Based On The TXNIP/NLRP3 Pathway To Study The Effect Of Yiqi Huoxue Drugs On Endoplasmic Reticulum Stress-induced Pyroptosis After Myocardial Infarction

Background:Affected by factors such as diet,lifestyle and social pressure,myocardial ischemia and myocardial infarction and the resulting heart failure still seriously affect the health and quality of life of residents,and the number of young and middle-aged patients is also increasing.Studies have found that myocardial damage can lead to dysfunction of endoplasmic reticulum and mitochondria,trigger a variety of cell death pathways including cell death,and lead to damage of cardiac function.Clinical use of Yiqi Huoxue Decoction can effectively improve patients' symptoms and cardiac function.The medicines for Yiqi Huoxue Decoction mainly include Astragalus 50g,Sunshine Ginseng 10g,Angelica 15g,Ligusticum chuanxiong Hort 15g,and Panax notoginseng 6g.They were established on the theoretical basis of the theory of qi and blood(Qi deficiency and blood stasis,qi stagnation and blood stasis)in traditional Chinese medicine.it is an effective prescription for the treatment of myocardial ischemia,hypoxia,myocardial injury,and various residual symptoms after myocardial infarction.Its effect is mainly to benefit the heart,promote blood circulation and clear the veins.The research team has proved that the Yiqi Huoxue Decoction can improve the symptoms of pain,chest tightness and shortness of breath in patients with myocardial infarction,significantly improve cardiac ejection function and cardiac morphology,decrease myocardial damage caused by inflammatory reaction in rats after myocardial infarction,and reduce the secretion of inflammatory cytokines in serum.Objective and significance:To explore the interaction and regulation mechanism of endoplasmic reticulum stress and pyroptosis after myocardial infarction,to study the pathophysiological process of myocardial infarction in detail,and to reveal the mechanism and targets of Yiqi Huoxue Decoction in the prevention and treatment of myocardial ischemia and myocardial infarction,which has important theoretical significance for the clinical treatment of myocardial infarction and the development of new drugs.Research methods:Animal experiment:Male SD rats(200±20g)were purchased and fed adaptively for three days.The left anterior descending coronary artery was ligated to establish the animal model of acute myocardial infarction.According to the electrocardiogram,the rats that did not meet the requirements were excluded.And then the rest of the animals were randomly divided into:acute myocardial infarction model group(Model,M),Yiqihuoxue group(YQHX,Y),Perindopril(P)and the sham operation group(Sham,S)with only threading and no ligation.After 28 days of myocardial infarction,the echocardiography and HE staining were used to observe the course of myocardial cells and evaluate the cardiac function of rats in each group.Rat heart function;transmission electron microscopy to observe the ultrastructure of myocardial tissue,immunofluorescence staining to observe the distribution and expression of TXNIP and GSDMD;Western blot was selected to observe GRP78,(P)IREla,(P)PERK,TXNIP,NLRP3,GSDMD,IL-1?,caspase-1,cleaved Caspase-1/11 in the marginal zone of myocardial tissue infarction.Revealing the changes of endoplasmic reticulum stress and pyroptosis in the marginal area of the tissues after myocardial infarction,as well as the regulatory effects of Yiqi Huoxue Decoction through the results of the previous studies.Cell experiment:The H9c2 cardiomyocyte cell line was used to replicate the hypoglycemia and hypoxia model,and the cardiomyocytes were divided into:normal control group(C);hypoglycemia and hypoxia model group(M);200?g/ml Yiqi Huoxue Decoction group(Y2);300?g/ml Yiqi Huoxue Decoction group(Y3);400?g/ml Yiqi Huoxue Decoction group(Y4).According to the previous research results of the team,the 12h time point of hypoglycemia and hypoxia was selected to observe the protective effect of Yiqi Huoxue Decoction on moderate to mild cardiomyocyte damage.The CCK-8 kit was used to observe the viability of myocardial cells in each group,determine the levels of LDH and ROS in the cells of each group,and screen out the optimal concentration of Yiqi Huoxue Decoction to regulate the level of oxidative stress as 300?g/ml.In order to further observe the relationship between endoplasmic reticulum stress and pyroptosis,the endoplasmic reticulum stress inhibitor 4-PBA was used,and cardiomyocytes were randomly divided into 5 groups at a density of 40×104:normal control group(C),Hypoglycemia and hypoxia model group(M),Yiqi Huoxue Decoction group(Y),4-PBA inhibitor group(4-PBA),Yiqi Huoxue Decoction+4-PBA inhibitor group(Y+4-PBA).Hoechst 33342/PI double staining was used to detect the pyroptosis rate of cardiomyocytes in each group.Western blotting was used to observe the protein expression of GRP78,(p)IRE1a,(p)PERK,TXNIP,NLRP3,GSDMD,IL-1?,Caspase-1,cleaved Caspase-1/11.Results:1.The left ventricular function in each group was evaluated by echocardiography.After 28 days of intervention in each group of rats,compared with the sham operation group,the left ventricular ejection fraction(EF)and left ventricular short axis rate(FS)of the M group rats were significantly reduced(p<0.01),LVAWd,LVIDd,LVIDs also increased significantly(p<0.01),LVPWs was increased too(p<0.05),but LVAWs was decreased(p<0.01).After the intervention of Yiqi Huoxue Decoction,the heart function of rats in each group can be significantly improved,and the corresponding echocardiogram indexes of each group can be restored to the normal range2.HE staining of cardiomyocytes in the marginal zone of infarction in each group.Compared with the S group,the myocardial cell morphology of the M group was damaged,the cell swelled,the nucleus was scattered with the morphology and position changed,the myocardial fiber was broken,the structure was destroyed,the cytoplasm was unevenly colored,the arrangement was disordered,and the intercellular space was significantly enlarged,and a large number of inflammatory cells were infiltrated;cardiomyocytes in the marginal zone of the Y and P groups have less swelling and less cell damage,a small amount of myocardial fiber breakage and a slight widening of the myocardial cell gap.3.The ultrastructural changes were observed by transmission electron microscope.In group S,the myocardial Z-line and M-line are clear,the thick and thin myofilaments are clearly arranged and neat,the mitochondrial structure is complete,the double membrane is clearly visible,the matrix particles and mitochondrial cristae in the mitochondria are clearly arranged,and the endoplasmic reticulum structure is normal.28 days after myocardial infarction,the ultrastructure of myocardial cells in group M was disordered,mitochondria were swollen and ruptured,mitochondrial cristae were blurred or even disappeared,thin myofilaments were broken,thick myofilaments were stacked,myofibrils were broken,endoplasmic reticulum was swollen,and many vacuoles were seen.In the drug intervention groups,the thick and thin filaments were fused,the boundary was unclear,the endoplasmic reticulum and mitochondria were swollen,the mitochondrial cristae was blurred,but the structure was still intact.5.Immunohistochemical staining of TXNIP and GSDMD.TXNIP was rarely expressed in the sham operation group,but increased significantly in the model group.Yiqi Huoxue Decoction and Perindopril groups can reduce the positive rate of TXNIP expression.GSDMD was widely expressed in the model group,and the rate of strong positive staining cells was significantly higher than that in the sham operation group.The positive rate of GSDMD protein in the drug group can be significantly reduced after intervention.6.The expression of endoplasmic reticulum stress and pyroptosis proteins in the marginal zone of infarction in each group of rats.Western blotting results showed that:Compared with S group,M group GRP78,(p)IRE1a,(p)PERK,TXNIP,NLRP3,GSDMD,IL-1? Caspase-1,cleaved Caspase-1/11 protein expression levels were significantly increased(p<0.01),while the Yiqi Huoxue Decoction and Perindopril could reduce the protein expression in different degrees and play a corresponding regulatory role(P<0.05).7.The protective effect of Yiqi Huoxue Decoction on H9c2 cardiomyocytes with hypoglycemia and hypoxia.Compared with group C,the vitality of myocardial cells was significantly reduced after 12h of hypoglycemia and hypoxia(p<0.01),while Y2,Y3,and Y4 could increase the vitality of myocardial cells and protect them from the damage of hypoglycemia and hypoxia.Y3 has the best effect.Continue to explore the effects of different concentrations of Yiqi Huoxue Decoction on LDH and ROS in each group of cells.The results showed that Yiqi Huoxue Decoction can reduce the levels of LDH and ROS in hypoglycemic and hypoxic cardiomyocytes.Among them,Y3 shows a better effect.In the next experiment of this subject,a concentration of 300?g/ml of Yiqihuoxue Decoction was used to explore the mechanism.We continued to observe the cell viability and LDH expression levels of cardiomyocytes in the five groups of C,M,Y(300?g/ml),4-PBA and Y(300?g/ml)+4-PBA.The results showed that compared with group C,the survival rate of M was significantly reduced(p<0.01),dropping to about 40%on average.The survival rates increased significantly under the intervention of drugs and inhibitors(p<0.05).Although the survival rates of Y,4-PBA and Y+4-PBA groups increased,there was no statistical difference between the three groups(p>0.05).The LDH in the cell supernatant of the M group was significantly increased(p<0.01),with an average of about 400 U/L.Y,4-PBA and Y+4-PBA could significantly reduce the release of LDH after intervention(p<0.01).Compared with Y+4-PBA group,the difference between Y group and Y+4-PBA group was statistically significant(p<0.01),The reduction of LDH in the Y+4-PBA group was more significant.8.The pyroptosis rate of cardiomyocytes.After double-staining with Hoechst 33342/PI,the cells of group C were light blue stained with Hoechst 33342/PI,with high cell density and normal cell size,and there were no red staining cells.In group M,the nucleus were stained brightly in blue,the cell density decreased,the cell volume increased,and a large number of cells were red-stained.After the use of inhibitors and Yiqi Huoxue Decoction intervention,the rate of pyroptosis cells could be reduced,and the cell density was also higher than that of the M group,but the inhibitor group had a better effect.9.The effect of Yiqi Huoxue Decoction on the endoplasmic reticulum stress and pyroptosis related proteins of hypoglycemia and hypoxia cardiomyocytes.Compared with group S,the protein expression levels of GRP78,(p)IRE1a,(p)PERK,TXNIP,NLRP3,GSDMD,IL-1?,and Caspase-1,cleaved Caspase-1/11 in group M were significantly increased(p<0.01).Inhibitor intervention can reduce the expression of related indicators,and the inhibitor had a better effect.When Yiqi Huoxue Decoction were used in combination with inhibitors,their expression levels could be further reduced(p<0.01).Conclusion:1.Yiqi Huoxue Decoction can improve the heart function after myocardial infarction by improving the ultrastructure damage of ischemic myocardial cells,improve the contractile function after myocardial infarction,and reduce the infarct area.2.Yiqi Huoxue Decoction can reduce the death of myocardial cells and save the ischemic myocardium by inhibiting endoplasmic reticulum stress and pyroptosis related proteins.3.4-PBA,the endoplasmic reticulum stress inhibitor,could significantly inhibit the expression of myocardial cell pyroptosis-related indicators,indicating that pyroptosis could be triggered by the endoplasmic reticulum stress pathway and aggravate the damage of pyroptosis,which may be related to TXNIP/NLRP3 passway.The Yiqi Huoxue Decoction could inhibit the endoplasmic reticulum stress TXNIP/NLRP3 pathway,alleviate the endoplasmic reticulum stress,inhibit cell pyroptosis,and reduce the damage of cardiomyocytes.The mechanism of Yiqi Huoxue Decoction is closely related to IRE1a,and this will be the focus of further research on this subject.4.Yiqi Huoxue Decoction alleviates the damage of endoplasmic reticulum,improves the state of "Qi Deficiency" and improves the power of damaged heart function,which provides a research basis for interpreting the theory of "Qi and blood" in Tradational Chinese Medicine from the perspective of modern science.