Study On The Mechanism Of Electroacupuncture Combined With Chinese Medicine Moxibustion In Delaying Myocardial Fibrosis In Prehypertensive Rat

Purpose:Using the method of syndrome differentiation and analysis of traditional Chinese medicine syndrome elements,this paper analyzes the syndromes and syndrome elements of patients with pre hypertension,and summarizes their common syndrome types.According to the syndrome types of prehypertension,which are mainly spleen deficiency and phlegm dampness syndrome,electroacupuncture combined with traditional Chinese medicine moxibustion was used to observe its effect of delaying myocardial fibrosis in prehypertension rats,with Sirt1 / TLR4 / NF-κ B signal pathway as the starting point,in order to try to explain the intervention mechanism of electroacupuncture combined with traditional Chinese medicine moxibustion on prehypertensive rats,and enrich the scientific connotation of "treating pre hypertension from the spleen".Materials and methods:1 Clinical research:This study uses the means of retrospective investigation and research,extracts the information of cases and medical records,and combines the form of questionnaire survey to design and establish the information collection table of traditional Chinese medicine syndrome characteristics of patients with prehypertension,so as to explore the distribution of traditional Chinese medicine syndrome of patients with prehypertension.According to the inclusion,exclusion and exclusion criteria,the general data,syndrome manifestations,tongue and pulse and other four diagnostic information of patients with prehypertension were collected by spss19.0 establish a database,use frequency analysis to summarize the four diagnosis information,extract the main disease location and disease syndrome elements by factor analysis,integrate the disease location and disease syndrome elements in combination with the basic theory of traditional Chinese medicine,summarize the main syndrome types and distribution rules in the prehypertension,and further explore the main etiology,pathogenesis,treatment principles and methods in the prehypertension.2 Experimental research:Thirty two 7-week-old salt sensitive rats were randomly divided into four groups with 8rats in each group,namely model group,acupuncture group,traditional Chinese medicine acupoint application group(hereinafter referred to as application group),acupuncture +traditional Chinese medicine acupoint application group(hereinafter referred to as acupuncture + application group).Eight 7-week-old salt resistant rats were the blank group.Rats in each group were fed adaptively in the animal experimental center for 1 week.The rats were fed with 8% high salt diet and the tail artery pressure was monitored.When the blood pressure reached 130 ～ 139 mm Hg and / or 85 ～ 89 mm Hg,the model was successfully established in the prehypertension.The rats in the blank group and the model group were bound with the fixator every day for 20 minutes,and the rats in the acupuncture group were bound with the fixator every day.The acupuncture were inserted into the "Zusanli" and "Quchi" points on both sides,and the needle handle was connected with the electroacupuncture instrument respectively.The intensity was based on the slight tremor of the local limbs of the rats(1-2 MA).The density wave was selected,the frequency was 4 / 20 Hz,and each waveform lasted 10 / 15 s respectively for 20 minutes a day,"Zusanli" is located at the posterolateral side of the rat knee joint,about 5mm below the fibular capitulum and7 mm straight;"Quchi" point is located in the depression in front of the outside of the joint at the proximal end of the radius,with a straight puncture of 4mm.In the application group,the traditional Chinese medicine particles were ground into powder and mixed with ginger juice to form a paste every day.The traditional Chinese medicine pills with a diameter of 2 mm were made and fixed on the bilateral "Pishu" points of the rats for application.Then the acupoints were applied on the traditional Chinese medicine pills.The nondrug part was isolated from the skin with a cotton pad.Finally,the rats were wrapped with adhesive tape and fixed for moxibustion every day for 6 hours,Positioning of "Pishu" point: between the lower ribs of the 12 th thoracic vertebra.In the acupuncture + application group,the rats were bound with the fixator every day,and electroacupuncture was applied to the "Zusanli" and "Quchi" points on both sides of the rats.The electroacupuncture method was the same as that in the acupuncture group.After electroacupuncture,the drug containing points were applied to the "Pishu" points on both sides of the rats,and the method was the same as that in the application group.The above intervention measures were once a day,6 days as a course of treatment,a total of 4 courses of treatment.After treatment,all rats were fasted for 24 hours,the body weight of rats in each group was measured,and 10% chloral hydrate was injected intraperitoneally into anesthetized rats according to 0.6ml/100g;After successful anesthesia,the rats were fixed in the supine position on the operating table,the chest skin was prepared,the chest skin was disinfected with 0.5% iodophor,the sternum was cut to expose the heart,part of the heart was taken and placed in a clean vessel,washed with normal saline,and the connective tissue around the heart was trimmed.The heart tissue was digested with neutral protease and collagenase to prepare a single-cell suspension,and the cell surface antigen was labeled with corresponding fluorescent antibody,The rest of the hearts were placed on ice and the excess tissues were cut off.After washing with normal saline,some of them were fixed with 4% paraformaldehyde,stored away from light at room temperature and some in-80 ℃ refrigerator.The systolic pressure of rat tail artery was measured by intelligent noninvasive sphygmomanometer,the left ventricular mass index and left ventricular ejection fraction were evaluated by echocardiography,the pathological changes of heart tissue were observed by HE staining,the volume fraction of collagen fibers in heart tissue was semi quantitatively analyzed by masson staining,the percentage of macrophages in heart tissue was detected by flow cytometry,and the IL-6 and IL-1β、TNF-α in heart tissue were measured by ELISA.The content of protein expression of Sirt1,NF-κB,TLR4,My D88,TRAF6 and IKKβ in heart tissue were detected by Wes all-in-one machine,The expression of m RNA of Sirt1,NF-κ B,TLR4,My D88,TRAF6 and IKKβ were detected by RT-PCR,The positive expression density of Sirt1 and TLR4,p-NF-κB protein in heart tissue was detected by immunohistochemistry.The experimental data were collected by spss19.0,evaluate whether the data is normal distribution,and test the variance homogeneity of the normal distribution data.The measurement data are expressed as(?)± s.The comparison between groups adopts one-way ANOVA.If the data does not conform to the normal distribution,the nonparametric test is adopted.P < 0.05 means that the difference is statistically significant.Results:1 Study on traditional Chinese medicine syndrome elements in prehypertension1.1 General information: among 151 patients with prehypertension,68 were male,accounting for 45.03%.The distribution of patients with prehypertension was different in different age groups,but most of them were 45-59 years old,with a total of 86 cases,accounting for56.95%.The patients with smoking,drinking and high salt diet were most distributed in the age group of 45-59 years old,with 31 cases,accounting for 52.54%,43 cases,accounting for64.18%,and 14 cases,accounting for 51.85%.1.2 Distribution characteristics of four diagnostic information: According to the frequency statistics,the most common symptoms of prehypertension were dizziness(64.24%),headache(54.97%),bitter mouth(54.03%),dizziness(49.01%),laziness(44.37%),light fat tongue(43.05%)and fatigue(41.06%).1.3 Distribution characteristics of syndrome elements: in the early stage of hypertension,the syndrome elements are spleen,liver and kidney,and the disease elements are phlegm dampness,Qi deficiency,heat and yin deficiency.Among them,spleen was the most common syndrome element,92 cases,accounting for 45.32%,and phlegm dampness was the most common syndrome element,92 cases,accounting for 32.97%.1.4 Syndrome distribution characteristics: prehypertension syndromes are divided into four categories: spleen deficiency and phlegm dampness,liver fire hyperactivity,yin deficiency and yang hyperactivity,qi and yin deficiency,of which the proportion of spleen deficiency and phlegm dampness syndrome is the highest,accounting for 92 cases,accounting for36.08%.2 Effect of electroacupuncture combined with traditional Chinese medicine moxibustion on delaying myocardial fibrosis in rats with prehypertension2.1 Effect of electroacupuncture combined with traditional Chinese medicine moxibustion on blood pressure in pre hypertensive ratsAfter 4 courses of treatment,the average systolic blood pressure of tail artery in each treatment group was lower than that in the model group,and the blood pressure in the acupuncture + application group decreased significantly compared with the other two groups(P < 0.05).2.2 Effect of electroacupuncture combined with traditional Chinese medicine moxibustion on myocardial fibrosis in pre hypertensive ratsCardiac ultrasound showed that the left ventricular mass index of acupuncture group,application group and acupuncture + application group decreased compared with the model group(P < 0.05).He staining showed that the arrangement of cardiomyocytes in the treatment group was more orderly than that in the model group,and the infiltration of inflammatory cells in myocardial stroma was less,which was more obvious in the acupuncture + application group.Masson staining showed that the formation of myocardial collagen fibers in the treatment group was less than that in the model group,and it was more obvious in the acupuncture + application group(P < 0.05).Immunohistochemistry showed that all groups in the treatment group The positive expression density of α-SMA was lower than that in the model group,and it was significantly higher in the acupuncture + application group(P <0.05).3 Study on the mechanism of electroacupuncture combined with traditional Chinese medicine moxibustion in delaying myocardial fibrosis in pre hypertensive rats based on Sirt1 / TLR4 /NF-κB signal pathway3.1 The proportion of macrophages in myocardial tissue of rats was detected by flow cytometryCompared with the blank group,the percentage of macrophages in the myocardial tissue of the model group increased significantly(P < 0.05).Compared with the model group,the percentage of macrophages in the myocardial tissue of the acupuncture group,the application group and the acupuncture + application group decreased(P < 0.05),and the percentage of macrophages in the acupuncture + application group decreased more significantly(P < 0.05).3.2 Detection cotent of IL-6 and IL-1β、TNF-αin rat myocardial tissue by ELISACompared with the blank group,the content of IL-6 and IL-1β、TNF-α in myocardial tissue of model group increased.Compared with the model group,the content of IL-6 and IL-1β 、 TNF-α in myocardial tissue of rats in acupuncture group and acupuncture +application group decreased(P < 0.05).The content of IL-1β decreased in the application group comparing with the model group(P < 0.05),and the decrease was more significant in the acupuncture + application group(P < 0.05).3.3 The expression levels of SIRT1,TLR4,My D88,TRAF6,NF-κB and IKKβ m RNA in rat myocardium were detected by RT-PCRCompared with the blank group,the expression levels of TLR4,My D88,TRAF6,NF-κB and IKKβ m RNA in the model group were up-regulated,and the expression level of Sirt1 m RNA decreased compared with the blank group(P < 0.05).Compared with the model group,the expression levels of TLR4,My D88,TRAF6,NF-κB and IKKβ m RNA in the treatment group decreased,and the expression level of Sirt1 m RNA increased(P < 0.05),The change of m RNA expression level in acupuncture + application group was significantly higher than that in acupuncture group and application group(P < 0.05).3.4 Wes all-in-one machine was used to detect SIRT1,TLR4,My D88,IKKβ,TRAF6 and NF-κB protein expression level in rat myocardial tissueCompared with the blank group,the expression of TLR4,My D88,TRAF6,NF-κB and IKKβ protein in the model group increased and the expression of Sirt1 protein decreased(P <0.05).Compared with the model group,the expression of TLR4,My D88,TRAF6,NF-κB and IKKβ protein in the treatment group decreased and the expression of Sirt1 protein increased(P < 0.05).The change of protein expression in acupuncture + application group was significantly higher than that in acupuncture group and application group(P < 0.05).3.5 The positive density of Sirt1,TLR4 and p-NF-κB protein expression in rat myocardium was detected by immunohistochemistryCompared with the blank group,the positive density of TLR4 and p-NF-κB protein expression in the model group increased and the positive density of Sirt1 protein expression decreased(P < 0.05).Compared with the model group,the positive density of TLR4 and p-NF-κB protein expression in the treatment group decreased and the positive density of Sirt1 protein expression increased(P < 0.05).Among them,the change of protein expression positive density in the acupuncture + application group was significantly higher than that in the acupuncture group and application group(P < 0.05).Conclusion:1 Men aged 45-59 were more common in patients with prehypertension,and patients with smoking,drinking and high salt diet were also most distributed in this age group.The most common symptoms of prehypertension are dizziness,headache,bitter mouth,dizziness,laziness,light fat tongue and fatigue.Disease location syndrome element spleen is the most common,disease nature syndrome element phlegm dampness is the most common.Pre hypertension syndrome types are divided into four categories: spleen deficiency and phlegm dampness,liver fire hyperactivity,yin deficiency and yang hyperactivity,Qi and yin deficiency,among which spleen deficiency and phlegm dampness syndrome types account for the highest proportion.2 Electroacupuncture combined with traditional Chinese medicine moxibustion can reduce the blood pressure of pre hypertensive rats,delay the process of myocardial fibrosis and improve the prognosis,which is more significant than single acupuncture or traditional Chinese medicine moxibustion.3 Electroacupuncture combined with traditional Chinese medicine moxibustion may play a role in delaying myocardial fibrosis by regulating Sirt1 / TLR4 / NF-κB signal pathway,reducing the content of macrophages in myocardial tissue and reducing the release of IL-6and IL-1β,TNF-α.