To Explore The Efficacy And Mechanism Of The Qi-invigorating And Qi-strengthening Method On Patients With Cerebral Infarction After Intravenous Thrombolysis Based On The Apelin Pathwa

Objectives1.Preliminary analysis of the syndrome distribution characteristics of patients with acute cerebral infarction with intravenous thrombolysis.The research method of meta-analysis was used to clarify the distribution law of syndromes and the evolution of syndrome elements in patients after intravenous thrombolysis.2.To evaluate the clinical efficacy and safety of Shenmai injection,which is the representative drug of the yiqi fuzheng method.A randomized controlled single-blind clinical trial was conducted to include patients at the acute stage of cerebral infarction after intravenous thrombolysis,to compare the effects of routine basic treatment and the addition of shenmai injection on the clinical outcomes of patients,observing and recording the effects of Shenmai injection on the syndromes of patients.3.To observe the changes of apelin and its common subtypes apelin-12,apelin-13,apelin-36 in serum at different time points in patients with acute cerebral infarction,and their differences from healthy people.The prognostic value of apelin and its subtypes in acute cerebral infarction and its effects on neurological impairment and prognosis were also explored.Meanwhile,the effects of Shenmai injection on the content of apelin and its subtypes were observed.4.The oxidative stress model of highly differentiated PC 12 cells induced by hydrogen peroxide(H2O2)was used to simulate the pathological process of cerebral infarction reperfusion injury.From the perspective of apoptosis,the neuroprotective effect of Shenmai injection on PC 12 cells was explored in vitro,and the mechanism of its action on apelin receptor APJ and its downstream molecules was explored.Methods1.Implemented computer retrieval of Chinese and English databases,including PubMed,China National Knowledge Infrastructure(CNKI),Wanfang Data,VIP,and China Biomedical Literature Service System.The retrieval date of each database is from the establishment of the database to 2021.12.31.Searched for journal papers,academic papers,and conference papers on the classification of syndromes in patients with acute cerebral infarction by intravenous thrombolysis.After screening the eligible literature according to the inclusion and exclusion criteria,the effective data was extracted,and the bias of the literature was evaluated at the same time.Statistical analysis was performed using Review Manager 5.4.1 software.2.Patients with acute cerebral infarction within 12 hours after intravenous thrombolysis with recombinant tissue plasminogen activator(rt-PA)were enrolled and randomly divided into control group and treatment group.On the foundation of conventional basic treatment,the treatment group was given Shenmai injection 100ml/day,and the control group was given normal saline 100ml/day,the course of treatment was 10 days.The clinical efficacy of Shenmai injection was evaluated by the National Institutes of Health Stroke Scale(NIHSS)score,modified Rankin Scale(mRS)score,blood pressure,and peripheral blood biological indicators;liver and kidney function were used as safety indicators,and the incidence of adverse events to evaluate the safety;observed and recorded the syndrome distribution of patients from onset to 30 days,and analyzed the effect of Shenmai injection on the evolution of syndromes in patients.3.Double-antibody one-step sandwich enzyme-linked immunosorbent assay(ELISA)was used to detect the serum apelin and its subtypes in patients with acute cerebral infarction within 24 hours and 10 days after the onset,and compared with healthy people.The correlation between the content of apelin and its subtypes and the degree of neurological deficit and prognosis of patients at onset was analyzed,and the prediction model of apelin and its subtypes for the diagnosis and prognosis of acute cerebral infarction was constructed by using machine learning methods.Meanwhile,the effects of Shenmai injection on serum apelin and its subtypes were observed.4.In vitro cell experiments,highly differentiated PC 12 cells were treated with H2O2 to form an oxidative stress model,and then Shenmai injection was added for intervention,and small interfering RNAs(siRNA)were transfected to silence the gene expression of APJ.Cells were divided into 6 groups,including the blank control group,Shenmai injection control group,H2O2 model group,H2O2+Shenmai injection group,H2O2+Shenmai injection+Si-APJ group,H2O2+Shenmai injection+Si-NC(negative control)group.TUNEL staining was used to detect the effect of Shenmai injection on cell apoptosis.The contents of intracellular malondialdehyde(MDA),superoxide dismutase(SOD),and reactive oxygen species(ROS)were used to evaluate the antioxidant capacity of Shenmai injection.Western blot and immunofluorescence were used to detect the effects of Shenmai injection on the APJ/AMPK/GSK-3β signaling pathway.Results1.The results of systematic review and meta-analysis show that the distribution frequency of single syndrome elements before intravenous thrombolysis in patients with acute cerebral infarction,from high to low,is in the order of wind syndrome 0.92[95%CI(0.79,1.05)],stasis syndrome 0.70[95%CI(0.49,0.90)],phlegm syndrome 0.65[95%CI(0.44,0.86)],qi deficiency syndrome 0.37[95%CI(0.03,0.71)],fire syndrome 0.29[95%CI(0.24,0.33)],yin deficiency syndrome 0.20[95%CI(0.11,0.28)].After 7±3 days of intravenous thrombolysis,-the distribution frequency of individual syndrome elements,from high to low,qi deficiency syndrome 0.62[95%CI(0.36,0.88)],stasis syndrome 0.50[95%CI(0.29,0.72)],phlegm syndrome 0.38[95%CI(0.19,0.57)],fire syndrome 0.21[95%CI(0.16,0.25)],yin deficiency syndrome 0.21[95%CI(0.07,0.34)],wind syndrome 0.05[95%CI(0.01,0.08)].Before and after thrombolytic therapy,the most significant single syndrome elements were wind syndrome and qi deficiency syndrome respectively.2.After Shenmai injection treatment,there was no significant difference in the NIHSS score on the 10th day between the two groups,but the mRS score on the 30th day in the treatment group was significantly lower than that in the control group(P=0.041).There were no differences in triglyceride,total serum cholesterol,low-density lipoprotein,high-density lipoprotein,homocysteine,blood uric acid,urea,and other peripheral blood indexes between the two groups before and after treatment.At the same time,there was no difference in the safety indicators of liver and kidney function and the incidence of adverse events between the two groups.After treatment,the systolic blood pressure(P=0.000)and diastolic blood pressure(P=0.000)of the two groups of patients were significantly decreased,but there was no difference between the groups.On the 10th day(P=0.008)and the 30th day(P=0.039)after the onset of the patients,the number of patients with qi deficiency syndrome in the treatment group was significantly lower than that in the control group.The patient-originating syndrome elements had no significant effect on the degree of neurological deficit at onset,but the short-term prognosis of patients with established wind(P=0.038)and internal fire(P=0.003)syndromes within the 30th day was poor.3.The levels of serum apelin(P=0.001)and apelin-13(P=0.000)in patients with acute cerebral infarction were significantly lower than those in the healthy group within 24 hours of the onset and rose significantly after 10 days of onset,but apelin-12 and apelin-36 was not different from the healthy group at both time points.Logistic regression analysis found that serum apelin(P=0.002),and apelin-13(P=0.001)were statistically associated with the incidence of acute cerebral infarction,and after adjusting for risk factors,the statistical association still existed.Using the random forest model,according to the level of serum apelin and its subtypes within 24 hours of the onset of the patient,the onset of acute cerebral infarction can be better predicted,of which apelin-13 has the best predictive performance.Meanwhile,the lower the level of serum apelin-13 within 24 hours of onset,the more severe the neurological deficit was(P=0.033).Support vector machine model was used to predict the short-term prognosis of patients according to the levels of serum apelin and its subtypes 10 days after onset.After 10 days of Shenmai injection treatment,the serum apelin content was significantly increased(P=0.026),but the subtype content was not affected.4.After H2O2 treatment of PC 12 cells,the cell survival rate was significantly decreased,the TUNEL positive cells were significantly increased,the ROS and MDA reflecting the oxidative stress process were significantly increased,and the SOD was significantly decreased.Shenmai injection can alleviate H2O2-induced oxidative damage of PC 12 cells and improve cell survival rate.Further mechanism studies have shown that Shenmai injection can reduce the positive rate of TUNEL and reduce cell apoptosis by regulating the levels of MDA,SOD,and ROS in cells.And Shenmai injection can significantly increase the expression of apelin receptor APJ,and can promote the phosphorylation of APJ downstream molecules AMPK and GSK-3β;after transfection of Si-APJ,silencing the gene expression of APJ,Shenmai injection for The regulating ability of MDA,SOD,and ROS,as well as the effects on AMPK and GSK-3β were inhibited,which further verified that the neuroprotective effect of Shenmai injection on PC 12 cells was partly achieved through the APJ/AMPK/GSK-3βsignaling pathway.Conclusions1.In patients with acute cerebral infarction,wind syndrome is the most prominent in the initial state syndrome.After 7±3 days of intravenous thrombolysis,qi deficiency syndrome becomes the most important syndrome element.The early addition of Shenmai injection,representative drug of the yiqi fuzheng method,can significantly improve the short-term prognosis of patients without obvious adverse reactions.The mechanism of action may be partly achieved by increasing the serum apelin content of patients.2.Compared with healthy people,the serum apelin and apelin-13 levels in patients with acute cerebral infarction showed a dynamic trend of first decreasing and then increasing from 24 hours to 10 days after the onset of the disease.And the content of apelin and its subtypes can better predict the incidence and short-term prognosis of acute cerebral infarction.In the oxidative stress model of PC12 cells induced by H2O2,Shenmai injection could increase the expression of apelin receptor APJ,partially reduce oxidative damage and apoptosis,simultaneously improve cell survival rate through APJ/AMPK/GSK-3β signaling pathway.