| Purpose:To observe the intervention effect of moxibustion paste with traditional Chinese medicine at "Pishu" combined with electroacupuncture at "Quchi" and "Zusanli" on the spontaneous hypertensive rats and the effect of MAPKs pathway related proteins,so as to clarify that the mechanism of moxibustion paste combined with electroacupuncture on the spontaneous hypertensive rats was revealed from the MAPKs pathway and theory of preventive treatment of disease.We hope to provide some experimental basis for clinical treatment of hypertension from spleen.Material and method:1.Experimental animalsThirty-two male 14 week old spontaneous hypertensive rats(SHR)and eight male 14 week old Wister-Kyoto(WKY)rats,SPF grade,weighing 220g～250g.(WKY)rats were the normal group,and SHR were randomly divided into 4 groups:the model group,the MP and EA group,the MP group and the EA group,n=8 rats in each group.2.Intervention methodsThe MP and EA group:rats were fixed by fixator and electroacupunctured at“Quchi”and“Zusanli”.The disperse-dense wave with frequency of 4 Hz/20Hz was used and lasted for 20 min.Treatments were repeated daily for 15 days.Rats were also treated by moxibustion paste at"Pishu.Treatment was lasted for 6 hours daily for 15 days.The EA group:rats were fixed by fixator and electroacupunctured at“Quchi”and"Zusanli".The disperse-dense wave with frequency of 4 Hz/20Hz was used and lasted for 20 min.Treatments were repeated daily for 15 days.The MP group:rats were fixed by fixator and treated by moxibustion paste at "Pishu".Treatment was lasted for 6 hours daily for 15 days.The normal group and the model group:rats were fixed by fixator daily for 15 days,but did not receive any treatment.3.Moxibustion paste medicineGastrodia elata,Achyranthes bidentata,Uncaria rhynchophylla,Concha haliotidis,Fructus Gardeniae,Eucommia ulmoides,Scutellaria baicalensis,Leonurus heterophyllus,Parasitic loranthus,Caulis polygoni multiflori,Poria with hostwood and Ginger-juice.4.Detection Indicators(1)Measurement of blood pressure of rat tail artery(systolic and diastolic pressure):the systolic and diastolic blood pressure were measured on the first day before the experiment and the first,third,fifth,seventh,ninth,eleventh and thirteenth days after the experiment.(2)The levels of ET-1 and NO in rats serum were detected by ELISA.(3)HE staining of rat heart tissue:to observe the pathological changes of myocardial tissue in rats in each group.(4)The left ventricular structure and function were evaluated by echocardiography.(5)Western blot was used to determine the expression of p38-MAPK、p-p38-MAPK、ERK1/2、p-ERK1/2、JNK、p-JNK proteins in heart tissue of rats.(6)The expression of p-p38-MAPK、p-ERK1/2、p-JNK proteins in heart tissue of rats was observed by immunohistochemistry.(7)qPCR was used to determine the expression of p38-MAPK and ERK1/2、JNK mRNA in heart tissue of rats.5.Statistical processingAll data was analysed by SPSS 22.0.The measured data indicated(x±s)that among the groups using single factor analysis of variance?and the comparison among groups was performed with LSD method.The differences of P<0.05 were statistically significant.Results:1.Analysis of blood pressure of rats in each group before and after treatmen:Before the experiment,the blood pressure pressure of the model group,the MP and EA group,the MP group and the EA group was significantly higher than that of the normal group.The blood pressure did not change significantly in the first 3 days.On the fifth day of the experiment,the blood pressure of the MP and EA group,the MP group and the EA group was lower than that of the model group,and the MP group and the EA group was the most obvious.On the seventh day of the experiment,the blood pressure of the MP and EA group,the MP group and the EA group continued to drop,and the MP group and the EA group was significantly lower than that of the MP group and the EA group.2.Comparison of ET-1 and NO in rats serum of rats in each groupCompared with the normal group,the level of ET-1 in other four groups was significantly increased and the level of NO was significantly decreased(P<0.05).After the intervention treatments,the level of ET-1 in the MP and EA group,the MP group and the EA group was lower than that of the model group,and the level of NO was increased significantly(P<0.05).Compared with the MP and EA group,ET-1 of the MP group and the EA group increased,while NO decreased(P<0.05).3.Pathological changes of myocardial tissue in rats in each groupCompared with the normal group,the pathological changes of myocardial tissue in the model group were obvious hypertrophy,disordered arrangement of muscle fibers,obvious infiltration of inflammatory cells and thickening of vascular wall.Compared with the model group,the arrangement of myocardial fibers in the MP and EA group was more orderly,the number of inflammatory cells was significantly reduced,and the thickening of vascular wall was significantly improved.In the EA group,the arrangement oof muscle fibers was disordered,the vascular wall was slightly thickened,and the infiltration of inflammatory cells was slightly improved.There was no obvious improvement in the MP group.4.Changes of left ventricular structure and function in rats of each groupCompared withthe normal group,the HW/BW and LVWI of other groups were significantly increased(P<0.05),which clarified that SHR had myocardial hypertrophy.The increase of HW/BW and LVWI of the MP and EA group and the EA group could be significantly inhibited(P<0.05).The results showed that the MP and EA group and the EA group could effectively inhibit myocardial hypertrophy of rats,and the effect of the MP and EA group was more obvious.The LVEF of the model group was significantly lower(P<0.05),which suggested that the cardiac function of SHR was weakened.Compared with the model group,the LVEF of the MP and EA group and the EA group were increased(P<0.05),and the effect of the MP and EA group was more obvious.5.Changes of p38 MAPK and p-p38 MAPK protein expression in heart tissue of rats in each groupCompared with the normal group,the protein expression of p38 MAPK and p-p38 MAPK in other four groups was significantly increased(P<0.05).The protein expressions of p38 MAPK and p-p38 MAPK in the MP and EA group and the EA group were significantly lower than those in model group(P<0.05),but there were no significant differences between with the MP group and the model group.The protein expression of p38 MAPK and p-p38 MAPK of the MP and EA group was lower than that of the EA group and the MP group(P<0.05).6.Changes of ERK1/2 and p-ERK1/2 protein expression in heart tissue of rats in each groupCompared with the normal group,the protein expression of ERK1/2 and p-ERK1/2 in other four groups was significantly increased(P<0.05).The protein expressions of p-ERK1/2 in the MP and EA group,the EA group and the MP group were significantly lower than those in model group(P<0.05),but there were no significant differences of ERK1/2 between with the MP group and the model group.The protein expression of ERK1/2 and p-ERK1/2 of the MP and EA group was lower than that of the EA group and the MP group(P<0.05).7.Changes of JNK and p-JNK protein expression in heart tissue of rats in each groupCompared with the normal group,the protein expression of JNK and p-JNK in other four groups was significantly increased(P<0.05).There were no significant differences of JNK and p-JNK in the MP and EA group,the EA group and the MP group.8.The positive density of p-p38-MAPK,p-ERK1/2 and p-JNK protein expression in heart tissue of rats in each groupCompared with the normal group,the positive density of p-p38 MAPK,p-ERK1/2 and p-JNK protein expression in other four groups was significantly increased(P<0.05).The positive density of p-p38 MAPK protein expressions in the MP and EA group and the EA group were significantly lower than those in model group(P<0.05),but there were no significant differences between with the MP group and the model group.The positive density of p-ERK1/2 protein expressions in the MP and EA group,the MP group and the EA group were significantly lower than those in model group(P<0.05).The positive density of p-p38 MAPK protein expressions in the MP and EA group was lower than that in the EA group and the MP group(P<0.05).There were no significant differences of p-ERK1/2 in the MP and EA group,the EA group and the MP group.There were no significant differences of p-JNK in model group,the MP and EA group,the EA group and the MP group.9.Changes of p38 MAPK mRNA expression in heart tissue of rats in each groupCompared with the normal group,the mRNA expression of p38 MAPK in other four groups was significantly increased(P<0.05).The mRNA expressions of p38 MAPK in the MP and EA group and the EA group was significantly lower than that in model group(P<0.05),but there were no significant differences between with the MP group and the model group.The mRNA expression of p38 MAPK of the MP and EA group was lower than that of the EA group and the MP group(P<0.05).10.Changes of ERK1/2 mRNA expression in heart tissue of rats in each groupCompared with the normal group,the mRNA expression of ERK1/2 in other four groups was significantly increased(P<0.05).The mRNA expressions of ERK1/2 in the MP and EA group,the EA group and the MP group was significantly lower than that in model group(P<0.05).The mRNA expression of ERK1/2 of the MP and EA group was lower than that of the EA group and the MP group(P<0.05).11.Changes of JNK mRNA expression in heart tissue of rats in each groupCompared with the normal group,the mRNA expression of JNK in other four groups was significantly increased(P<0.05).There were no significant differences of JNK mRNA in the MP and EA group,the EA group and the MP group.Conclusion:1.The blood pressure of early stage of spontaneous hypertensive rats could be significantly reduced by moxibustion paste combined with electroacupuncture.2.The effect of early stage of spontaneous hypertension on target organs could be reduced by moxibustion paste combined with electroacupuncture.3.The damage of vascular endothelial cells in early stage of spontaneous hypertension could be reduced by moxibustion paste combined with electroacupuncture.4.The mechanism of moxibustion paste combined with electroacupuncture on early stage of spontaneous hypertensive rats was revealed from the MAPKs pathway.The intervention effect of moxibustion paste combined with electroacupuncture on early stage of spontaneous hypertensive rats may be reducing the phosphorylation of p38 MAPK and ERK,inhibiting the activation of p38 MAPK and ERK,and effectively inhibiting the activation of related pathway. |
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