Study On The Role Of Calreticulin In Hepatic Fibrosis Through Ca²⁺-mediated Endoplasmic Reticulum Stress

Objectives The changes of calreticulin（CALR）,fibrosis and endoplasmic reticulum stress（ERS）indexes in the progression of hepatitis B to cirrhosis were observed in population study and cytological study,and further explore the effect of CALR/Ca²⁺/ERS signaling pathway on human hepatic stellate cell（LX-2）apoptosis,so as to provide theoretical basis for cytological treatment of hepatic fibrosis（HF）.Methods 1 Population study:109 patients with hepatitis B and hepatitis B cirrhosis diagnosed in Tangshan infectious disease hospital from September to December 2020 were selected.General demographic data,clinical characteristics and laboratory indicators was collected.Fasting venous blood was collected in the morning on the first day of admission.The supernatant was collected and centrifuged.The m RNA expression of PERK,e IF2α,ATF4 and CHOP in serum was detected by real-time PCR.The protein expression of CALR in serum was detected by ELISA.The data was analyzed by SPSS20.0,and the results were expressed as（?）.One-way ANOVA was used to compare the mean of multiple groups,LSD-t test was used to compare the pairwise mean between groups,X²test was used to compare the categorical variables,and Pearson test was used to analyze the correlation between the measurement data,P<0.05 showed that the difference was statistically significant.2 Cytological study:LX-2 cells were stored in liquid nitrogen,resuscitated and passaged.The transfection efficiency of different concentrations of FAM-siRNA was observed by fluorescence microscope.The expression of CALR with different siRNA target sequences（CALR-siRNA 1,CALR-siRNA 2,CALR-siRNA 3）was detected by real-time PCR According to the appropriate concentration and target of the silencing effect of CALR-siRNA,the experiment was divided into five groups:（1）blank control group:put serum-free medium into LX-2 for 48 h;（2）Negative-siRNA group:Nc-siRNA was transfected into LX-2 for 48 h;（3）CALR-siRNA group:CALR-siRNA2 was transfected into cells for 48 h;（4）TGF-β1 group:5 ng/m L TGF-β1 cultured cells for 24 h;（5）CALR-siRNA+TGF-β1 group:CALR-siRNA2 was transfected into the cells for 24 h,and then 5 ng/m L TGF-β1 was added for 24 h.Cell morphology was observed under inverted microscope,cell proliferation was detected by CCK-8 method,apoptosis was detected by Annexin V-FITC/PI double staining and TUNEL method,intracellular Ca²⁺level was detected by laser confocal microscopy,m RNA expression levels of CALR,GRP78,caspase-12 and caspase-3 were determined by real-time PCR,and Western blot analysis was used to determine the expression levels of CALR,GRP78,caspase-12,caspase-3,Bax and Bcl-2.Results 1 Population study:in the serum of patients with mild,moderate and severe hepatitis B,ALT,AST expression,fibrosis index（CⅣ,HA,LN,PCⅢ）and HBV-DNA gradually increased（P<0.05）,and these indexes decreased in the stage of cirrhosis（P<0.05）.With hepatitis B is aggravated,PERK,e IF2α,ATF4,and CHOP expression in serum was gradually increased（P<0.05）,and the hepatitis B cherrifination group reached its highest.CALR expression in severe hepatitis B was significantly higher than that in other periods（P<0.05）.Related analysis results showed that ALT,AST and lg（HBV-DNA）,fibrosis index,CALR were significantly positively correlated（P<0.05）,CALR and lg（HBV-DNA）,fibrosis index showed a strong positive correlation（P<0.05）.2 Cytological study:the relative proliferation rates of blank control group,Negative-siRNA group,CALR-siRNA group,TGF-β1 group and CALR-siRNA+TGF-β1 group were 100%,98.56%,66.11%,115.95%and 83.25%respectively,and the differences among the groups were statistically significant（F=444.421,P<0.05）;the apoptosis rates of each group with Annexin V-FITC/PI double staining were（4.02±0.35）%,（4.55±0.47）%,（29.81±3.59）%,（1.91±0.10）%,and（18.57±2.06）%,（F=124.626,P<0.05）;with TUNEL assay were（12.33±2.75）%,（14.25±1.63）%,（53.74±3.82）%,（7.45±2.99）%and（28.05±2.64）%,（F=129.157,P<0.05）;Ca²⁺concentration in each group was（94.656?9.331）,（95.464?9.490）,（206.408?18.339）,（70.202?7.643）,（153.042?15.271）,（F=56.870,P<0.05）,respectively.In the CALR-siRNA group,CALR expression was significantly lower than that in the blank control and Negative-siRNA group（P<0.05）,and the expression of GRP78,Caspase-12,Caspase-3 and Bax were significantly increased（P<0.05）,and Bcl-2 protein expression was also significantly reduced（P<0.05）;compared with TGF-β1 group,the CALR-siRNA+TGF-β1 group had weakened cell proliferation,increased apoptotic rate,increased Ca²⁺concentration,and decreased CALR expression（P<0.05）,Bcl-2 protein expression decreased significantly（P<0.05）,and the expression of GRP78,Caspase-12,Caspase-3 and Bax all increased significantly（P<0.05）.Conclusions 1 The pathological process of hepatitis B involves ERS;2 CALR is a characteristic index in hepatitis B lesions;3 CALR-siRNA inhibits the activation of TGF-β1 on LX-2,4 Silence the expression of CALR,causes the imbalance of Ca²⁺homeostasis,induces ERS,and then promotes cell apoptosis and inhibits its proliferation.5The apoptosis of LX-2 induced by CALR/Ca²⁺/ERS signaling pathway is an important target of anti-HF.Figure13;Table15;Reference 159...