| Purpose:In this experiment,salt sensitive rats were used as animal models.Under the guidance of the theory of prevention of disease in traditional Chinese medicine,the effects of electroacupuncture at"Zusanli"（ST36）and"Quchi"（LI11）on blood pressure and renal function of rats with normal high blood pressure were explored,and the regulation of Electroacupuncture on water metabolism and hypotension mechanism of rats with normal high blood pressure were discussed through the water regulation mediated by"AVP-V2R-c AMP-PKA-AQP2"pathway,To provide theoretical support for the prevention and treatment of hypertension and target organ injury with traditional Chinese medicine.Material and method:Eight male SPF grade 8-week-old salt resistant rats（DR）were included in the blank group;24 male SPF 8-week-old salt sensitive rats（DS）were randomly divided into model group,acupuncture group and non acupoint group,with 8 rats in each group.After one week of adaptive feeding with ordinary feed,each group was fed with 8%high salt feed for 4 weeks.During this period,the tail artery pressure of rats in each group was measured by intelligent noninvasive sphygmomanometer,and the average value was taken for 3 times.The model was evaluated after 4 weeks.The rats with successful model evaluation were treated with electroacupuncture for 4 weeks.During the treatment,rats in each group were bound and fixed in the same way.Blank group:no intervention.Model group:high salt induced normal high blood pressure rat model was prepared.Acupuncture group:take the acupoints of ST36 and LI11 on both sides of the rats for acupuncture,and connect the electroacupuncture instrument.The stimulation parameters:density wave,2Hz.The current should be a slight twitch（1-2ma）of the lower limbs of the rats,once a day,20minutes each time,6 times a week for 4 weeks.The rats in the non acupoint group were acupunctured 0.5cm from the tail to the tail root,and connected with the electroacupuncture instrument.The stimulation parameters were density wave,2Hz.The current was suitable for the slight twitch（1-2ma）of the lower limbs of the rats,once a day,20 minutes each time,6times a week for 4 weeks.During the treatment period,the changes of blood pressure of rats in each group were monitored by intelligent noninvasive sphygmomanometer from 8 to 11a.m.,and the mean value was taken for three consecutive measurements,which were recorded and counted.After treatment,the rats were anesthetized intraperitoneally with 10%chloral hydrate,fixed,disinfected and depilated.The abdominal aorta was exposed,and the blood was taken.After centrifugation,the supernatant was taken and frozen in a sterile EP tube for standby.The levels of serum creatinine（CR）,blood urea nitrogen（BUN）,serum sodium,potassium and chloride were measured by automatic biochemical instrument;The contents of ET-1,AVP in serum and AngⅡin renal tissue were measured by enzyme-linked immunosorbent assay.After blood collection,both kidneys of rats were quickly removed,and some tissues were rinsed and fixed with 4%paraformaldehyde solution.Hematoxylin eosin staining method was used to repair,embed,slice and slice.The pathological changes of renal glomerulus and renal tubules were observed under optical microscope;Some tissues were immersed in 10%formalin solution and fixed for immunohistochemical detection of AQP1and AQP2 proteins in renal tissue.Take the kidney tissue,add PBS buffer,grind it fully on the mortar,centrifuge,take the supernatant,extract the protein from the kidney tissue,measure the concentration of the extracted protein with BCA kit,detect the protein with Wes all-in-one machine,check the results with compass for SW software,calculate the gray value with PS software,and quantitatively analyze the protein expression of AQP1,AQP2,camp and PKA in the kidney of rats in each group.Take the kidney tissue and extract the m RNA after full grinding.Reverse and amplify V2R,c AMP,PKA and AQP2 by RT-PCR.The data were counted by 2-△△CTmethod.The experimental data were analyzed and compared by statistical software SPSS 26.0,and the measurement data were expressed in the form of mean±standard deviation.The measurement data meet the normal distribution,and the comparison between groups uses one-way ANOVA.The homogeneous variance is tested by the least significant difference method,and the uneven variance is tested by the Kruskar Wallis rank sum test.P<0.05 indicates that the difference is statistically significant,and P<0.01indicates that the difference is statistically significant.Results:1.Blood pressure changes of rats before and after treatment:before treatment,the systolic and diastolic blood pressure of model group,acupuncture group and non acupoint group were significantly higher than that of blank group（P<0.001）,suggesting that the model was successful,but there was no significant difference in blood pressure among the three groups（P>0.05）;After treatment,the systolic and diastolic blood pressure of the acupuncture group were significantly lower than those of the model group and the non acupoint group（P<0.001）,indicating that acupuncture can reduce the blood pressure of salt sensitive rats;Compared with the acupuncture group,the systolic and diastolic blood pressure in the non acupoint group were significantly higher（P<0.01）.2.Comparison of serum ion levels of rats in each group:the blood biochemical results showed that there was no significant difference in serum potassium ion levels of rats in each group（P>0.05）.Compared with the blank group,the level of serum sodium ion in the model group and non acupoint group was significantly higher（P<0.01）;Compared with the model group,the content of sodium ion in the acupuncture group decreased significantly（P<0.01）;Compared with the acupuncture group,the level of sodium ion in the non acupoint group was significantly higher（P<0.05）.Compared with the blank group,the level of serum chloride ion in the model group and non acupoint group was significantly higher（P<0.01）;Compared with the model group,the level of chloride ion in the acupuncture group decreased significantly（P<0.01）;Compared with the acupuncture group,the level of chloride ion in the non acupoint group was significantly higher（P<0.01）.3.Changes of water and salt metabolism hormone levels in rats in each group:compared with the blank group,the content of renal AngⅡin the model group and non acupoint group was significantly higher,the difference was statistically significant（P<0.001）;Compared with the model group,the content of AngⅡin the kidney of the acupuncture group was significantly lower（P<0.01）;Compared with the acupuncture group,the content of renal AngⅡin the non acupoint group was significantly higher（P<0.05）.Compared with the blank group,the content of serum AVP in the model group and non acupoint group was significantly higher（P<0.001）;Compared with the model group,the content of serum AVP in the acupuncture group decreased significantly（P<0.01）;Compared with the acupuncture group,the content of serum AVP in the non acupoint group was significantly higher（P<0.05）.4.Analysis of serum endothelin-1 level of rats in each group:compared with the blank group,the content of serum ET-1 in the model group and non acupoint group was significantly higher（P<0.001）;Compared with the model group,the content of serum ET-1 in the acupuncture group decreased significantly（P<0.01）;Compared with the acupuncture group,the content of serum ET-1 in the non acupoint group was significantly higher（P<0.01）.5.Analysis of renal function and renal histomorphological changes of rats in each group:compared with the blank group,the levels of blood creatinine and blood urea nitrogen in the model group and non acupoint group were significantly higher（P<0.01）;Compared with the model group,the levels of serum creatinine and blood urea nitrogen in the acupuncture group were significantly lower（P<0.05）;Compared with the acupuncture group,the levels of blood creatinine and blood urea nitrogen in the non acupoint group were significantly higher（P<0.01）.The results of HE staining showed that there were no obvious pathological changes in the glomerulus of normal rats under light microscope,the size of renal tubular lumen was moderate,the renal tubular epithelial cells were not swollen,the nuclei were evenly arranged and did not fall off;In the model group,there were some abnormal changes in the kidney,such as compensatory dilation of renal tubules,edema of renal tubular epithelial cells,nuclear abscission,micro interstitial inflammatory cell infiltration and so on;After treatment,the edema of renal tubular epithelial cells and the abscission of nuclear cells in the acupuncture group were significantly reduced;The kidney of rats in the non acupoint group showed abnormal changes such as unclear boundary of renal tubular cells,edema and nuclear abscission of renal tubular epithelial cells,powder stained particles in the cytoplasm of cells and so on.6.The expression level of renal AQP1 in each group:compared with the blank group,the expression content of renal AQP1 protein and its gene in the model group and non acupoint group were significantly higher,and the difference was statistically significant（P<0.01）;Compared with the model group,the expression of renal AQP1 protein and its gene in the acupuncture group decreased significantly（P<0.05）;Compared with the acupuncture group,the content of renal AQP1 protein and its gene expression in the non acupoint group was significantly higher（P<0.05）.7.Gene expression of signal molecules in"V2R/c AMP/PKA/AQP2"signal pathway of rats in each group:compared with the normal group,the expression level of renal AQP2m RNA in model group,acupuncture group and non acupoint group was significantly up-regulated（P<0.01）;Compared with the model group,the expression level of renal AQP2m RNA in the acupuncture group was significantly decreased（P<0.01）;Compared with the acupuncture group,the expression level of renal AQP2 m RNA in the non acupoint group was significantly up-regulated（P<0.01）.Compared with the normal group,the expression levels of V2R,c AMP and PKA m RNA in kidney of model group and non acupoint group were significantly up-regulated（P<0.05）;Compared with the model group,the expression levels of renal V2R,c AMP and PKA m RNA in the acupuncture group were significantly decreased（P<0.05）;Compared with the acupuncture group,the expression levels of renal V2R,c AMP and PKA m RNA in the non acupoint group were significantly up-regulated（P<0.05）.8.Expression of signal molecule proteins in"c AMP/PKA/AQP2"signal pathway of rats in each group:compared with the normal group,the expression of c AMP and PKA proteins in kidney of rats in model group and non acupoint group were significantly up-regulated,and the difference was statistically significant（P<0.05）;Compared with the model group,the expression of renal c AMP and PKA protein in the acupuncture group decreased significantly（P<0.05）;Compared with the acupuncture group,the expressions of renal c AMP and PKA protein in the non acupoint group were significantly up-regulated（P<0.05）.Conclusion:1.Electroacupuncture ST36 and LI11 can reduce the blood pressure of salt sensitive normal high blood pressure rats.2.Salt sensitive hypertensive rats had pathological changes of water metabolism and water and sodium retention.Electroacupuncture at ST36 and LI11 can inhibit the high expression of renal AQP1 and AQP2,and adjust the secretion of water and salt metabolism hormones to correct the disorder of water and sodium metabolism in salt sensitive pre hypertensive rats.3.Salt sensitive normal high blood pressure rats have pathological changes such as renal function injury,edema and abscission of renal tubular epithelial cells.Electroacupuncture ST36 and LI11 can inhibit the contraction of renal arterioles and improve vascular endothelial function by down regulating the levels of ET-1 and AngⅡ,so as to protect the renal function of salt sensitive normal high blood pressure rats,Improve the abnormal pathological changes of renal histomorphology.4.Electroacupuncture ST36 and LI11 can reduce blood pressure by inhibiting the over activation of"AVP-V2R-c AMP-PKA-AQP2"pathway,reducing the reabsorption of water by renal tubules and improving the body’s water and salt metabolism. |
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