibrosis activity of elution part from hydrolyzate was investigated. RESULTS： The hydrolysis degree of PA and NE were 14.15％ and 15.70％， showing strong enzymatic hydrolysis ability. The yield of 95％ ethanol elution part from PA， NE and AL hydrolyzate were （0.73±0.04）％，（0.65±0.01）％ and（0.64±0.05）％， improving 30.36％， 16.07％， 14.29％ compared with degreasing ointment without enzyme. Results of inhibition test in vitro showed that inhibitory rate of 50％， 60％， 70％ ethanol elution parts isolated and purified from hydrolyzate had a low inhibition rate or a growth-promoting effect on HSC-T6 cells. Inhibition rates of 95％ ethanol elution parts to HSC-T6 cells were all more than 20％. IC50 of 95％ ethanol elution part isolated and purified from PA and NE hydrolyzate for 24-72 h were 94.5-112.3 and 117.1-120.0 μg/mL， which were lower than that （116.1-123.0 μg/mL） of degreasing ointment without enzyme. CONCLUSIONS： PA is the best hydrolyzate for enzymolysis technology of active parts against liver fibrosis in degreasing ointment from P. americana， followed by NE and AL； PE and TR， which have poor effect， are not suitable for the enzymatic hydrolysis technology.
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Objective: To establish the composition-activity relationship (CAR) model based on study of chemical composition and relating proliferation inhibitory rate of Corydalis soxicola and recognize the anti-hepatic fibrosis active compounds of C. soxicola. Methods: Nine orthogonal C. soxicola extract were analyzed by HPLC, and 21 characteristic peaks were profiled. Anti-hepatic fibrosis activity was investigated by MTT assays on HSC-T6, and the potential active components were identified by scores plot and variable importance in projection (VIP) values by means of orthogonal partial least squares (OPLS) analysis, and the activities of identified components were verified by MTT and flow cytometry. LDH kit was used to detect the effect of dehydrocavidine, palmatine, and berberine on LDH activity. Results: The results showed that six peaks including peaks 13, 14, 16, 18, 19, and 20 were significantly related to anti-hepatic fibrosis activity among nine orthogonal extract from C. soxicola. Peaks 18, 19, and 20 were characterized as dehydrocavidine, palmatine, and berberine, respectively. MTT assay showed that dehydrocavidine, palmatine, and berberine with various concentration significantly inhibited the proliferation of HSC-T6 cells. It was also found in flow cytometry that the apoptotic rates of dehydrocavidine, palmatine, and berberine (0.10 mg/mL) on HSC-T6 were 42.12%, 42.22%, and 36.73%, respectively, which were obviously higher than that of control (1.69%, P < 0.01). No obvious cytotoxic effect was found when the final concentration of dehydrocavidine, palmatine, and berberine were less than 0.15, 0.10, and 0.10 mg/mL, respectively. Conclusion: In this study, it was for the first time found that dehydrocavidine, palmatine, and berberine in C. soxicola extract can effectively inhibit the proliferation and induce apoptosis of HSC-T6 based on composition-activity relationship, and there was no obvious cytotoxic effect of the effective concentration, which showed that they may be the active components with potential anti-hepatic fibrosis effect and have a certain security in the application in C. soxicola. At the same time, it also suggests that the research ideas based on CAR can provide an effective method for the identification of active components in natural plants.
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Objective To observe the possible mechanism of total lignans from Herpetospermum pedunculosum seeds (TLHPS) in fighting against hepatic fibrosis of hepatic stellate cell-T6 (HSC-T6) by studying the effects of TLHPS on inhibiting proliferation and inducing apoptosis of HSC-T6. Methods The cells were divided into totally six groups such as control group, TLHPS groups (10, 20, 30, 40 μg/mL), and colchicine positive control group (0.1 μg/mL). HSC-T6 cells were cultivated for 24, 48, and 72 h by six groups of culture medium containing different drugs. The proliferation inhibitory rate of HSC-T6 cells was detected by CCK8 method at 24, 48, and 72 h respectively. The apoptotic rate and cell cycle were detected by flow cytometry. Protein expression levels of B cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax) and transcription factors-κB (NF-κB) were detected by Western blotting. Results Compared with control group, 24, 48, and 72 h proliferation inhibitory rate of HSC-T6 was obviously elevated in TLHPS 10, 20, 30, 40 μg/mL groups (P 0.05), cell numbers in S phase obviously decreased (P < 0.01). Compared with control group, Bal-2 protein expression obviously decreased in TLHPS 10, 20, 30, 40 μg/mL groups (P < 0.05, 0.01), Bax protein expression obviously increased in TLHPS 40 μg/mL group (P < 0.01) and NF-κB protein expression obviously decreased in TLHPS 20, 30, 40 μg/mL groups (P < 0.01). Conclusion The mechanism of total lignans from Tibetan medicine Herpetospermum fighting against hepatic fibrosis might be associated with inhibiting proliferation and inducing apoptosis of HSC-T6. The inhibition of hepatic stellate cells proliferation and induction of its apoptosis may be related to lowering the expression of Bcl-2 and NF-κB.
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Aim To observe the effects of cytokine signaling inhibition protein-3(SOCS3) on the liver fibrosis progression and reverse.Methods C57BL/6 mouse model was established by subcutaneous injection of carbon tetrachloride(CCl4).After a successful model of fibrosis, one-month normal diet was given to induce the reverse fibrosis model, while normal mice of the same gender and weight were as control group.Mice were sacrificed at 1, 2, 3, 4, 5, 6, 7, 8 weeks, respectively, then the liver tissue was harvested for the observation of its injury by hematoxylin and eosin(HE) staining.Then Masson staining was applied for the detection of changes in collagen, and the immunohistochemistry(IHC) for the observation of type Ⅰ Collagen(Colla-1), alpha smooth muscle actin(α-SMA), transforming growth factor beta 1(TGF-β1) and SOCS3 protein expression.In vitro formation of fibrosis was induced by TGF-β1 stimulating HSC-T6 cell lines, which was then reversed by MDI medium, with co-incubation of HSC-T6 cells with plasmid in the process of the reverse.Western blot was employed to detect SOCS3, Colla-1, α-SMA, TGF-β1 expression.Results The expression of SOCS3 and TGF-β1 increased in mouse model of fibrosis with the worsening fibrosis process and decreased in the reverse process.Over-expression SOCS3 in the reverse process reduced the development of liver fibrosis;meanwhile, the expression of TGF-β1 was also reduced accordingly.Conclusion SOCS3 may influence the development of the liver fibrosis and its reverse via regulating the expression of TGF-β1.
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OBJECTIVE: To observe the effect of ligustrazine hydrochloride on 3HSC-T6 apoptosis and to explore the possible mechanism. METHODS: LDH kit was used to detect the effect of ligustrazine hydrochloride on LDH activity. 3H-TdR incorporation assay was used to detect the effect of drug on HSC-T6 proliferation. Apoptosis was measured by using flow cytometry (FCM) analysis with Annexin-V/PI dual staining for apoptotic cell ratio. Western blotting and Real-time PCR were used to detect the protein and mRNA expression of Bcl-2, Bax and caspase-3. RESULTS: No obvious cytotoxic effect was found when the final concentration of ligustrazine hydrochloride was less than 80 μmol · L-1. 3H-TdR incorporation assay showed that ligustrazine hydrochloride inhibited HSC-T6 proliferation at the concentrations of 50 and 70 μmol · L-1. FCM detection showed that ligustrazine hydrochloride dose-dependently stimulated apoptosis of HSC-T6. Western blotting results showed that ligustrazine hydrochloride promoted HSC-T6 apoptosis by inhibiting Bcl-2, and increasing Bax and caspase-3 protein expression. Further use of real-time PCR showed that ligustrazine hydrochloride inhibited Bcl-2 mRNA expression, and promoted the mRNA expression of Bax and caspase-3. CONCLUSION: Ligustrazine hydrochloride has the function of promoting the apoptosis of HSC-T6 by inhibiting Bcl-2, and promoting Bax and caspase-3 at both protein and mRNA levels.
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Objective: To investigate the induction effect of oxymatrine on the apoptosis process in rat hepatic stellate cell line HSC-T6 and to define its impact on telomerase activity and mRNA expression of subunit telomerase reverse transcriptase (rTERT). Methods: HSC-T6 cells were cultivated with different concentration of oxymatrine for different time periods. Effect of oxymatrine on the growth inhibition of HSC-T6 cells was analyzed by MTT assay. Apoptosis of HSC-T6 cells was detected by flow cytometry. Telomerase activity was determined by TRAP-PAGE-silver staining and the expression of rTERT mRNA was examined by RT-PCR assay. Results: Oxymatrine significantly suppressed the growth of HSC-T6 cells and induced apoptosis, and also reduced the activity of telomerase and inhibited the rTERT-mRNA expression in HSC-T6 cells. Conclusion: The function that oxymatrine inhibits the proliferation of HSC-T6 cells may be associated with its action on the cellular telomerase and telomerase rTERT-mRNA activity.
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Objective: To study the optimum particle size of Cordyceps sinensis for liver fibrosis in combination with in vitro dissolution experiment from serum pharmacology. Methods: To prepare the powder samples with different grinding degrees, Cordyceps sinensis was crushed through 100-, 150-, 200-, and 300-mesh sieves. The in vitro dissolution of adenosine was measured at different time points. Meanwhile, the powder sample was ig administered to rats, and pharmacodynamic approach was adopted to study the inhibition of medicated serum on HSC-T6 proliferation. Results: The accumulative in vitro dissolution of C. sinensis by 200-300 meshes was higher than that of other meshes. Medicated serum could significantly inhibit HSC-T6 cell proliferation. The AUC of HSC-T6 inhibition kinetics of medicated serum crushed to 200-300 meshes was significantly higher than that in other groups. Conclusion: The in vitro dissolution and pharmacodynamic method could be used for the study on different particle sizes of C. sinensis for anti-hepatic fibrosis, and 200-300 meshes are the optimal particle size.
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Objective:To investigate the effect of sulfasalazine on proliferation and apoptosis of HSC-T6 and L02 in culture. Methods:HSC-T6 and L02 were incubated with different concentration of sulfasalazine.Cell proliferation was assessed by CCK-8 assay,cell apoptosis was analyzed by annexin Ⅴ FITC/PI flow cytometry;and apoptotic morphology was examined by vital staining of acridine orange/ethidium bromide(AO/EB). Results:Compared with the control group,the proliferation of HSC-T6 was significantly inhibited by sulfasalazine in a time and dose-dependant manner,when the concentration of sulfasalazine in the medium reached a certain level range. The HSC-T6 apoptotic induced by sulfasalazine was confirmed by AO/EB and annexin Ⅴ FITC/PI.compared with the control group,the proliferation and apoptosis rate of L02 treated by group had no statistical significance,and AO/EB staining showed no apoptosis. Conclusion:Sulfasazinea at the same concentration can specifically inhibit the proliferation of HSC and induce HSC apoptosis.
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Objective:To investigate the effects of salidroside on the proliferation,activation,Collagen Ⅰ secretion as well as expression of ROCK-Ⅰand TIMP-1 in HSC-T6 cell,and to elucidate the molecular mechanism of salidroside against hepatic fibrosis with Y-27632 as the positive control. Methods: HSC-T6 cell line was employed as the model for following study. MTT colorimetric assay was used to detect the proliferation of HSC-T6 cell. ELISA was employed to value the content of collagen Ⅰ in supernatant of HSC-T6 cells treated with salidroside and Y-27632. The effects of salidroside and Y-27632 on expressions of smooth muscle actin (?-SMA)、ROCK-Ⅰand TIMP-1 expression were analyzed by Real-time PCR and Western blot. Results(:1)Both Salidroside(3、6、12 mmol/L)and Y-27632(5、 10、20 ?mol/L)had dose-dependent inhibitory effects on ?-SMA expression which represents the activated state of HSC-T6(P
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Objective To determine whether sulfasalazine stimulates hepatic stellate cell (HSC-T6) apoptosis and its possible mechanism. Methods CCK-8 assay, acridine orange/ethidium bromide(AO/EB) and Annexin Ⅴ FITC/PI were used to determine cell growth and cell apoptosis. The expression of NF-?B P65, phospho-IKK and phospho-I?B was detected by Western blotting. The nuclear translocation of HSC-T6 P65 was observed with laser confocal microscopy. Results Sulfasalazine displayed a strong growth inhibition and promoting apoptosis effect on HSC-T6 cells in a dose and time-dependent manner. Sulfasalazine, but not 5-aminosalicylic acid or sulfapyridine, inhibited the activation of NF-?B by down-regulating the expressions of P-IKK, P-I?B and the nuclear translocation of P65. Conclusion Sulfasalazine can inhibit NF-?B activity and promote apoptosis in HSC-T6 cells, where the Rel/NF-?B/I?B/IKK pathway plays an important role in HSC survival.