Study Of Activating Blood And Resolving Stasis Formula Of SHXXT(Sanhuangxiexin Decoction) On Anti-atherosclerotic Effects And The Mechanism

Research objectAS(Atherosclerosis)is the main pathological basis of cardiovascular and cerebrovascular diseases,which seriously threatens the health of human being.It is one of the main causes of death in the developed countries.In China,especially in recent years,the incidence of AS has showing a rising trend.Therefore,it is very important to study the pathogenesis and treatment of AS,but the pathogenic factors and pathogenesis of atherosclerosis is very complex,and so far,there is no exact theory to prove its mechanism.Since 1880s,there have been many mechanisms to explain the pathogenesis of AS,including lipid infiltration,inflammatory immune response,oxidative stress,endothelial damage,abnormal proliferation of smooth muscle cells and so on.With the development of the research,inflammation and oxidative stress have been recognized as the most important pathogenesis.The drug on the treatment of atherosclerosis,currently the most widely used statins,the moderating effect of blood lipid significantly,especially in reducing LDL,and studies have shown that statins can reduce blood lipids in addition,anti-inflammatory,antioxidant,repair blood vessels and inhibit platelet aggregation.However,with the deepening of clinical application,the study found that statins have hepatotoxicity,muscle damage,gastrointestinal reactions and neurological diseases and other adverse reactions.The traditional Chinese medicine compound as a composition of traditional medicine,many targets,which can effectively reduce the adverse reactions after treatment,reduce the incidence of drug resistance,diversity and the pathogenesis of atherosclerosis is consistent,effective treatment of AS.From the perspective of traditional Chinese medicine,atherosclerotic disease because of yin and yang qi deficiency,phlegm stasis stasis,block thread,clinical therapy method to blood stasis,phlegm detoxification.Sanhuang Xiexin Decoction is small compound clinical classic,was contained in the "Golden Chamber",by the composition of rhubarb,Coptis chinensis and Radix Scutellariae,mainly for detoxification,promoting blood circulation and removing blood stasis.In previous research for the effect of San Huang Xie Xin Decoction Combined with processing nine components of the role of screening experiments,prescription 7 for blood stasis rats has good curative effect,so this experiment takes advantage of Sanhuang Xiexin Decoction Huoxue Huayu Recipe(prescription 7)for the tested drug,study its therapeutic effect on AS and its mechanism.Research content1.Effect on the atherosclerosis ratsUsing high fat feed in combination with vitamin D3 injection and stimulating the body’s immune response to copy the rat model of atherosclerosis,they were divided into 6 groups,that is control,model,positive drug and Sanhuang Xiexin Decoction and small dose.After 8 weeks,the rats were administrated for 4 weeks.At the end of 12th weeks,blood was collected from the abdominal aorta,the determined the following indicators:total cholesterol(Total,Cholesterol,T-CHO),triglycerides(Triglyceride,TG),high density lipoprotein cholesterol(High-density lipoprotein-cholesterol,HDL-C),low density lipoprotein cholesterol(Low-density lipoprotein-cholesterol LDL-C),superoxide dismutase(superoxide dismutase,SOD)(Malondialdehyde,MDA,MDA),nitric oxide(Nitric oxide,NO),high sensitive C reactive protein(hypersensitive C-reactive,protein,hs-CRP),tumor necrosis factor alpha(Tumor necrosis factor-TNF-a),interleukin 1 beta(interleukin-1 beta,beta IL-1),interleukin 6(interleukin-6,IL-6)and interleukin 8(interleukin-8,IL-8),interleukin 18(interleukin-18,IL-18),RIA determination of 6-ketone Prostaglandin 1 alpha(6-keto-PGFla),endothelin(Endothelin,ET),thromboxane B2(Thromboxane B2,TXB2).At the same time,the aorta was fixed and stained with HE.The pathological changes of blood vessels were observed under microscope.2.Protective effect on HUVECsCultured the HUVECs in vitro to determine the maximum non-toxic concentration of Sanhuang Xiexin Decoction by MTT.The cell injury model was established by 200 μg·mL-1 Ox-LDL,the cell survival rates and the following indicators were measured:ET,6-keto-PGF1α,TXB2,MDA,SOD,NO,ICAM-1,VCAM-1,MCP-1.And another cell injury model was established by 60 ng·mL-1 TNF-a,and the cell survival rates and the following indicators were measured:ICAM-1,VCAM-1,MCP-1,IL-1β,IL-6,IL-8,NO.3.Study on the mechanism of anti ASHUVECs cultured in vitro,with 200μg·mL-1 Ox-LDL to establish a cell model of oxidative stress,ROS content in cell supernatant were measured,as well as the apoptosis rate and mitochondrial membrane potential by flow cytometry,the expression of Bax,Bcl-2,Caspase-3,Caspase-9 were calibrated by western blot.The expression level of VE-Cadherin,E-Selectin,ICAM-1 and VCAM-1 was detected by Western Blot with 60 ng·mL-1 TNF-α cell injury model.Research result1 Effect of the atherosclerosis in rats1)The effect on blood lipid level:it can effectively reduce the content of TC,TG,LDL-C,increase the content of HDL-C.2)The effect of anti oxidation:It can increase SOD,reduce MDA has a certain role.3)Effect on the anti-inflammatory:it can reduce Hs-CRP,TNF-a,IL-1β,IL-6,IL-8,IL-18 content.4)The effects of endothelial function:it had a significant effect on the content of NO,6-keto-PGF1α,and ET,TXB2.5)The effect on the morphology and structure of vascular pathological changes:it can reduce lipid deposition and cholesterol crystallization,and improve calcification.2.Protective effect on the HUVECs1)Maximum non-toxic concentration of drugs:the maximum non-toxic concentration is 62.5μg mL-1.62.5,31.25,15.625μg mL-1 were selected as the concentration of drug delivery.2)On cell survival rate:it can significantly improve the survival rate of HUVECs injured by Ox-LDL and TNF-a.3)Effect on the content of the active substances in the cell:in cell injured by Ox-LDL,it can significantly decrease the content of MDA,ET,TXB2,ICAM-1,VCAM-1,MCP-1,increase SOD,NO,6-keto-PGF1α content.The ICAM-1,VCAM-1,MCP-1,IL-6,IL-1,IL-8,and NO contents in the HUVECs induced by TNF-were significantly decreased and the content of NO increased.3.Study on the mechanism of anti AS1)Apoptosis induced by anti oxidative stress:it can reduce the content of ROS,the rate of apotosis and regulate the expression of apoptosis-associated proteins(decrease the expression of Bax,caspase-3,caspase-9,increase the expression of Bcl-2).2)Anti-inflammatory effect:it can effectively reduce the expression level of proteins(VE-Cadherin,E-Selectin,ICAM-1,VCAM-1)related inflammation.Conclusion1.It can play an important role in the protection of AS rats from regulating blood lipids,anti oxygen,anti-inflammatory,improving endothelial function and so on.2.It can effectively protect HUVECs injured by Ox-LDL and TNF-α,and the formula can reduce the expression of proteins related to inflammation to inhibit the development of inflammation,and inhibit the endothelial cell apoptosis caused by oxidative stress by regulating the expression of proteins related to apoptosis,Its mechanism of protecting HUVECs may be associated with the above.