Role Of Calpain-2 In Endoplasmic Reticulum Stress-Induced HSC Apoptosis In Hepatic Fibrosis

Objectives Calcium ionophore A23187 and calpain inhibitor（N-acetyl-Leu-Leu-Norleucinal,ALLN）were applied to HSC stimulated by transforming growth factor beta 1（TGF-β₁）.The changes of death and ERS aim to explore the role of Calpain-2 in the apoptosis of hepatic stellate cell at the cellular level,providing theoretical basis for further elucidating the mechanism of ERS-mediated HSC apoptosis in hepatic fibrosis in this study.Methods HSCs were removed from liquid nitrogen and routinely resuscitated.After synchronization,they were divided into 5 groups:control group（HSC）,TGF-β₁ group,ALLN group,ALLN+A23187 group and A23187 group.Except for the control group,the other four groups were stimulated with TGF-β₁ for 24 h,and the final concentrations were25μmol/l ALLN and 2μmol/l A23187 respectively.ALLN+A23187 group was treated with ALLN for 1 h and then added A23187,to perform various indexes after 24 h.The ultrastructural changes of HSC were used to observe by transmission electron microscopy;the proliferation of HSCs was used to detect by MTT assay;the changes of cell cycle was used to detect by flow cytometry;the changes of calpain-2 activity in HSCs of each group by ELASA kit;Apoptosis of HSCs was detected by V-FITC&PI double staining and Hoechst33342 staining;Ca²⁺concentration was observed by laser confocal microscopy;GRP78,calpain-2 and caspase-12 expression was detected by real-time quantitative PCR;I-type collagen was detected by immunohistochemical staining;Calpain-2,Caspase-3,Caspase-12,Caspase-9,GRP78 protein expression were used to detect by Western Blot.Results 1 Morphology:Control group and TGF-β₁ group showed chromatin distribution,ALLN cells showed cytoplasm contraction,ALLN+A23187 group showed chromatin lightly concentrated and edge set,A23187 group of collagen fibers in the presence of a small amount,marked edge set of chromatin under electron microscopy;2 Cell proliferation:The comparations of proliferation rates（PR）of HSCs among each groups had statistical significance（P<0.05）,the proliferation rate in TGF-β₁ group was higher than control group（P<0.05）.The PR value in remaining three groups were lower than that in TGF-β1 group（P<0.05）.The PR value of ALLN+A23187 group was significantly higher than that of A23187 group,which was lower than that of ALLN group（P<0.05）;3 Cell cycle:The comparations of proliferation rates of HSCs among each groups had statistical significance（P<0.05）.Compared with control group,the proportion of G₁ phase cells in the TGF-β₁ group decreased,the proportion of S+G₂ cells increased（P<0.05）.The proportion of G1 phase cells in ALLN+A23187 group was lower in A23187 group and the proportion of S+G2 cells was increased（P<0.05）.Compared with ALLN group,the proportion of G1 phase cells increased and the proportion of S+G2 cells decreased（P<0.05）;4 Calpain-2 activity:The changes of calpain-2 activity were statistically different among the groups（P<0.05）.There was no significant difference in the activity of Calpain-2（6.280±2.466）in the TGF-β₁ group compared with control group（6.362±1.160）.The activity of Calpain-2 in the remaining three groups were higher than that in the TGF-β₁group;the activity of Calpain-2 in HSCs in the ALLN+A23187 group（122.90±2.180）was significantly lower than that of Calpain-2 in HSCs of A23187 group（221.723±11.383）,higher than the activity of Calpain-2 in HSCs of ALLN group（21.763±2.466）,which had statistically significant different（P<0.05）;5 Ca²⁺:The comparations of Ca²⁺among groups had statistical significance（P<0.05）,It was no significant difference that in Ca²⁺concentration between TGF-β₁ group（136.117±13.448）and control group（133.623±12.507）（P>0.05）.The concentrations of Ca²⁺in ALLN+A23187 group and A23187 group were increased significantly than that in TGF-β₁ group（P<0.05）,The comparations of Ca²⁺（610.373±26.143）in ALLN+A23187 group was lower than A23187group（987.227±60.341）,higher than ALLN group（P<0.05）,which had statistically significant different（P<0.05）;6 Hoechst33342 staining:There were significant differences in the nucleus of HSC cells in each group.Control group and TGF-β₁ group showed normal blue color.More nuclei of pyknosis or granular fluorescence were observed after the administration of A23187.In the ALLN+A23187 group,less pyknosis or granular fluorescence was seen with respect to the A23187 group,and more pyknotic fluorescence than in the ALLN group;7 Apoptosis:There were statistically significant difference in apoptotic rate among groups（P<0.05）.The apoptotic rate in TGF-β₁ group（1.70±0.14）%was significantly lower than that in control group（3.32±0.29）%（P<0.05）,The apoptotic rate of the remaining three groups were significantly higher than that in TGF-β₁group（P<0.05）.The apoptotic rate in ALLN+A23187 group（8.45±0.35）%was lower than A23187 group（14.51±0.37）%,higher than ALLN+A23187 group（5.67±0.52）%（P<0.05）;8 Changes in mRNA of ERS:GRP78,Calpain-2 and caspase-12 mRNA groups were statistically differenent（P<0.05）.It was no significant difference that in three mRNA expression of TGF-β₁ group and control group（P>0.05）.The expression of mRNA in the other three groups was higher than that in TGF-β₁ group.The mRNA expression of signal molecules in ALLN+A23187 group was lower than that in A23187 group,higher than ALLN group（P<0.05）;9ICollagen protein expression:The expression ofIcollagen in TGF-β₁ group was higher than that in control group（P<0.05）.The expression ofIcollagen in the other three groups was lower than that in TGF-β₁ group.The expression ofIcollagen in ALLN+A23187 group was lower than that in A23187 group,higher than than inALLN group（P<0.05）;10 Changes in ERS Proteins:There were significant differences in the expression of GRP78,caspase-9,caspase-12,caspase-3,and calpain-2 proteins in each group（P<0.05）.The ERS protein levels in TGF-β₁ group were higher than those in the control group（P<0.05）,the expression of ERS protein in the other 3 groups was higher than that in TGF-β₁ group.The protein expression of ERS in ALLN+A23187 group was lower than that in A23187 group and higher than ALLN group（P<0.05）.Conclusions 1 The imbalance of Ca²⁺in HSC induced the occyrrence of ERS and promoted the apoptosis of HSC.2 Blocking the calpain-2 pathway in the ERS pathway could reduce Ca²⁺concentration and ERS-related index changes,inhibit cell apoptosis and accelerate the progression of fibrosis.