Mechanism Of The Apoptosis Of Hepatic Stellate Cells Induced By Ca²⁺/CaMK Ⅱ Signaling Pathway Regulating ERS

Objectives Calmodulin kinase Ⅱ（Ca MK Ⅱ）inhibitor KN-62 and inositol 1,4,5-trisphosphate（IP3）agonist uridine triphosphate（UTP）were used to act on human hepatic stellate cells（HSC）,then the apoptosis of HSC was observed.The aim is to explore the mechanism of Ca²⁺/Ca MK Ⅱ on HSC apoptosis from the endoplasmic reticulum stress（ERS）,and provide a scientific basis for finding new targets for the treatment of liver fibrosis（HF）.Methods HSCs stored in liquid nitrogen tank,were divided into 5 groups: blank control group,Transforming growth factor beta-1（TGF-β₁）group,KN-62 group,UTP group,KN-62+UTP group.The cells in the blank control group was cultured with serum-free medium for 24 h,which in the TGF-β₁ group was added with 5 ng/m L TGF-β₁ for 24 h,in the other three groups were cultured with 5 ng/m L TGF-β₁ for 24 h,and the medium was discarded,the KN-62 group was replaced with 10 μmol/L KN-62 for 6 h,and the UTP group was replaced with 3.2 mmol/L UTP for 24 h,the KN-62+UTP group was treated with 10 μmol/L KN-62 for 6 h,discarded the medium in the bottle,and replaced with 3.2 mmol/L UTP for 24 h.After HSCs were treated,HSC morphology was observed by the inverted microscope,the cell proliferation was measured with CCK-8 method,cell cycle was measured with flow cytometry,Ca MK Ⅱ activity was examined by ELISA method,the apoptosis of HSC was examined by one-step TUNEL experiment and Annexin V-FITC & PI double staining method,intracellular Ca²⁺ concentration was examined by laser confocal microscopy,Ca MK Ⅱ m RNA level was measured by real-time quantitative PCR method,and the proteins of Ca MK Ⅱ,GRP78,caspase-12,Caspase-3,Bcl-2 and Bax were detected with Western Blot.Results 1 HSC morphological changes: the cells the blank control group were irregularly star-shaped and closely connected;In the TGF-β₁ group,the intercellular space was widened,showing long fusiform changes,and transformed into flat myofibroblast-like cells,some of them were still star-like.The cell density and volume of KN-62 group,UTP group and KN-62+UTP group became smaller,apoptotic cells appeared,and HSC apoptosis in KN-62+UTP group was severe.2 HSC proliferation: The relative growth rate（RGR）of HSC in each group was significantly different（P<0.05）.After adding TGF-β₁,its RGR value is higher than that of the blank control group.When compared with TGF-β₁ group,RGR in KN-62 group,UTP group and KN-62+UTP group were significantly reduced（P<0.05）,and RGR in KN-62+UTP group was the lowest（P<0.05）.3 Cycle distribution: There were significant differences in the distribution of cell cycles among different groups（P <0.05）.Compared with the blank control group,TGF-β₁ group decreased in G1 and G2 phases,and S phase increased（P<0.05）;Compared with TGF-β₁ group,S phase in KN-62 group continued to increase（P<0.05）,but after UTP was given,G1 phase increased and S phase decreased（P<0.05）.4 Changes in Ca MK Ⅱ activity: There were significant differences in Ca MK Ⅱ activity among 5 groups（P<0.05）.Ca MK Ⅱ activity in TGF-β₁ groupwas significantly higher than that of the blank control group（P<0.05）;Compared with TGF-β₁ group,Ca MK Ⅱ activity in the KN-62 group decreased significantly（P<0.05）,while in the UTP group increased significantly（P<0.05）,and in the KN-62+UTP group was higher than that in KN-62 group and lower than in UTP group（P<0.05）.5 TUNEL results showed there were significant differences in the apoptosis rate among different groups（P<0.05）.The blank control group（19.00±4.52）% had no significant difference from the TGF-β₁ group（17.90±4.45）%（P> 0.05）.Compared with the TGF-β₁ group,the apoptosis rates in the KN-62 group,UTP group and KN-62+UTP group [（35.42±3.78）%,（40.07±2.88）%,（68.27±2.66）%] all increased（P<0.05）;the apoptosis rate in the KN-62+UTP group was significantly higher than that in the KN-62 group and UTP group（P<0.05）.6 Annexin VFITC&PI double staining showed that the difference in apoptosis rate between cell groups was statistically significant（P<0.05）,the blank control group（3.47±0.40）% was not significantly different from the TGF-β₁ group（2.52±0.48）%（P>0.05）.The apoptosis rates in KN-62 group,UTP group and KN-62+UTP group [（14.20±1.31）%,（13.77±1.57）%,（32.40±2.42）%] were higher than that in TGF-β₁ group and which of KN-62+UTP group was significantly higher than that of KN-62 group and UTP group（P<0.05）.7 Intracellular Ca²⁺ in HSC cells: Ca²⁺ concentrations in each cell group were significantly differents（P<0.05）.There was no significant difference in Ca²⁺ concentration between blank control group and TGF-β₁ group（P>0.05）;Ca²⁺ concentrations in KN-62 group,UTP group and KN-62+UTP group were significantly higher than that in TGF-β₁ group,and which in KN-62+UTP group was higher than that of KN-62 group and UTP group（P<0.05）.8 Ca MK Ⅱ m RNA and protein expression: the expression levels among different groups were significantly differents（P<0.05）.Compared with the blank control group,the m RNA and protein levels of TGF-β₁ group increased（P<0.05）;Compared with TGF-β₁ group,the m RNA and protein levels of the KN-62 group decreased（P<0.05）,the results of the UTP group increased significantly（P<0.05）,the KN-62+UTP group showed no significant difference（P>0.05）;The Ca MK Ⅱ level in the KN-62+UTP group was between the KN-62 group and the UTP group,and there was a difference between the groups（P <0.05）.9 Western Blot: There was significant difference in protein expression among different groups（P<0.05）,while there was no significant difference between the blank control group and TGF-β₁ group（P>0.05）The protein expression（except Bcl-2）and Bax/Bcl-2 ratio in TGF-β₁ group were lower than the other three groups.The levels of GRP78,caspase-12,caspase-3 and Bax / Bcl-2 in the KN-62+UTP group were significantly higher than those in the KN-62 group and UTP group（P<0.05）.The levels of GRP78,caspase-12,caspase-3 and Bax / Bcl-2 in the KN-62+UTP group were significantly higher than those in the KN-62 group and UTP group（P<0.05）.Conclusions 1 Inhibition of Ca MK Ⅱ can make the imbalance of Ca²⁺ homeostasis in HSC.2 Induced Ca²⁺ homeostasis imbalance can inhibit HSC proliferation and promote its apoptosis.3 Ca²⁺/Ca MK Ⅱ signaling pathway may regulate ERS to induce the apoptosis of HSC.Figure13;Table14;Reference 149...