Effects Of Heat Stress On ApoE^-/- Mouse Atherosclerosis And HSP70,TLR4 Signaling Pathway And The Treatment And Prevention Of Wang’s Qingjie Yiqi Decoction

Objective High temperature heat wave has obvious influence on the morbidity and mortality of cardiovascular system diseases,so the prevention and treatment of heat stress is of great significance to the impact of people on cardiovascular diseases.And atherosclerosis is the basic pathologic basis of cardiovascular disease,therefore this study applies the climate environment simulation technology,compare the mice in the thermal environment of aortic atherosclerotic plaques and HSP70,TLR4,MyD88,NF-κB gene expression level and quantity of protein expression,to study the stress of thermal environment for the influence of the degree of atherosclerotic lesions in mice and explore the HSP70 and HLR4 signaling pathways in the influence of heat stress on atherosclerosis role mechanism.In addition,the changes of the above indexes were observed after the intervention of wang’s qingjie yiqi decoction group,and the above mechanism was proved to provide experimental basis for the prevention and treatment of cardiovascular and cerebrovascular diseases under high temperature heat wave by traditional Chinese medicine.Methods1 Effects of different heat stress patterns on mice and observation of therapeutic effect of traditional Chinese medicine1.1 To observe the effect of high temperature with different rising rates on the mortality of mice:16 kunming mice were randomly divided into two groups:slow rising group and sudden rising group.They were fed in an artificial climate box to make the two groups warm faster and slower.The death number and time of the mice in the two groups were observed and recorded,and the death rate was calculated.1.2 Effect of traditional Chinese medicine on the prevention and treatment of heat stress on mice:40 SPF male C57BL/6J mice were randomly divided into five groups:blank group,high temperature group,wang’s qingjie yiqi decoction group,shengmai powder group,and dendrobium shengmai powder group.The artificial climate box was placed for feeding,and the drug group was gavaged with the responding drugs.The mortality rate and lipid profile of the mice were observed,so as to select the traditional Chinese medicine group with the strongest control effect.2 Effects of heat stress on atherosclerotic plaques.HSP70 and TLR4 signaling pathways in ApoE-/-mice and the preventive and therapeutic effects of wang’s qingjie yiqi decoction.2.1 To investigate the effects of heat stress on the atherosclerotic plaque.HSP70 and TLR4 signaling pathways in ApoE-/-mice:16 C57BL/6J mice were randomly divided into blank group and high temperature group.16 ApoE-/-atherosclerosis mice were randomly divided into AS group and high temperature AS group,and were fed high-fat diet.All 4 groups were raised in a normal environment.After 12 weeks of modeling,the blank group and AS group were still kept in the ordinary environment.The mice in the high temperature group and the high temperature AS group were fed in an artificial climate box and exposed to heat.After the experiment,the mice were sacrificed and samples were collected to detect the anal temperature,visceral index,atherosclerotic plaque size,lipid level,mRNA and protein expression levels of HSP70,TLR4,MyD88,and NF-κB.2.2 To investigate the effects of heat stress on ApoE-/-mouse atherosclerosis and HSP70.TLR4 signaling pathway and the treatment and prevention of wang’s qingjie yiqi decoction.8 C57BL/6J mice were fed with normal diet.40 ApoE-/-mice were randomly divided into model group,high dose group,medium dose group and low dose group and were fed high-fat diets throughout the whole process.After 11 weeks of modeling,the normal group was given 0.9%sodium chloride solution at the same dose and fed with normal environment and feed.The model group was given 0.9%sodium chloride solution at the same dose.High dose group,middle dose group,low dose group were given 2 g/ml,1 g/ml,1 time/day the wang qingjie yiqi decoction lavage 1 time/day,a week later in addition to the normal group were placed in the artificial climate box feeding,to accept heat exposure,after the death in mice,collecting specimens,detection of anal temperature and visceral index of mice,atherosclerotic plaque size,four,blood lipids and HSP70,TLR4,MyD88,NF-κB mRNA and protein expression level.Results1 Effects of different heat stress patterns on mice and observation of therapeutic effect of traditional Chinese medicine.1.1 To observe the effect of high temperature with different rising rates on the mortality of mice:When the climate box temperature was 35℃ and 38℃,no mice died in the slow rising group and the sudden rising group within 96h,and the mortality rate of both groups was 0%.When the temperature of the climate box increased to 40℃,the mortality rate of the sudden rise group within 24 hours was 87.5%,and that of the slow rise group was 25%.1.2 Effect of traditional Chinese medicine on the prevention and treatment of heat stress on mice:Mortality rate:mice were placed in a climate box at 35℃ for 72h and a climate box at 38 ℃ for 24h.The mortality rate of mice in each group was 0%.The death rate of the blank group and the wang’s qingjie yiqi decoction group was 0%,and that of the high temperature group was 100%.The mortality rate of the three drug treatment groups decreased,among which no mice died in wang’s qingjie yiqi decoction group and 42.9%in shengmai powder group.The mortality rate of dendrobium shengmai powder group was 14.3%.Blood lipid:compared with the blank group,HDL-C in the high temperature group was significantly lower(***P<0.001).Compared with the high temperature group,shengmai powder group.HDL-C in wang’s qingjie yiqi decoction group and dendrobium shengmai powder group were all significantly increased(#P<0.05).2 Effects of heat stress on atherosclerotic plaques.HSP70 and TLR4 signaling pathways in ApoE-/-mice and the preventive and therapeutic effects of wang’s qingjie yiqi decoction.2.1 To investigate the effects of heat stress on the atherosclerotic plaque.HSP70 and TLR4 signaling pathways in ApoE-/-mice:Mortality rate:when the climate box temperature was 35℃ and 38℃,no mice died in each group,and the mortality rate was 0%.Anal temperature:after 72h in the 35℃ climate box,the anal temperature in the high temperature group increased significantly(***P<0.001)compared with the blank group.To compare with the AS group,the rise height of anal temperature in the high temperature AS group was significantly higher(ΔΔΔP<0.001).When the mice were placed in a climate box at 38℃ for 24h,compared with the blank group,the rise height of anal temperature in the high temperature group was significantly decreased(***P<0.001).Compared with the AS group,the rise height of anus temperature in the high temperature AS group was significantly lower(ΔΔP<0.01).Body mass:after 12 weeks of modeling,compared with the blank group,the body mass of the AS group and the high temperature AS group increased significantly,AS group*P<0.05.and the high temperature AS group**P<0.01.After mice were put into the climate box,compared with the blank group,the body mass of mice in the AS group still increased significantly(***P<0.001).Viscera index:Compared with the blank group,the liver index of the AS group increased significantly(***P<0.001).Compared with the AS group,the liver index of the high temperature AS group was significantly lower(#P<0.05).Oil red O staining:compared with the blank group,The aortic intima of ApoE-/mice in the AS group and the high temperature AS group showed round diffuse thickening and formed typical atherosclerotic plaque.Compared with the AS group,intimal thickening was more severe in the high temperature AS group,and the area ratio of atherosclerotic plaque and lumen increased significantly(**P<0.01).Blood lipid:compared with the blank group,TG and LDL-C increased in the high temperature group,AS group and high temperature AS group,while HDL-C and TC decreased.Compared with the high temperature group,TG,TC,LDL-C and HDL-C increased in the high temperature AS group.Compared with the AS group,TG and LDL-C increased in the high temperature AS group,while HDL-C and TC decreased significantly.mRNA level and protein expression:compared with the blank group,the mRNA levels of HSP70,TLR4,MyD88,NF-κB in the high temperature group,AS group and high temperature AS group increased to different degrees and showed statistical differences.Compared with the high temperature group,the mRNA levels of HSP70,TLR4,MyD88 and NF-κB in the high temperature AS group were significantly increased,with statistical differences.Compared with AS group,mRNA levels of HSP70,TLR4,MyD88 and NF-κB were significantly increased in AS group with high temperature,showing statistical difference.The results of protein expression level and mRNA level were consistent.2.2 To investigate the effects of heat stress on ApoE-/-mouse atherosclerosis and HSP70,TLR4 signaling pathway and the treatment and prevention of wang’s qingjie yiqi decoction:Mortality rate:when the climate box temperature was 35℃ and 38℃,no mice died in each group,and the mortality rate was 0%.Anal temperature:after 72h in the 35℃ climate box,compared with the blank group,the elevation of anal temperature in the model group was significantly higher(***P<0.001)compared with the blank group.To compare with the model group,the rise height of anal temperature in the high-dose group and the medium-dose group was significantly decreased,with the high-dose group##P<0.01 and the medium-dose group#P<0.05.When the mice were placed in a climate box at 38℃ for 24h,the rise height of anal temperature in the model group was lower than that in the blank group significantly(**P<0.01).Body mass:after 12 weeks of modeling,compared with the blank group,the body mass of the model group,the medium dose group and the low dose group increased significantly(**P<0.01),while that of the high dose group increased significantly(#P<0.05).Oil red O staining:compared with the model group,the three TCM control groups all reduced the area ratio of plaque and vascular lumen(###P<0.001).Blood lipid:TG,TC,LDL-C and HDL-C increased significantly in the model group compared with the blank group.Compared with the model group,TG,TC,LDL-C and HDL-C in the three drug groups decreased to varying degrees.mRNA levels and protein expression:mRNA levels of HSP70 and TLR4,MyD88,NF-κB in the model group increased to varying degrees compared with the control group.Compared to the model group,mRNA levels of HSP70 and TLR4,MyD88,NF-κB were decreased in the three drug groups significantly.The results of protein expression level and mRNA level were consistent.Conclusions1.When heat stress reaches a certain intensity(such as 41℃),the mortality and anal temperature of mice were increased,and the body mass of mice was reduced.2.The effects of slowly rising heat stress model on mortality of mice was less than that of the rapidly rising heat stress model.3.shengmai powder,wang’s qingjie yiqi decoction and dendrobium shengmai powder can alleviate the effects of heat stress on death rate and anal temperature of mice to some extent,among which,wang’s qingjie yiqi decoction has the best effect,followed by dendrobium shengmai powder,and shengmai powder has a slightly worse effect than the former two.4.Heat stress-induced overexpression of HSP70 may also be a ligand of TLR4,binding to activate NF-κB through the Myd88-dependent or non-dependent pathways,promoting the synthesis and release of inflammatory factors,thereby promoting the rupture and thrombosis of atherosclerotic plaques.5.Wang’s qingjie yiqi decoction can inhibit the overexpression of HSP70,negatively regulate TLR4/MyD88/NF-κB signaling pathway,reduce inflammatory response,prevent and cure the cardiovascular damage of ApoE-/-mice caused by high temperature,reduce atherosclerosis,and stabilize the vulnerable plaque and inflammation of ApoE-/-mice.