ABSTRACT
Objective To investigate the effects and mechanisms of receptor-interacting serine-threonine kinase 3 (RIP3) in the formation of cholestatic hepatic injury.Methods The experimental study was conducted.(1) Processing and viability of hepatic stellate cell line HSC-T6:HSC-T6 cells were transfected by RIP3-siRNA and NC-siRNA,respectively.The viabilities of un-transfected,RIP3-siRNA-transfected and NC-siRNA-transfected HSC-T6 cells were respectively measured by cell-counting kit-8 (CCK-8).HSC-T6 cells were treated by 100 μmol/L Glycochenodeoxycholic acid (GCDCA) at 0,2,4,8 and 12 hours,and then were extracted and stored,12-hour cell viability was measured by CCK-8.RIP3 that was treated by 100 μmol/L GCDCA knocked down HSC-T6 cells to establishment RIP3 knockdown HSC-T6 cells (RIP3-KD cells).RIP3-KD cells were cultured for 12 hours,and cell viability was measured.(2) Mice model of bile duct ligation (BDL):40 adult mice were randomly divided into 8 groups,5 mice in each group.Sham group:bravery manager was only separated,without ligation,and bloods of inferior vena cava and liver tissues were extracted at 7 days postoperatively.The BDL-1,-3,-5,-7,-14,-21 and-28 d groups:bloods of inferior vena cava and liver tissues were extracted at 1,3,5,7,14,21 and 28 days postoperatively,respectively.(3) The relative expressions of RIP3,α-SMA and TNF-αmRNA in the cells and liver tissues were detected by quantitative real-time polymerase chain reaction (RT-PCR).(4) The relative expressions of RIP3,α-SMA and TNF-α proteins were detected by Western blot.Measurement data with normal distribution were represented as-x±s.The ANOVA was used for data analysis in different time gradient.Comparisons among groups were analyzed using the ANOVA.Pairwise comparison was done by the t test.Results (1) The HSC-T6 cells viability and expressions of RIP3,α-SMA,TNF-α mRNA and proteins:results of CCK8 test showed that 12-hour viabilities of GCDCA-treated HSC-T6 cells,GCDCA-treated RIP3-KD cells,HSC-T6 cells and RIP3-KD cells were 61.3％ ±0.3％ and 83.2％ ±0.4％ and 98.4％ ±0.7％ and 97.4％ ±0.7％ respectively,showing statistically significant differences in the viabilities among them (F =115.200,P＜ 0.05),and showing no statistically significant difference in the viabilities between HSC-T6 cells and RIP3-KD cells (t =1.283,P＞ 0.05).There were statistically significant differences in the viabilities between HSC-T6 cells and GCDCA-treated HSC-T6 cells or GCDCA-treated RIP3-KD cells (t =17.910,6.604,P＜ 0.05) and between GCDCA-treated HSC-T6 cells and GCDCA-treated RIP3-KD cells (t=7.186,P＜0.05).Results of RT-PCR test showed relative expressions of RIP3 mRNA in un-transfected,RIP3-siRNA-transfected and NC-siRNA-transfected HSC-T6 cells were respectively 0.012 1±0.001 3,0.011 2±0.003 1 and 0.002 8±0.000 5,with a statistically significant difference (F =20.410,P ＜ 0.05).There was no statistically significant difference in relative expressions of RIP3 mRNA between un-transfected and NC-siRNA-transfected HSC-T6 cells (t =0.483,P ＞0.05).The relative expression of RIP3 mRNA in RIP3-siRNA-transfected HSC-T6 cells was significant different from that in un-transfected and NC-siRNA-transfected HSC-T6 cells (t =11.760,4.586,P＜0.05).The relative expressions of RIP3 mRNA,α-SMA mRNA and TNF-α mRNA in GCDCA-treated HSC-T6 cells at 0,2,4,8 and 12 hours were 0.012 1±0.001 3,0.011 2±0.003 1,0.021 2±0.002 2,0.027 8±0.002 1,0.029 8±0.002 3 and 0.571±0.012,0.611±0.024,0.691±0.021,0.711±0.021,0.752±0.031 and 0.873±0.022,0.912± 0.024,1.015±0.031,1.210±0.042,1.471±0.041,respectively,showing an increased trend over time and statistically significant differences (F=70.720,30.050,166.700,P＜0.05).The relative expressions of RIP3 mRNA in HSC-T6 cells and GCDCA-treated HSC-T6 cells were 0.012 1±0.001 3 and 0.029 8±0.002 3,with a statistically significant difference (t=13.970,P＜0.05).Results of Western blot showed that relative expressions of RIP3 protein in un-transfected,RIP3-siRNA-transfected and NC-siRNA-transfected HSC-T6 cells were respectively 0.054 ± 0.012,0.013 ± 0.008 and 0.052± 0.021,with a statistically significant difference (F =7.410,P＜0.05).There was no statistically significant difference in relative expressions of RIP3 protein between un-transfected and NC-siRNA-transfected HSC-T6 cells (t =0.143,P ＞ 0.05),and statistically significant differences were found in relative expressions of RIP3 protein between RIP3-siRNA-transfected HSC-T6 cells and un-transfected or NC-siRNA-transfected HSC-T6 cells (t =4.924,3.006,P＜0.05).The relative expressions of RIP3,α-SMA and TNF-oα proteins in GCDCA-treated HSC-T6 cells at 0,2,4,8 and 12 hours were 0.045±0.024,0.047±0.034,0.062±0.025,0.121±0.015,0.154±0.034 and 0.064±0.031,0.072±0.017,0.097±0.035,0.078±0.031,0.254±0.051 and 0.078±0.025,0.094±0.037,0.129±0.041,0.198±0.011,0.324±0.061,respectively,showing an increased trend over time and statistically significant differences (F =9.658,15.810,20.090,P＜0.05).The relative expressions of RIP3 protein in HSC-T6 cells and GCDCA-treated HSC-T6 cells at 12 hours were 0.045±0.024 and 0.154±0.034,with a statistically significant difference (t =4.536,P＜0.05).(2) Expressions of RIP3,α-SMA and TNF-α mRNA in hepatic tissues of mice in each group:the results of RT-PCR showed that relative expressions of RIP3 mRNA,α-SMA mRNA and TNF-α mRNA in the Sham,BDL-1 d,BDL-3 d,BDL-5 d,BDL-7 d,BDL-14 d,BDL-21 d,BDL-28 d groups were 0.047 3±0.003 1,0.041 2±0.007 8-0.339 7±0.017 1 and 2.948±0.612,2.654± 1.032-8.387±0.910 and 0.563±0.078,0.610±0.113-1.078± 0.289,respectively,with statistically significant differences (F =25.180,27.820,7.425,P＜0.05).The results of western blot showed that relative expressions of RIP3,α-SMA and TNF-α proteins in Sham,BDL-1 d,BDL-3 d,BDL-5 d,BDL-7 d,BDL-14 d,BDL-21 d,BDL-28 d groups were 0.245±0.011,0.228±0.023-1.018±0.052 and 0.424±0.057,0.392±0.041-0.985±0.081 and 0.551 ±0.052,0.588±0.087-0.962±0.074,respectively,with statistically significant differences (F=19.160,94.410,22.750,P＜0.05).Conclusion Cholestasis promotes hepatic injury and fibrosis by inducing TNF-α pathway activation and upregulation RIP3.