The Role And Relative Mechanism Of Calcium-sensing Receptor In Ischemia-reperfusion Injury Of Rat Liver

Calcium-sensing receptor（CaSR）is a G-protein coupled receptor. The mostimportant physiologic function of the CaSR is to regulate the systemic calciumhomeostasis. CaSR is also involved in cell proliferation, differentiation, apoptosis,gene expression and hormonal secretion. Liver ischemia-reperfusion （I/R） injury is acommon pathological change accompanying liver surgery, and also be a major focusin the hepatology. However, the functional involvement of CaSR in liver I/R injuryremains less well characterized.Objective To observe the expression of CaSR during liver I/R and analyze therelationship between CaSR expression and liver injury; to investigate the role ofCaSR in BRL cells apoptotsis induced by anoxia-reoxygenation （A/R）; to exploreintracellular signaling pathways of CaSR’s downstream.Methods （1） The experimental liver I/R model was established by the45minligating and1h、2h、4h、6h hour reperfusing the left branch of portal vein,hepaticartery and bile duct in rats. Expression of CaSR mRNA and protein was evaluated byReverse Transcription-Polymerase Chain Reaction （RT-PCR） and Western blottingrespectively. The levels of plasma ALT, AST, SOD, MDA and NO were measured.The change of ultrastructure was observed using electron microscopy.（2） Theexpression and distribution of CaSR in BRL cells were detected by RT-PCR, Westernblotting and immunofluorescence; and the intracellular calcium concentration ([Ca²⁺]_i)was measured using Laser Confocal Scanning Microscopy.（3） BRL cells wereincubated in ischemia-mimetic solution for4h, and re-incubated in normal culturemedium for10h to establish a model of A/R. Then, with GdCl₃（a specific activator ofCaSR） and NPS-2390（an antagonist of CaSR） for the intervention factors, we assayed the viability and apoptosis of BRL cells by MTT, Hoechest33342staining and flowcytometry; observed morphological alterations with electron microscope; measured[Ca²⁺]_i by Laser Confocal Scanning Microscope; explored intracellular signalingpathways of CaSR’s downstream by detecting the P38and ERK pathway. The proteinexpression of caspase-3, Bax/Bcl-2and Cyt-c were analyzed using Western blotting.Results （1） In rat liver I/R model in vivo, SOD activity decreased gradually withthe reperfusion time, ALT, AST, NO activity and MDA content increased, while liverultrastructure injury was more serious. The expression of CaSR increased moresignificantly after reperfusion of1h and2h, and decreased after4h and6h.（2） BothCaSR mRNA and protein were expressed in BRL cells and mainly distributed in cellmembrane and cytoplasm. Increased extracellular calcium or GdCl₃could increase[Ca²⁺]_iand CaSR expression. Moreover, this increase of [Ca²⁺]_icould be inhibited oreven abolished by U73122（a specific inhibitor of PLC）,2-APB （an inhibitor of IP3receptor） and thapsigargin （an inhibitor of endoplasmic reticulum calcium pump）.（3）A/R increased the expression of CaSR and the BRL cells apoptosis. GdCl₃furtherenhanced CaSR expression and apoptotic BRL cells during A/R. Activation of CaSRdown-regulated Bcl-2expression, up-regulated Bax, P53and caspase-3expressionsand cytochrome c release, meanwhile, stimulated P38, and ERK1/2phosphorylation.Conclusions （1） The expression of CaSR was increased in the early stage ofreperfusion and then decreased, the injury of liver was severer with the reperfusiontime prolonging.（2） CaSR is functionally expressed in BRL cells, and activation ofCaSR involves in increased intracellular calcium through Gq-PLC-IP3pathway.（3）CaSR could induce A/R injury and apoptosis of BRL cells through inducing calciumoverload and activating mitochondrial apoptosis pathway. Meanwhile, MAPKpathway was activated, and resulted in promoting or inhibiting apoptosis, in which theformer was dominant. Regulation of CaSR activity may serve as a novelpharmacological target to prevent and treat liver disease.