Based On APN Regulation Of AMPK/AKT/eNOS Pathway, The Effects And Mechanisms Of Banxia Baizhu Tianma Decoction On Blood Pressure In SHR Fed With High-fat Diet Were Explore

Purpose:In this study,we took SHR and 3T3-L1 adipocytes fed with high fat as the research object,and took adiponectin level and its polymerization process as the starting point,to explore whether Banxia Baizhu Tianma Decoction can mediate the expression of adiponectin and the initiation process of polymerization through the PPARγ around the aorta,further affect the AMPK/AKT/eNOS signal pathway of the aorta,promote the release of NO and play a role in regulating blood pressure.To reveal the molecular biological mechanism of Banxia Baizhu Tianma Decoction in treating hypertension with dyslipidemia.Material and method:Experiment 1: We randomly divided 48 SHRs into hypertension(SHR-ND)group,high-fat feeding hypertension(SHR-HF)group,low,medium and high dose of Banxia Baizhu Tianma Decoction(BBTD-L,M,H)group,and telmisartan(TMST)group with 8 rats in each group.Ten Wistar Kyotorats served as control(WKY-ND)group.The rats in WKY-ND group and SHR-ND group were fed with basic diet,and the other five groups were fed with high-fat diet for 8 weeks.BBTD-L group,BBTD-M group and BBTD-H group were given Banxia Baizhu Tianma Decoction and gavage at the concentrations of 2.52g/kg/d,5.04g/kg/d and 10.08g/kg/d,while TMST group was given telmisartan at the concentration of 8.4mg/kg/d.WKY-ND group,SHR-ND group and SHR-HF group were given equal volume of normal saline by gavage once a day for 12 weeks.After 12 weeks of gavage,we used an intelligent non-invasive blood pressure meter to measure the systolic and diastolic pressure of the tail artery of rats in each group,and calculate the mean arterial blood pressure.Scored the irritability of rats in each group.Measured the body mass,abdominal circumference and body length of rats,and calculate Lee’s index.Two-variable curve regression was used to analyze the influence of body mass on systolic blood pressure,diastolic blood pressure and mean arterial blood pressure of rats in each group,and the regression relationship expression was established.The contents of TG,TC,LDL-C and HDL-C in serum of rats in each group were detected by automatic biochemical analyzer;HE staining was used to observe the pathological and morphological changes of rat aorta.Experiment 2: The animal modeling and intervention methods in this part are the same as those in paper 1.BBTD-H group was selected from the intervention effect of Banxia Baizhu Tianma Decoction in paper 1 for follow-up experiment.We used HE staining to detect the pathomorphological changes of the adipose tissue around the aorta of rats in each group.The serum levels of APN,TNF-α and IL-6 were detected by ELISA;RT-qPCR was used to detect the relative expression level of adiponectin polycondensation-related factors(PPARγ,APN,DsbA-L)mRNA in the adipose tissue around the aorta of rats in each group;Western blot method was used to detect the relative expression level of adiponectin polycondensation-related factors(PPARγ,APN,DsbA-L)in the adipose tissue around the aorta of rats in each group;RT-qPCR was used to detect the relative expression level of AdipoR and APPL mRNA in the aorta of rats in each group;Western blot method was used to detect the relative protein expression level of AdipoR,APPL and AMPK/AKT/eNOS pathway related factors in the aorta of rats in each group.Experiment 3: We used IBMX solution,DEX solution and insulin solution to induce the differentiation of 3T3-L1 preadipocytes.Oil red O staining was used to observe the maturation of 3T3-L1 preadipocytes.We prepared blank serum and serum containing Banxia Baizhu Tianma Decoction.We set different concentrations(0,1.25%,2.5%,5%,10%,15%,20%,25%)of Banxia Baizhu Tianma Decoction medicated serum,and used the CCK-8method to screen the best dose concentration of BBTD medicated serum intervention cells,and set different time(0,12 h,24h,48 h,72h),and used the CCK-8 method to screen the best dose time of BBTD medicated serum intervention cells.RT-qPCR was used to detect the APN mRNA expression under the intervention of BBTD drug-containing serum with different concentrations(0,10%,15%,20%),and Western blot was used to detect the APN protein expression under the intervention of BBTD drug-containing serum with different concentrations(0,10%,15%,20%).Set T0070907 with different concentrations(0,6.25,12.5,25,50,100,200,500 μM),and use CCK-8 method to screen the best drug concentration of T0070907 intervention cells.The mature adipocytes were divided into normal control group(Control group),blank serum group(Blank group),Banxia Baizhu Tianma Decoction medicated serum group(BBTD group)and PPARγ inhibitor group(T0070907 group).The normal control group was only given complete medium culture,the blank serum group was given complete medium culture with 15% blank serum,the Banxia Baizhu Tianma decoction containing serum group was given complete medium culture with 15% Banxia Baizhu Tianma decoction containing serum,and the PPARγ inhibitor group was given T0070907 with a concentration of 100μM on the basis of the Banxia Baizhu Tianma decoction containing serum group,and the intervention lasted for 24 hours.The expression level of APN in adipocytes was detected by immunofluorescence method,and the relative expression level of PPARγ,APN,DsbA-L,ERp44,Ero1-L mRNA and protein in adipocytes was detected by RT-qPCR and Western blot.Results:Experiment 1:1.Changes in blood pressure: Compared with WKY-ND group,the levels of systolic blood pressure,diastolic blood pressure and mean arterial blood pressure in SHR-ND group and SHR-HF group were significantly higher(P<0.01);Compared with SHR-ND group,systolic blood pressure,diastolic blood pressure and mean arterial blood pressure in SHR-HF group were increased(P<0.05,P<0.01);Compared with SHR-HF group,the levels of systolic blood pressure,diastolic blood pressure and mean arterial blood pressure in BBTD-L group,BBTD-M group and BBTD-H group decreased significantly(P<0.01).2.Change of irritability score: the irritability score of WKY-ND group was mainly 0,a total of 9;The irritability score of SHR-ND group increased significantly by 2 and 3 points,respectively 5 and 2;The irritability score of SHR-HF group was mainly 3 points,a total of 6animals.After the intervention of Banxia Baizhu Tianma Decoction,the irritability score decreased to different degrees.3.Changes in body mass,abdominal circumference and Lee’s index: compared with WKY-ND group,the body mass,abdominal circumference and Lee’s index of SHR-ND group showed a downward trend,but the difference was not statistically significant(P>0.05);Compared with SHR-ND group,the body mass,abdominal circumference and Lee’s index of SHR-HF group increased significantly(P<0.01);Compared with SHR-HF group,the body mass,abdominal circumference and Lee’s index of BBTD-M group and BBTD-H group decreased(P<0.01,P<0.05),while the body mass,abdominal circumference and Lee’s index of BBTD-L group showed a downward trend,but the difference was not statistically significant(P>0.05).4.Regression analysis of body mass and blood pressure: The body mass of SHR was positively correlated with the changes of systolic blood pressure,diastolic blood pressure and mean arterial blood pressure.The relationship between body mass and systolic blood pressure was Y=-92.57+0.72 * X(R=0.664,R2=0.441,P<0.01);Between body mass and diastolic blood pressure Y=-65.43+0.52 * X(R=0.579,R2=0.336,P<0.01);Between body mass and mean arterial blood pressure Y=-76.61+0.60 * X(R=0.634,R2=0.402,P<0.01).5.Changes in serum lipid level: compared with SHR-ND group,the levels of TG,TC and LDL-C in serum of SHR-HF group increased(P<0.05,P<0.01),while the level of HDL-C decreased significantly(P<0.01);Compared with SHR-HF group,the levels of TG,TC and LDL-C in BBTD-L group,BBTD-M group and BBTD-H group decreased significantly(P.6.Pathological and morphological changes of aorta: compared with WKY-ND group,the intima of aorta in SHR-ND and SHR-HF group was rough,the endothelial cells swelled and even fell off,the elastic fibers and smooth muscle cells in the middle membrane were disordered and proliferated,and some smooth muscle cells were watery degeneration;Compared with SHR-ND group,the inner membrane edge of SHR-HF group was rougher,the elastic fibers and smooth muscle cells in the middle membrane were more disordered and proliferated significantly,and the elastic fibers were indistinct;Compared with SHR-HF group,BBTD-L,M,H groups had more complete aortic intimal margin,reduced endothelial cell swelling and shedding,and restored the disorder and proliferation of elastic fibers and smooth muscle cells in the middle membrane.Experiment 2:1.Pathological changes of adipose tissue around the aorta: The adipose tissue around the aorta in WKY-ND group and SHR-ND group was evenly stained,the adipocytes were round or oval,the nuclei were oblate,and the size of adipocytes was relatively uniform.Compared with SHR-ND group,the diameter of single cell of adipocytes in SHR-HF group increased,the area of fat vacuoles increased significantly,the nuclei were squeezed to one side,and the arrangement was more chaotic.2.Changes of serum APN,TNF-α,IL-6 and NO levels: Compared with WKY-ND group,the levels of APN and NO in SHR-ND and SHR-HF groups decreased(P<0.05,P<0.01),and the levels of TNF-α and IL-6 water increased on average(P<0.05,P<0.01);Compared with SHR-ND group,the levels of APN and NO in SHR-HF group were significantly decreased(P<0.01),and the levels of TNF-α and IL-6 were increased on average(P<0.05,P<0.01);Compared with SHR-HF group,the levels of APN and NO in BBTD group were significantly increased(P<0.01),while the levels of TNF-α and IL-6 were decreased on average(P<0.05,P<0.01).3.Changes of adiponectin polymer-related factors in peri-aortic adipose tissue: Compared with WKY-ND group,the mRNA expression levels of APN,PPAR,and DsbA-L in SHR-ND group decreased(P<0.05,P<0.01),and the protein expression of APN,PPARγ,and DsbA-L showed a downward trend,in which the expression of APN protein changed significantly(P<0.01);The mRNA and protein expression of APN,PPARγ and DsbA-L in SHR-HF group decreased significantly(P<0.01).Compared with SHR-ND group,the mRNA expression of APN,PPARγ and DsbA-L in SHR-HF group decreased(P<0.05,P<0.01),and the protein expression of APN,PPARγ and DsbA-L showed a downward trend,in which PPARγ and DsbA-L changed significantly(P<0.05,P<0.01).Compared with SHR-HF group,the mRNA expression of APN,PPARγ,and DsbA-L in BBTD group increased(P<0.05,P<0.01),and the protein expression of APN,PPARγ,and DsbA-L showed an upward trend,with significant changes in APN and PPARγ(P<0.05,P<0.01).4.Changes in the expression of AdipoR and APPL in aorta: Compared with WKY-ND group,the expression of AdipoR and APPL mRNA in SHR-ND group decreased significantly(P<0.01),and the expression of AdipoR and APPL protein showed a downward trend,of which the change of APPL was significant(P<0.01);The expression of AdipoR and APPL mRNA and protein in SHR-HF group decreased significantly(P<0.01);Compared with SHR-ND group,the expression of AdipoR mRNA and protein in SHR-HF group decreased significantly(P<0.01),and the expression of APPL mRNA and protein showed a downward trend,but the difference was not statistically significant(P>0.05);Compared with SHR-HF group,the expression of AdipoR and APPL mRNA in BBTD group increased(P<0.05),the expression of APPL protein increased significantly(P<0.01),and the expression of AdipoR protein increased,but the difference was not statistically significant(P>0.05).5.Changes of AMPK/AKT/eNOS pathway in aorta: Compared with WKY-ND group,the expression of p-AMPK,p-AKT and p-eNOS protein in SHR-ND group showed a downward trend,in which p-AKT and p-eNOS protein changed significantly(P<0.01),while the expression of p-AMPK,p-AKT and p-eNOS protein in SHR-HF group decreased significantly(P<0.01);Compared with SHR-ND group,the expression of p-AMPK,p-AKT and p-eNOS protein in SHR-HF group showed a downward trend,in which p-AKT decreased significantly(P<0.05);Compared with SHR-HF group,the expression of p-AMPK,p-AKT and p-eNOS protein in BBTD group showed an upward trend,and the changes of p-AKT and p-eNOS were significant(P<0.05,P<0.01).Experiment 3:1.Oil red O staining: 3T3-L1 preadipocytes before induction of differentiation were mostly spindle or rhomboid fibroblasts;After induction of differentiation,more than 90% of cells have a large number of red lipid droplets in their cells,and some small lipid droplets fuse into larger lipid droplets.2.The best intervention concentration of BBTD medicated serum: compared with the control group,the activity of adipocytes increased significantly when the concentration of BBTD medicated serum was 10% and 15%(P<0.01),and decreased significantly when the concentration of BBTD medicated serum was 25%(P<0.01).3.The best intervention time of BBTD medicated serum: after 12 h and 24 h of BBTD medicated serum intervention,the activity of adipocytes showed an upward trend;Compared with 24 h,the cell viability showed a downward trend after 48 h and 72 h of BBTD drug-containing serum intervention,but the difference was not statistically significant(P>0.05).4.The expression of APN in BBTD drug-containing serum of different concentrations:compared with the control group,the expression of APN mRNA and protein in BBTD drug-containing serum of 10%,15% and 20% concentrations increased after intervention(P<0.05,P<0.01);Compared with 15% BBTD serum group,the APN mRNA and protein expression of 20% BBTD serum group decreased(P<0.05,P<0.01).5.Changes of adiponectin in each group: Compared with the control group,the expression of APN mRNA and protein in the Blank group had no significant difference(P>0.05);Compared with Blank group,the expression of mRNA and protein in BBTD group increased(P<0.05,P<0.01);Compared with BBTD group,the expression of APN mRNA and protein in T0070907 group decreased significantly(P<0.01).Immunofluorescence detection showed the same trend.6.Changes of adiponectin polycondensation-related factors in each group: Compared with the control group,the expression of DsbA-L,ERp44,Ero1-L mRNA and protein in Blank group did not differ significantly(P>0.05);Compared with Blank group,the expression of DsbA-L,Ero1-L mRNA and protein in BBTD group increased(P<0.05,P<0.01),and the expression of ERp44 mRNA decreased significantly(P<0.01).The expression of ERp44 protein showed a downward trend,but the difference was not statistically significant(P>0.05);Compared with BBTD group,the expression of APN,DsbA-L,Ero1-L mRNA and protein in T0070907 group decreased significantly(P<0.01),while the expression of ERp44 mRNA and protein increased(P<0.05,P<0.01).Conclusion:1.Banxia Baizhu Tianma Decoction can reduce the blood pressure and body mass of SHR fed with high fat,regulate the disorder of blood lipids,and improve the dysfunction of adipose tissue around the aorta.2.Banxia Baizhu Tianma Decoction can regulate the AMPK/AKT/eNOS signal pathway of the aorta,stimulate the release of NO,and play a role in regulating blood pressure by influencing the expression of PPARγ in the adipose tissue around the aorta,mediating the level of adiponectin and the process of aggregation.