Ginsenoside Rg1 Ameliorates Chronic Intermittent Hypoxia-induced Vascular Endothelial Dysfunction Through The Calpain-1 Pathway

ObjectiveEstablishing an animal model of chronic intermittent hypoxia(CIH)simulate the pathophysiological process of patients with obstructive sleep apnea(OSA),to observe the vascular function under CIH model,to investigate the role of calpain-1 signaling pathway and mitochondrial reactive oxygen species formation in CIH-induced vascular endothelial dysfunction(VED),and to explore the effect of ginsenoside Rg1 on CIH-induced VED,so as to provide a new treatment direction for the clinical prevention and treatment of cardiovascular complications caused by OSA.MethodsIn vivo,the CIH model was established by placing healthy male C57BL/6J wild-type mice and calpain-1 knockout mice in an animal chamber with autoregulating oxygen concentration.After 4 weeks,the fluctuation of arterial oxygen saturation between 60% and 85% was considered as a successful CIH model.Wild-type mice were randomly divided into 4 groups: control group;CIH group;ginsenoside Rg1 low-dose and medium-dose groups(10,20 mg·kg-1·d-1).Calpain-1 knockout mice were randomly divided into two groups: control group and CIH group.During the CIH interval,ginsenoside Rg1 groups received ginsenoside Rg1 gavage for 4 weeks.The function of vasodilation and vasoconstriction were observed by vascular reactivity detection.The levels of calpain-1,PP2 A and e NOS Ser1177 phosphorylation(p-e NOS)were detected by immunofluorescence and immunohistochemistry.The levels of intracellular reactive oxygen species(ROS)and mito ROS were observed by DHE and Mito SOX staining,and the levels of malondialdehyde(MDA)and nitric oxide(NO)in serum were detected by kits.The protein expressions of calpain-1,PP2 A,p-e NOS,e NOS,Drp1 and Mfn2 were detected by Western blotting.In vitro,human coronary endothelial cells(HCAECs)were incubated in a hypoxic chamber with autoregulated oxygen concentration for 24 h and pretreated with ginsenoside Rg1,MLL-28170,okadaic acid(OKA),NAC and TEMPOL for 48 h.the CAPN1-lentiviral vector was added to HCAECs,and then cells were divided into 9 groups: control group;CIH group;MDL-28170 group;OKA group;NAC group;TEMPOL group;ginsenoside Rg1 groups;p LVCAPN1-NC group;PLV CAPN1-CIH group.Cell viability was detected by CCK8.DHE and Mito SOX staining were used to observe the intracellular ROS and mito ROS levels.The contents of MDA and NO in cell supernatant were detected by kits.Immunofluorescence was used to detect the level of p-e NOS in cells.Mitochondrial membrane potential was detected by JC-1 staining.The protein expressions of calpain-1,PP2 A,p-e NOS,e NOS,Drp1 and Mfn2 were detected by Western blotting.ResultsIn vivo,compared with the control group,the vascular endotheliumdependent relaxation function was decreased,the vasoconstriction function was enhanced in CIH group.The levels of ROS,mito ROS and MDA in CIH group were increased.The protein levels of calpain-1 and PP2 A were increased,while the serum NO content and the protein level of p-e NOS were decreased in CIH group.Compared with the CIH group,the damaged vascular endothelium-dependent relaxation function and vasoconstriction function were improved in calpain-1 knockout group and ginsenoside Rg1 groups.The levels of ROS,mito ROS and MDA were decreased in ginsenoside Rg1 groups.The protein levels of calpain-1 and PP2 A were decreased,while the levels of NO and p-e NOS were increased in ginsenoside Rg1 groups.Moreover,scavengers of O2·-,H2O2,complex I or mito KATP abolished CIH-induced impairment in endothelial-dependent relaxation.In vitro,compared with the control group,the cell viability of HCAECs in CIH group was decreased.The levels of ROS,mito ROS,mitochondrial membrane potential and MDA were increased in CIH group.The protein levels of calpain-1 and PP2 A were increased,while the levels of NO and p-e NOS were decreased in CIH group.Compared with CIH group,ginsenoside Rg1,calpain-1 knockout,OKA,NAC and TEMPOL treatment increased the cell viability and levels of NO,p-e NOS in HCAECs,while decreased the levels of ROS,mito ROS,mitochondrial membrane potential,MDA and the protein levels of calpain-1,PP2 A in HCAECs.In addition,overexpression of calpain-1 decreased the cell viability,the levels of NO and p-e NOS in HCAECs,while increased the levels of ROS,mito ROS,mitochondrial membrane potential,MDA and the protein levels of calpain-1,PP2 A in HCAECs.Conclusions1.Ginsenoside Rg1 can alleviate vascular endothelial dysfunction,oxidative stress and mitochondrial dysfunction induced by chronic intermittent hypoxia.2.Calpain-1 knockout can improve vascular endothelial dysfunction,oxidative stress and mitochondrial dysfunction induced by chronic intermittent hypoxia.3.Calpain-1 overexpression can accelerate the activation of oxidative stress and mitochondrial dysfunction induced by chronic intermittent hypoxia.4.Ginsenoside Rg1 can protect vascular endothelial dysfunction induced by chronic intermittent hypoxia by inhibiting the calpain-1/PP2 A signaling pathway and reducing the formation of mitochondrial reactive oxygen species.