| Part I Role of TLR4/PI3K/AKT/ GSK3β signaling pathway inapoptosis of BRL-3A cellsObjective This study is to investigate whether toll like receptor 4(TLR4)- mediated PI3K/AKT/GSK3β signaling pathway exists in BRL-3A cells and explore the effect and mechanism of this pathway in apoptosis in BRL-3A cells, thus providing a new target for prevention and treatment of acute liver failure(ALF).Methods The apoptosis model of BRL-3A cells was established by lipopolysaccharide(LPS). TLR4 inhibitor(CLI-095), AKT inhibitor(LY294002) and GSK3β inhibitor(Li Cl) pretreated BRL-3A cells respectively before LPS stimulation in order to weaken or strengthen the effect of TLR4/PI3K/AKT/GSK3β signaling pathway. Cell viability was detected by CCK-8 method, and apoptotic rate was detected by flow cytometry. Hoechst 33342 staining method was used to observe the morphology of apoptotic cells after LPS stimulation and TLR4 inhibitor blocking. Western-blot method was used to detect the expression of AKT, P-AKTSer473, GSK3β, P-GSK3βSer9, Bax, Bcl-2 and active-caspase-3 protein. Real-time quantitative PCR(RT-q PCR) was used to detect m RNA of Bax, Bcl-2 and caspase-3 in rat hepatocytes. GSK3β nuclear translocation was observed by immunofluorescence assay.Results(1) The results of CCK-8 assay showed that, cell viability at different time(1, 3, 6, 12, 24 h) after LPS(10μg/ml) stimulation in BRL-3A cells was significantly lower than that in control group(P<0.05), cell viability at 24 h after LPS stimulation in BRL-3A cells was 58% of the control group(P<0.05).(2) Hoechst 33342 staining showed that, with prolongation of stimulation time, the apoptotic cells increased, and necrotic cells appeared, nucleus fragmentation and nuclear pyknosis were visible at 24 h. The apoptotic cells in CLI-095 pretreatment group were significantly decreased, and no apoptotic body was found.(3) The results of flow cytometry showed that the apoptotic rate of BRL-3A cells gradually increased after LPS(10μg/ml) stimulation at different times(1, 3, 6, 12, 24 h)(P<0.01), the apoptotic rate of BRL-3A cells was 68.30% at 24 h. Compared with the LPS group, CLI-095 preconditioning can attenuate apoptotic rate of BRL-3A cells stimulated by LPS(P<0.05), the apoptotic rate in LY294002 + LPS group increased(P<0.05); the apoptotic rate in Li Cl + LPS group decreased(P<0.05).(4) Western-blot determined that AKT, P-AKTSer473, GSK3β, P-GSK3βSer9 protein express in control group. The expression of P-AKTSer473 and P-GSK3βSer9(P<0.05) decreased in LPS group; compared with the LPS group, the expression of P-AKTSer473 and P-GSK3βSer9 up-regulated in CLI-095 + LPS group; however, the expression of P-AKTSer473 and P-GSK3βSer9 decreased in LY294002 + LPS group(P<0.05); P-GSK3βSer9 expression upregulated in Li Cl + LPS group(P<0.05).(5) Immunofluorescence showed that GSK3β mainly expressed in cytoplasm of BRL-3A cells in control group; GSK3β translocated to the nucleus in LPS group; nuclear translocation of GSK3β weakened in CLI-095 + LPS group and Li Cl + LPS group, while enhanced in LY294002 + LPS group.(6) The results of RT-PCR showed that the levels of Bax/Bcl-2 and caspase-3 m RNA up-regulate in LPS group(P<0.05). Compared with LPS group, the levels of Bax/Bcl-2 and caspase-3 m RNA decreased in CLI-095 + LPS group and Li Cl + LPS group(P<0.05), while increased in LY294002 + LPS group(P<0.05).(7) The results of Western-blot showed that the expression of Bax/Bcl-2 and active-caspase-3 upregulate in LPS group compared with the control group(P<0.05). Compared with LPS group, the expression of Bax/Bcl-2 and active-caspase-3 decreased in CLI-095 and Li Cl + LPS group(P<0.05), while increased in LY294002 + LPS group(P<0.05).Conclusion(1) TLR4-mediated PI3K/AKT/GSK3β signaling pathway exists in BRL-3A cells.(2) After TLR4 activation in BRL-3A cells, the expression of P-AKTSer473 and P-GSK3βSer9 down-regulated, while the expression of Bax/Bcl-2 and active-caspase-3 increased, so as to increase the apoptotic rate. TLR4 mediated PI3K/AKT/GSK3β signaling pathway was involved in apoptosis of BRL-3A cellsPart II Role of TLR4/P38/JNK signaling pathway inapoptosis of BRL-3A cellsObjective This study is to investigate whether TLR4/P38/JNK signaling pathway exists in BRL-3A cells and explore the effect and mechanism of this pathway in apoptosis in BRL-3A cells, thus providing a new target for prevention and treatment of acute liver failure.Methods The apoptosis model of BRL-3A cells was established by LPS. TLR4 inhibitor(CLI-095), P38 inhibitor(SB203580), JNK inhibitor(SP600125), ERK inhibitor(FR180204) were used to pretreat BRL-3A cells respectively before LPS stimulation, in order to affect TLR4/P38/JNK signaling pathway. Apoptotic rate was detected by flow cytometry. Western-blot method was used to detect the expression of JNK, P38, P-JNK, P-P38, Bax, Bcl-2, and active-caspase-3 protein. RT-q PCR was used to detect m RNA of Bax, Bcl-2 and caspase-3 in rat hepatocytes.Results(1) The results of flow cytometry showed that the apoptotic rate was higher in LPS group than that in the control group(P<0.05); in CLI-095 + LPS group, SB203580 + LPS group, and SP600125 + LPS group, apoptotic rates were lower than that in LPS group(P<0.05); there was no significant difference between LPS group and FR180204 + LPS group.(2) The results of Western-blot showed that the expression of P-P38 and P-JNK increased in LPS group than that in control group(P<0.05); in CLI-095 + LPS group, SB203580 + LPS group, SP600125 + LPS group, the expression of P-P38 and P-JNK decreased compared with LPS group(P<0.05), there was no significant difference between LPS group and FR180204 + LPS group.(3) RT-PCR showed that the levels of Bax/Bcl-2 and caspase-3 m RNA increased in LPS group(P<0.05); in CLI-095 + LPS group, SB203580 + LPS group and SP600125 + LPS group, the levels of Bax/Bcl-2 and caspase-3 m RNA decreased in contrast to LPS group(P<0.05); there was no significant difference between LPS group and FR180204 + LPS group.(4) Western-blot showed that, compared with LPS group, the expression of Bax/Bcl-2 and active-caspase-3 decreased in CLI-095 + LPS, SB203580 + LPS group and SP600125 + LPS group. There was no significant change in FR180204 + LPS group.Conclusion(1) TLR4- mediated P38/JNK signaling pathway exists in BRL-3A cells.(2) After TLR4 activation in BRL-3A cells, the expression of P-P38, P-JNK up-regulated, the expression of Bax/Bcl-2 and active-caspase-3 increased, thus promoting apoptosis of BRL-3A cells. TLR4/P38/JNK signaling pathway participated in apoptosis of BRL-3A cells.Part III Inhibitory effect and mechanism of oxymatrineon apoptosis of BRL-3A cellsObjective The study is to investigate the effects of oxymatrine(OMT) on LPS-induced apoptosis in BRL-3A cells and to explore the possible mechanisms, thus providing more experimental evidence of OMT.Methods The apoptosis model of BRL-3A cells was established by LPS. CCK-8 method was used to determine the effect of OMT on cell viability in BRL-3A cells. Biological chemical method were used to determine the release rate of lactate dehydrogenase(LDH) induced by LPS with different doses of OMT. The following experiments were carried out according to the LDH release rate, choosing OMT concentration as 0.75, 1.5 and 3.0 g/L. Then the experiment was divided into five groups: normal control group, LPS group, OMT-low dose + LPS group, OMT-middle dose + LPS group, OMT-high dose + LPS group. Hochest 33342 staining was used to observe the morphology of cell apoptosis. Flow cytometry was adopted to detect the cell apoptotic rate. RT-q PCR method was used to detect the Bax, Bcl-2 and caspase-3 m RNA expression in BRL-3A cells. Western-blot was used to detect AKT, P-AKTSer473, GSK3β, P-GSK3βSer9, P38 and P-P38, JNK, P-JNK, Bax, Bcl-2 and active-caspase-3. Immunofluorescence was used to observe GSK3β nuclear translocation.Results(1) Different doses of OMT(0.375 ~ 6 g/L) and BRL-3A cells were co-cultured for 24 h, the BRL-3A cells grew well.(2) After LPS stimulation, the LDH release rate of BRL-3A cells was increased(P<0.01), but the OMT pretreatment could significantly reduce the release of LDH(P<0.05 or P<0.01).(3) The results of CCK-8 method showed that the cell viability was significantly reduced(P<0.05) in LPS group, and the pretreatment of OMT could weaken the effect of LPS(P<0.05 or P<0.01).(4) The results of flow cytometry showed that the apoptotic rate of BRL-3A cells in LPS group was significantly higher than that in control group(P<0.05), and OMT pretreatment could significantly reduce the BRL-3A cell apoptotic rate induced by LPS stimulation(P<0.05).(5) With Hochest 33342 staining, the apoptotic cells in LPS group were significantly increased, which were significantly decreased after OMT pretreatment.(6) The results of Western-blot showed that expression of TLR4 in LPS group increased(P<0.05), the expression of P-AKTSer473 and P-GSK3βSer9 decreased(P<0.05), and the expression of P-P-38 and P-JNK increased(P<0.05). Compared with the LPS group, in OMT-M + LPS group and OMT-H + LPS group, TLR4 expression was down-regulated, the expression of P-AKTSer473 and P-GSK3βSer9 increased(P<0.05), the expression of P-P38 and P-JNK decreased(P < 0.05 or P<0.01).(7) Immunofluorescence showed that OMT pretreatment could significantly reduce the nuclear translocation of GSK3β induced by LPS stimulation.(8) The results of RT-q PCR indicate that the expression of Bax/Bcl-2 and caspase-3 m RNA in LPS group was significantly increased(P<0.05). Compared with the LPS group, OMT pretreatment could significantly reduce Bax/Bcl-2 and caspase-3 m RNA in BRL-3A cells(P<0.05 or P<0.01).(9) Western-blot showed that, compared with LPS group, OMT pretreatment could significantly reduce the expression of Bax/Bcl-2 and active-caspase-3 in BRL-3A cells.Conclusions(1) OMT pretreatment can significantly reduce the apoptosis of BRL-3A cells induced by LPS.(2) OMT can increase levels of P-AKTSer473 and P-GSK3βSer9, attenuate the LPS induced GSK3β nuclear translocation, reduce LPS activated P38 and JNK phosphorylation level, decrease the ratio of Bax/Bcl-2 and active-caspase-3 expression, thus inhibiting the apoptosis of BRL-3A cells.(3) OMT can inhibit apoptosis of BRL-3A cells through TLR4/PI3K/AKT/GSK3β and TLR4/P38/JNK signaling pathway.Part IV Inhibitory effect of oxymatrine on apoptosis ofhepatocytes in acute liver failureObjective To investigate the effects of oxymatrine(OMT) on hepatocyte apoptosis of LPS/D-Gal N induced acute liver failure in rats and search effective drugs for clinical prevention and treatment of acute iver failure through inhibiting hepatocyte apoptosis.Methods LPS/D-Gal N was adopted to establish the model of acute liver failure in rats. 100 rats were randomly assigned to five groups: normal control group, model group, OMT-low dose group, OMT-middle dose group, OMT-high dose group. The pathological changes of liver were observed under light microscope with HE staining. Cell apoptosis was observed by transmission electron microscope(TEM). Automatic biochemical analyzer was used to measure the serum alanine aminotransferase(ALT) and aspartate aminotransferase(AST). ELISA was used to determine the levels of tumor necrosis factor-α(TNF-α) and interleukin-1β(IL-1β) in the peripheral blood of rats. The apoptotic rate of rat hepatocytes was determined by flow cytometry. Western-blot was used to detect the expression of AKT, P-AKTSer473, GSK3βSer9, P-GSK3βSer9, P38, P-P38, JNK, P-JNK, Bax, Bcl-2, and active-caspase-3 in liver tissues. Immunohistochemistry S-P method was used to observe the expression of TLR4, Bax, Bcl-2 and active-caspase-3 in liver tissue.Results:(1) The mortality and gross observation of liver in each group: all rats in normal group and groups pretreated with OMT survived. The mortality rate in model group was 30%. Gross observation in model group: marked swelling of liver tissue, diffuse hemorrhage and a lot of ecchymosis on dark red liver surface. OMT preconditioning can reduce liver injury in different degrees.(2) HE staining showed obvious necrosis of liver cells, large patchy hemorrhage and necrosis, only a small amount of liver cell survived, no normal structure of hepatic lobules, fibrous mesh scaffolds collapsed, accompanied by periportal infiltration of a large number of inflammatory cells. TEM showed significant necrosis of liver cells, irregular shape of liver cells, visible pyknosis and apoptotic bodies, hepatocyte mitochondria damage, no obvious cell structure. OMT preconditioning reduced pathological changes of liver tissue in different degrees.(3) Biochemical analyzer showed that, compared with control group, the levels of AST and ALT in model group increased significantly(P<0.01), OMT pretreatment could significantly reduce levels of AST and ALT(P<0.05).(4) The results of ELISA showed that the expression of TNF-α and IL-1β in LPS/D-Gal N group increased significantly(P<0.01). Compared with the LPS/D-Gal N group, the expression of TNF-α and IL-1β pretreated with OMT was decreased(P<0.05 or P<0.01).(5) The results of flow cytometry showed that the apoptotic rate of hepatocytes in model group was significantly higher than that in control group(P<0.05), compared with the model group, OMT-middle dose and OMT-high dose pretreatment down-regulated apoptotic rate of hepatocytes(P<0.05).(6) Western-blot results showed that expression of TLR4 in model group increased(P<0.05), the expression of P-AKTSer473 and P-GSK3βser9 decreased(P<0.05), the expression of P-P38 and P-JNK increased(P<0.05). Compared with the model group, OMT-middle dose and OMT-high dose pretreatment down-regulated TLR4 expression(P<0.05), up-regulated the expression of P-AKTSer473 and P-GSK3βSer9(P<0.05), decreased the expression of P-P38 and P-JNK(P<0.05).(7) The expression of Bax/Bcl-2 ratio and active-caspase-3 protein in model group was significantly increased(P<0.05). Compared with the model group, OMT pretreatment could down-regulate the expression of Bax/Bcl-2 and active-caspase-3 protein(P<0.05).(8) Immunohistochemistry showed that the expression of TLR4, Bax and active-caspase-3 in model group increased, Bcl-2 expression decreased compared with normal control group(P<0.05). Compared with the model group, OMT pretreatment downregulated the expression of TLR4, Bax and active-caspase-3, while increased the expression of Bcl-2(P<0.05).Conclusions(1) OMT pretreatment protected liver cells by improving the liver pathological change and reducing serum aminotransferase in LPS/D-Gal N induced acute liver failure in rats.(2) OMT can reduce the expression of TLR4, up-regulate the expression of P-AKTSer473 and P-GSK3βSer9, down-regulate the expression of GSK3β, decrease the expression of P-P38 and P-JNK, reduce the levels of TNF-α and IL-1β, decrease the expression of Bax/Bcl-2 and active-caspase-3 in liver tissue of rats with acute liver failure, so as to inhibit the apoptosis of liver cells.(3) OMT inhibited the apoptosis of liver cells in rats with acute liver failure by TLR4/PI3K/AKT/GSK3β and TLR4/P38/JNK signaling pathway. |
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