Mechanism And Clinical Significance Of Tongnao Decoction Activating PI3K/Akt Signaling Pathway On Angiogenesis After Ischemic Stroke

Objective:Ischemic stroke(IS)is mostly caused by vascular occlusion,stenosis or rupture of the middle cerebral artery and its collateral circulation caused by a variety of predisposing factors,and then the blood flow of various cerebral arteries is blocked.Ischemia and hypoxia,patients mostly show transient or permanent neurological damage.IS accounts for 60%-80%of the total stroke cases,and has become one of the leading causes of death in adults worldwide,seriously affecting the quality of life of patients.So far,the clinical medical community still lacks effective methods that can reduce ischemia-induced nerve damage.Recombinant tissue plasmmogen activator(rt-PA)is currently the only one approved by China National Medical Products Administration that can treat ischemic stroke by dissolving blood clots and restoring blood reperfusion effective treatment.However,its application is limited due to the extremely narrow therapeutic time window and the need for rapid dosing in most patients within about 4.5 hours after the onset of ischemia.Induced angiogenesis can occur in the ischemic site of stroke animal models and clinical patients.The new blood vessels can further improve the supply of cerebral ischemia and improve neurological function,which is of great value in the prognosis of IS.The density of newborns has an important relationship with patient prognosis.In the end,this study is mainly based on the research concept of angiogenesis to explore the pathophysiological mechanism of the treatment of IS by Traditional Chinese Medicine(TCM)from three aspects:in vitro and in vivo experiments and clinical patients.Methods:The first part is the theoretical research,which reviews and studies the origin,etiology,mechanism and treatment of stroke through TCM and modern medicine respectively.The second part of the experimental study was divided into two parts.In the first part,effect and mechanism of Tongnao decoction(TND)on angiogenesis in transgenic zebrafish embryos:transgenic zebrafish were divided into groups after adaptive feeding and given TND(3.5,7.0,14.0 mg/kg)and placebo(3.5 mg/kg)twice a day for 14 days.The semi-lethal concentration(LC50)and maximum non-lethal concentration(MNLC)of TND in zebrafish embryos were calculated.The specific inhibitor test of zebrafish treated with VEGF receptor inhibitor(VRI)was conducted.Determination of cell viability in vitro;In vitro wound healing test;Test tube formation experiment;Evaluation of angiogenic vascular changes in zebrafish embryos after drug treatment;In the second part,gastrodin,the main active component of TND,affects the function of HUVECs by regulating miR-21 and induces angiogenesis through the following mechanism:EdU and MTT methods were used to detect the EdU labeling rate and inhibition rate of HUVECs cell proliferation,real-time quantitative RT-PCR was used to detect the expression of miR-21 in HUVECs,and scratch test and tube formation test were used to detect the migration and tube formation ability of HUVECs.The protein expression levels of p-PI3K/PI3K and p-Akt/Akt were detected by Western blotting.In the third part,80 patients with acute cerebral infarction were selected as the research objects,and 80 patients with acute cerebral infarction were divided into control group(n=40)and experimental group(n=40)according to different treatment methods.The control group was given routine basic treatment combined with clopidogrel intervention,and the experimental group was given TND intervention on the basis of the control group.The TCM syndrome scores,serum HIF-lα,VEGF expression,platelet-related parameters,degree of neurological deficit,daily living ability,clinical efficacy,and adverse reactions were observed in the two groups before and after treatment.Results:The second part TND can improve the apoptosis of HUVECs induced by VRI,protect the viability of HUVECs,promote the proliferation of HUVECs,promote the wound healing of HUVECs,significantly promote the vascularization of HUVECs(P<0.05),and improve the ISVs injury induced by VRI in zebrafish embryos.Enlarged blood vessels on SIVs and promoted CtAs recovery in VRI-treated zebrafish embryos.Inhibitors of downstream signaling molecules of VEGFR2 blocked the pro-angiogenic activity of TND in VRI-treated zebrafish embryos.Compared with the control group,the cell proliferation level of VEGF group,GSTD-10μM group and GSTD-25μM group was significantly increased(P<0.05),and GSTD was concentration dependent.The tube formation and cell migration ability of VEGF group and GSTD-10μM group were significantly higher than those of GSTD-25μM group,and the expression levels of MMP-2 and MMP-9 were significantly increased(P<0.05).Both VEGF and GSTD significantly up-regulated the expression of miR-21 in HUVECs,and the miR-21 in GSTD-25μM group was significantly higher than that in GSTD-10μM group(P<0.05).Compared with the control group,the protein levels of p-PI3K and p-Akt in VEGF and GSTD groups were significantly increased,and the ratio of p-PI3K/PI3K and p-Akt/Akt was significantly higher.Compared with the GSTD+NC inhibitor group,the protein levels of p-Akt and p-PI3K in the GSTD+miR-21 inhibitor group were significantly decreased,and the ratio of p-Akt/Akt and p-PI3K/PI3K were significantly decreased(P<0.05).Comparison of TCM syndrome scores between the two groups before treatment(P>0.05),the TCM syndrome scores in the two groups after treatment were significantly lower than those before treatment,and the TCM syndrome scores in the experimental group were lower than those in the control group(P<0.05).Comparison of the expression levels of serum VEGF and HIF-1α(P>0.05),the expression level of serum VEGF in the two groups after treatment was significantly higher than that before treatment,the expression level of HIF-la was significantly lower than that before treatment,and the expression level of serum VEGF in the experimental group was higher than that before treatment.The expression level of HIF-lα in the control group was lower than that in the control group(P<0.05);The platelet-related parameters PDW,MPV and P-LCR were compared between the two groups before treatment(P>0.05).The platelet-related parameters PDW,MPV and P-LCR after treatment were significantly lower than those before treatment,and the platelet-related parameters PDW,MPV and P-LCR in the experimental group were lower than those in the control group(P<0.05).Comparison of NIHSS and Barthel scores between the two groups before treatment(P>0.05),the NIHSS scores of the two groups of patients after treatment were significantly lower than those before treatment,and the Barthel index was significantly higher,and the NIHSS scores of the experimental group were lower than those of the control group,and the Barthel index was higher than the control group(P<0.05).The effective rate was 95.00%higher than that of the control group,which was 80.00%(P<0.05);after treatment,the total incidence of adverse reactions such as skin allergy,gastrointestinal reactions,nausea and increased blood pressure in the experimental group was 7.50%was slightly lower than the total incidence of adverse reactions in the control group 15.00%,but the comparison between groups(P>0.05).Conclusion:1.TND may promote cell proliferation,migration and angiogenesis in HUVECs by activating VEGFR2,PI3K/Akt,Raf/MEK/ERK dependent signaling pathways.2.Gastrodin,the main active component of TND can increase the expression of miR-21 in HUVECs by activating the PI3K/Akt signaling pathway,improve cell proliferation,migration and tube formation,and induce neovascularization.3.The clinical effect of TND in the treatment of patients with acute cerebral infarction is definite.It can effectively improve the neurological function and serum HIF-1α and VEGF of the patients,and has high safety.It is worthy of clinical promotion and wide application.