受体相互作用丝氨酸-苏氨酸激酶3在胆汁淤积性肝损伤中的作用及机制

Effects and mechanisms of receptor-interacting serine-threonine kinase 3 in the formation of cholestatic hepatic injury

目的：探讨受体相互作用丝氨酸-苏氨酸激酶3（RIP3）在胆汁淤积性肝损伤中的作用及机制。 方法：采用实验研究方法。（1）肝星状细胞株HSC-T6细胞处理和活性检测：采用RIP3-siRNA和NC-siRNA转染HSC-T6细胞。采用CCK8法检测未转染、转染RIP3-siRNA、转染NC-siRNA的HSC-T6细胞存活率。采用100 μmol/L甘氨鹅脱氧胆酸（GCDCA）试剂处理HSC-T6细胞后0、2、4、8、12 h取材细胞并保存，检测12 h细胞存活率。采用100 μmol/L GCDCA试剂处理RIP3敲低HSC-T6细胞后培养12 h，检测细胞存活率。（2）胆管结扎小鼠模型建立：采用随机数字表法将40只小鼠分为8组，每组5只。Sham组：仅游离胆总管，不结扎，于术后第7天取下腔静脉血和肝组织；BDL-1 d组、BDL-3 d组、BDL-5 d组、BDL-7 d组、BDL-14 d组、BDL-21 d组、BDL-28 d组：分别于行胆总管结扎术后第1、3、5、7、14、21、28天取材。（3）RT-PCR检测细胞和肝组织RIP3、α-平滑肌肌动蛋白（α-SMA）、TNF-α mRNA相对表达量。（4）Western blot检测细胞和肝组织RIP3、α-SMA、TNF-α蛋白相对表达量。正态分布的计量资料以±s表示，不同时间点数据检验采用方差分析，多组间比较采用方差分析，两组间比较采用t检验。 结果：（1）不同HSC-T6细胞活性和RIP3、α-SMA、TNF-α mRNA及蛋白表达情况： CCK8检测结果显示：采用100 μmol/L GCDCA试剂处理HSC-T6细胞和RIP3敲低HSC-T6细胞后12 h细胞存活率分别为61.3%±0.3%和83.2%±0.4%，与未采用100 μmol/L GCDCA试剂处理HSC-T6细胞和RIP3敲低HSC-T6细胞培养12 h存活率（98.4%±0.7%和97.4%±0.7%）4者比较，差异有统计学意义（F=115.200，P〈0.05）。其中后两者比较，差异无统计学意义（t=1.283，P〉0.05）；未采用100 μmol/L GCDCA试剂处理HSC-T6细胞存活率分别与采用100 μmol/L GCDCA试剂处理HSC-T6细胞和RIP3敲低HSC-T6细胞存活率比较，差异均有统计学意义（t=17.910，6.604，P〈0.05）；采用100 μmol/L GCDCA试剂处理HSC-T6细胞和RIP3敲低HSC-T6细胞存活率比较，差异有统计学意义（t=7.186，P〈0.05）。RT-PCR检测结果显示：未转染、转染RIP3-siRNA、转染NC-siRNA的HSC-T6细胞RIP3 mRNA相对表达量分别为0.012 1±0.001 3、0.011 2±0.003 1、0.002 8±0.000 5，3者比较，差异有统计学意义（F=20.410，P〈0.05）。其中未转染的HSC-T6细胞RIP3 mRNA相对表达量与转染NC-siRNA的HSC-T6细胞比较，差异无统计学意义（t=0.483，P〉0.05）；转染RIP3-siRNA的HSC-T6细胞RIP3 mRNA相对表达量分别与未转染、转染NC-siRNA的HSC-T6细胞比较，差异均有统计学意义（t=11.760，4.586，P〈0.05）。采用GCDCA试剂处理HSC-T6细胞后0、2、4、8、12 h RIP3 mRNA相对表达量分别为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，α-SMA mRNA相对表达量分别为0.571±0.012、0.611±0.024、0.691±0.021、0.711±0.021、0.752±0.031，TNF-α mRNA相对表达量分别为0.873±0.022、0.912±0.024、1.015±0.031、1.210±0.042、1.471±0.041，3者相对表达量均随时间延长升高，差异均有统计学意义（F=70.720，30.050，166.700，P〈0.05）。未采用GCDCA试剂处理HSC-T6细胞RIP3 mRNA相对表达量为0.012 1±0.001 3，采用GCDCA试剂处理HSC-T6细胞12 h后RIP3 mRNA相对表达量为0.029 8±0.002 3，两者比较，差异有统计学意义（t=13.970，P〈0.05）。Western blot检测结果显示：未转染、转染RIP3-siRNA、转染NC-siRNA的HSC-T6细胞RIP3蛋白相对表达量分别为0.054±0.012、0.013±0.008、0.052±0.021，3者比较，差异有统计学意义（F=7.410，P〈0.05）。其中未转染的HSC-T6细胞RIP3 蛋白相对表达量与转染NC-siRNA的HSC-T6细胞比较，差异无统计学意义（t=0.143，P〉0.05）；转染RIP3-siRNA的HSC-T6细胞RIP3蛋白相对表达量分别与未转染、转染NC-siRNA的HSC-T6细胞比较，差异均有统计学意义（t=4.924，3.006，P〈0.05）。采用GCDCA试剂处理HSC-T6细胞后0、2、4、8、12 h RIP3蛋白相对表达量分别为0.045±0.024、0.047±0.034、0.062±0.025、0.121±0.015、0.154±0.034，α-SMA蛋白相对表达量分别为0.064±0.031、0.072±0.017、0.097±0.035、0.078±0.031、0.254±0.051，TNF-α蛋白相对表达量分别为0.078±0.025、0.094±0.037、0.129±0.041、0.198±0.011、0.324±0.061，3者相对表达量均随时间延长升高，差异均有统计学意义（F=9.658，15.810，20.090，P〈0.05）。未采用GCDCA试剂处理HSC-T6细胞RIP3蛋白相对表达量为0.045±0.024，采用GCDCA试剂处理HSC-T6细胞12 h后RIP3蛋白相对表达量为0.154±0.034，两者比较，差异有统计学意义（t=4.536，P〈0.05）。（2）各组小鼠肝组织中RIP3、α-SMA、TNF-α mRNA及蛋白表达情况：RT-PCR检测结果显示：Sham组、BDL-1 d组、BDL-3 d组、BDL-5 d组、BDL-7 d组、BDL-14 d组、BDL-21 d组、BDL-28 d组小鼠肝组织中RIP3 mRNA相对表达量为0.047 3±0.003 1、0.041 2±0.007 8～0.339 7±0.017 1，α-SMA mRNA相对表达量为2.948±0.612、2.654±1.032～8.387±0.910，TNF-α mRNA相对表达量为0.563±0.078、0.610±0.113～1.078±0.289，各组小鼠上述指标比较，差异均有统计学意义（F=25.180，27.820，7.425，P〈0.05）。Western blot检测结果显示：Sham组、BDL-1 d组、BDL-3 d组、BDL-5 d组、BDL-7 d组、BDL-14 d组、BDL-21 d组、BDL-28 d组小鼠肝组织中RIP3蛋白相对表达量为0.245±0.011、0.228±0.023～1.018±0.052，α-SMA蛋白相对表达量为0.424±0.057、0.392±0.041～0.985±0.081，TNF-α蛋白相对表达量为0.551±0.052、0.588±0.087～0.962±0.074，各组小鼠上述指标比较，差异均有统计学意义（F=19.160,94.410,22.750，P〈0.05）。 结论：胆汁淤积通过诱导TNF-α通路活化，并上调RIP3，促进肝损伤和纤维化。

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-α mRNA 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 ±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-α 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）. Conclusions：Cholestasis promotes hepatic injury and fibrosis by inducing TNF-α pathway activation and up-regulation RIP3.