Effects And Mechanisms Of Ginsenoside Rc On Ameliorating Endothelial Insulin Resistance And Vascular Endothelial Dysfunction Based On Angiotensin-Converting Enzyme 2

Type 2 diabetes mellitus(T2DM)has been a major health concern worldwide,which exerts devastating effects on blood vessels,namely vascular complications,and vascular disease remains the leading cause of morbidity and mortality in individuals with diabetes.Insulin resistance(IR),regarded as the pathogenic hallmark of T2DM,not only plays a critical role in the development of T2DM,but also leads to endothelial dysfunction under conditions of metabolic syndrome.Endothelium is the first barrier of blood vessels,which carries on multiple functions including paracrine,autocrine and endocrine.Endothelial dysfunction is an early pathogenic event of vascular dysfunction,and it interacts with IR and they promote each other,both of which play a major role in the development of diabetic cardiovascular complications.As a peptidergic endocrine system,renin-angiotensin system(RAS)is mainly responsible for maintaining the balance of blood pressure and water-electrolyte,whose classic component,angiotensin-converting enzyme(ACE)/angiotensin II(Ang II)/angiotensin type 1 receptor(AT₁R)axis plays an important role in the occurrence and development of diabetes and its complications.Indeed,the key effector molecule of this axis,Ang II,is involved in a variety of pathophysiological processes including oxidative stress,inflammatory response,fibrosis,hypertrophy and endothelial dysfunction.Angiotensin-converting enzyme 2(ACE2)/angiotensin-(1-7)[Ang-(1-7)]/Mas axis is a physiological antagonist of ACE/Ang II/AT₁R axis,whose protective effects not only rely on the degradation of Ang II,but also the production of Ang-(1-7)which exerts actions including anti-oxidation,anti-inflammation,antihypertrophy,vasodilation and improving endothelial dysfunction by interacting with Mas.Although it has been reported that ACE2/Ang-(1-7)/Mas axis could ameliorate metabolic IR,and Ang-(1-7)could improve endothelial dysfunction in db/db mice as well as Ang II induced IR in endothelial cells,literatures about ACE2/Ang-(1-7)/Mas axis emphasizing endothelial IR remain scarce.Hence further exploration into its relationship with IR will be conducive to the in-depth understanding of the molecular mechanisms underlying IR with subsequent prevention and treatment for diabetic cardiovascular complications from a new perspective.Ginsenoside Rc is a protopanaxadiol-type saponin and one of the major components of ginsenosides with relatively less research on it.Only a few studies have demonstrated that ginsenoside Rc exhibits potent anti-inflammatory and antioxidative activities.However,the potential benefits of ginsenoside Rc against vascular complications have not been explored.As the isomerides of ginsenoside Rc,ginsenoside Rb2 and ginsenoside Rb3 have been shown to display significant cardiovascular protective effects.Moreover,it has been indicated that ginsenosides are intimately associated with RAS.Considering the well-established role of inflammation and oxidative stress in IR as well as the essential part played by ACE2/Ang-(1-7)/Mas axis in cardiovascular complications,we speculated that ginsenoside Rc could contribute to the alleviation of endothelial IR and endothelial dysfunction via activating ACE2/Ang-(1-7)/Mas axis.Thus preliminary validation was performed in this study.First,high glucose(HG)(30 mmol/L D-glucose for 24 h)was used to establish the IR model in human umbilical vein endothelial cells(HUVECs).The experiment results showed that HG did not affect cell viability and morphology.HG induced a significant decrease of NO as well as a remarkable augmentation of ET-1 mRNA in HUVECs,suggesting that HG disrupted the balance of vasomotor factors.HG significantly reduced the levels of p-IRS1^Tyr896,p-PI3K p85^Tyr607,p-Akt^Ser473and p-eNOS^Ser1177while increased the level of p-IRS1^Ser307,suggesting that the insulin signal transduction was impaired by HG.HG remarkably reduced the protein expression of ACE2 and Mas,suggesting that HG impaired the protective effects of ACE2/Ang-(1-7)/Mas axis.As was shown by the results above,HG managed to induce IR as well as reduce the protein expression of ACE2 and Mas in HUVECs.To address whether ginsenoside Rc could ameliorate IR in HUVECs,we next treated HUVECs with HG and ginsenoside Rc(25,50 mmol/L)synchronously.The experiment results showed that the decrease of NO and increase of ET-1 mRNA induced by HG were markedly inhibited by ginsenoside Rc,suggesting that ginsenoside Rc corrected the imbalance of vasomotor factors.Ginsenoside Rc induced an obvious reduction of intracellular ROS as well as MDA content in the culture medium,and SOD activity was elevated by ginsenoside Rc,indicating the enhanced antioxidative capacity of HUVECs.Moreover,Ginsenoside Rc significantly reduced mRNA levels of TNF-?,IL-1?and IL-6,suggesting that ginsenoside Rc reinforced the anti-inflammatory ability of HUVECs.An obvious decrease of Ang II production as well as a further increase of Ang-(1-7)content was observed in the HG groups treated with ginsenoside Rc.Besides,ginsenoside Rc markedly attenuated the reduction of ACE2 and Mas protein expressions induced by HG without affecting mRNA levels,suggesting that ginsenoside Rc activated ACE2/Ang-(1-7)/Mas axis in a non-transcriptional regulation manner.Ginsenoside Rc induced a remarkable elevation of the levels of p-IRS1^Tyr896,p-PI3K p85^Tyr607,p-Akt^Ser473and p-eNOS^Ser1177accompanied by a lowered level of p-IRS1^Ser307,suggesting that the impaired insulin signal transduction was restored by ginsenoside Rc.The increase of NOX2 protein expression,the enhanced activation of IKK?/NF-?B and the augmentation of JNK phosphorylation were all inhibited by ginsenoside Rc,suggesting that ginsenoside Rc suppressed oxidative stress and inflammatory pathways related to IR.As was shown by the results above,ginsenoside Rc was able to ameliorate endothelial IR via anti-oxidation,anti-inflammation and upregulation of ACE2/Ang-(1-7)/Mas axis.To further elucidate the potential mechanisms of ginsenoside Rc on improving endothelial IR in HG-treated HUVECs,we compared the effects of ginsenoside Rc with those of the selective ACE2 inhibitor MLN-4760(100 nmol/L).The experiment results showed that the effects of ginsenoside Rc on correcting the imbalance of vasomotor factors were significantly inhibited by MLN-4760.In addition,the anti-inflammatory effects of ginsenoside Rc and ginsenoside Rc-mediated activation of ACE2/Ang-(1-7)/Mas axis were also abrogated by MLN-4760.In the insulin signaling pathway,MLN-4760 abolished the effects of ginsenoside Rc on activating IRS-1/PI3K/Akt/eNOS.Meanwhile,Inhibition of ACE2 by MLN-4760 reversed the effects of ginsenoside Rc on NOX2 inhibition and inflammation pathways in HUVECs.However,MLN-4760 failed to inhibit the effects of ginsenoside Rc on reducing intracellular ROS as well as MDA content in the culture medium,and improving SOD activity,which indicated that ginsenoside Rc possessed the anti-oxidative capacity independent of ACE2.As was shown by the results above,the anti-oxidative and anti-inflammatory effects of ginsenoside Rc relied on the upregulation of ACE2 to a certain extent,through which ginsenoside Rc ameliorated endothelial IR.Based on the findings of our in-vitro experiments,we next conducted in vivo studies applying db/db mice of the T2DM model with MLN-4760 to further determine the role of ACE2 in the effects of ginsenoside Rc in vivo where endothelial dysfunction occurred.The experiment results showed that ginsenoside Rc did not affect body weight,fasting blood glucose,lipid profiles(TC,TG,LDL-C and HDL-C)and insulin levels in serum,suggesting that ginsenoside Rc had no hypoglycemic and lipid-regulating effects.Ginsenoside Rc treatment induced a significant reduction of TNF-?in serum,indicating its anti-inflammatory action in vivo,which was abolished by MLN-4760.Additionally,there was a remarkable decline of local Ang II production in aortas from db/db mice benefiting from ginsenoside Rc treatment,which was reversed by MLN-4760,though Ang II and Ang-(1-7)in serum as well as aortic Ang-(1-7)levels were similar among each group.Ginsenoside Rc significantly increased ACE2 and Mas expressions in aortas,accompanied by strong inhibitory effects of MLN-4760,which suggested that ginsenoside Rc also upregulated ACE2and Mas in vivo.Furthermore,we observed a restoration of the impaired endothelium-dependent relaxation and Akt/eNOS pathway in aortas,suggesting the improvement of endothelial function in vivo,whereas MLN-4760 markedly attenuated the effects above.The group treated with ginsenoside Rc produced a marked decrease in NOX2 and NOX4 expression in aortas,indicating its anti-inoxidative action in vivo,which was inhibited by MLN-4760 to some exent.As was shown by the results above,ginsenoside Rc ameliorated aortic endothelial dysfunction in db/db mice,whose mechanisms depended more on the degradation of Ang II than the production of Ang-(1-7)without blood glucose and lipid regulation.In conclusion,the current study demonstrated that ginsenoside Rc effectively ameliorated endothelial IR in HUVECs and endothelial dysfunction in db/db mice via upregulation of ACE2.Our findings not merely reveal a novel action with its possible mechanism of ginsenoside Rc,but also provide new insight into the potential clinical application of ginsenoside Rc which holds promise for the treatment of diabetic vascular complications.