Study On Antihypertensive Effect And Mechanism Of Compound Gaoziban Tablet

Objective:Uyghur medicine and pharmacy are important components of traditional medicine in our country.Uyghur medicine believes that hypertension is caused by the imbalance of four body fluids in the human body.Therefore,adjustment,maturation and elimination therapies are adopted to improve abnormal body fluids in the body to treat hypertension.Clinical studies have shown that the compound gaoziban tablet（CGT）can effectively treat hypertension caused by bile substantia abnormality,but there is no research report on its pharmacodynamics,and its mechanism of action and effective material basis are still unclear.In this thesis,the volatile components in the CGT and the main medicinal flavour bugloss and bugloss flower were analyzed by headspace-solid phase microextraction-gas chromatography-mass spectrometry（HS-SPME-GC-MS）.The CGT was explored through the spontaneously hypertensive rat model.The study on the pharmacodynamics of CGT and its mechanism of improving kidney injury provides theoretical and experimental basis for the treatment of hypertension with CGT.Methods:Analyze the volatile components in CGT and bugloss and bugloss flowers by HS-SPME-GC-MS technology,and explore its potential effective substance basis.Forty 12-week-old spontaneous hypertension rats（SHR）were taken as the disease model animal study object and randomly divided into five groups,including disease model group,positive drug（Metoprolol tartrate tablet）group,CGT high dose group,CGT middle dose group and CGT low dose group.Another normal group is 8 Wistar-Kyoto（WKY）rats of the same age.The drug distilled water(10 m L·kg^-1)were orally administered continuously for 9 weeks.Systolic blood pressure was measured at the same time point weekly.After 9 weeks of administration,the rats in each group were sacrificed and samples were taken.The serum levels of triglyceride（TG）,total cholesterol（TC）,glucose（Glu）,high-density lipoprotein cholesterol（HDL-C）and low density lipoprotein cholesterol（LDL-C）were detected in blood lipid levels,while also detecting alanine aminotransferase（ALT）,aspartate aminotransferase（AST）,glutathione S-transferase（GST）liver function level.The level of renin（Renin）,angiotensinⅡ（AngⅡ）,aldosterone（ALD）and endothelin 1（ET-1）were analyzed via ELISA.The pathological changes of organ tissues were observed by hematoxylin-eosin（HE）staining and masson staining and statistically analyze the collagen area.RT-PCR method for detection of m RNA relative expression of angiotensin converting enzyme（ACE）,angiotensinⅡtype 1 receptor（AT1R）,endothelial nitric oxide synthase（e NOS）,phosphoinositide3-kinase（PI3K）and protein kinase B（AKT）.Western blotting was used to detect the relative expression levels of PI3K,AKT,and p-AKT（Phospho-protein kinase B）in rat kidneys.Results:1.The HS-SPME-GC-MS technology combined with database matching was used to identify the volatile components in CGT,bugloss and buglo ss flowers.Among them,a total of 39 volatile components have been i dentified in CGT.The higher content components are cis-α-Santalol acco unting for 15.29%,β-Santalol accounting for 10.49%,trans-α-Bergamotol accounting for 8.42%,（E）-Nuciferol accounted for 6.96%and Geranylg eraniol accounted for 4.80%.A total of 31 volatile components were ide ntified in bugloss.The higher content components are Heptacosane for 14.06%,Phytone for 13.62%,2,6,10-trimethyltetradecane for 6.71%,and c is-α-Santalol accounts for 6.39%.A total of 27 volatile components were identified in the flowers of bugloss.The higher content components are Tricosane for 25.96%,cis-α-Santalol for 21.37%,trans-β-Santalol for 8.41%,Phyton for 6.08%and trans-α-Bergamotol accounted for 5.92%.2.The blood pressure results showed that compared with the disease model group,the systolic blood pressure of the rats in the low dose group decreased significantly after 2 weeks of administration of the CGT（P<0.05）.After gavage for 9 weeks,the systolic blood pressure of the rats in the high dose group and the middle dose group decreased significantly（P<0.05）.The blood lipid level in the rat serum was detected,and compared with the disease model group,the levels of TG and HDL-C in the rat serum of the CGT high-dose group and the middle-dose group had a significant reduction effect（P<0.05）,but it had no significant effect on Glu,TC and LDL-C in the rat serum（P>0.05）.Transaminase levels in serum samples were also evaluated.Compared with the disease model group,CGT has a different lowering effect on the level of AST,ALT and GST in serum samples of rats（P<0.05,P<0.01）.Histopathological observations show that,compared with the disease model group,CGT can effectively improve the morphology of glomeruli and renal tubules in spontaneously hypertensive rats,reduce the infiltration of inflammatory cells,and make the morphology closer to the normal group.At the same time,CGT can also reduce the apoptosis of myocardial cells,reduce the breakage and proliferation of myocardial fibers,and improve the damage of myocardial cells.3.The levels of Renin,AngⅡ,ALD and ET-1 in rat serum were detected by ELISA.The results showed that compared with the disease model group,after treatment with CGT,both the high dose group and the middle dose group could Significantly reduce the content of Renin,AngⅡ,ALD and ET-1 in rat serum（P<0.05）.RT-PCR results showed that compared with the disease model group,the ACE m RNA and AT1R m RNA in the kidney tissue of the CGT middle dose group and the low dose group were down-regulated（P<0.05）,and e NOS m RNA was significantly up-regulated（P<0.05）.4.MASSON staining results showed that compared with the disease model group,after CGT administration,the collagen fiber area in the kidney of the high dose group and the middle dose group was significantly reduced（P<0.05）,and the collagen fiber surface of the middle dose group was significantly reduced（P<0.01）,which was close to the normal group.Western blot results showed that,compared with the normal group,the expression of PI3K protein and p-AKT protein in the kidney of the model group were significantly reduced（P<0.05）.After CGT treatment,the expressions of PI3K protein and p-AKT protein in the kidneys of rats in the high dose group,middle dose group and low dose group were all up-regulated（P<0.05）,and they were dose-dependent.The results of RT-PCR showed that,compared with the normal group,the expression of PI3K m RNA and AKT m RNA in the kidney of the disease model group was significantly reduced（P<0.05）.After CGT treatment,the expressions of PI3K m RNA and AKT m RNA in the kidneys of rats in the high dose,middle dose and low dose groups were all up-regulated（P<0.05）,and they were dose-dependent.Conclusions:1.Most of the medicinal flavors in the CGT are aromatic medicinal materials,and its volatile components are mainly sandalol,and also the main volatile components in bugloss,bugloss flower and sandalwood.Pharmacological research shows that sandalwood Aroma compounds have good anti-inflammatory and sedative effects;β-bisabolol and trans-α-bergamot are the volatile components in bugloss flowers,among whichβ-bisabolol has anti-inflammatory and sedative effects;ten Pentaxane andα-cedarene are all derived from bugloss;α-curcumene can be attributed to bugloss and sandalwood,and has a good antioxidant effect;cis-β-farnesene and oxycaryophyllene come from lavender Medium;Cooperol is a volatile component in Pandan;α-santalene can be attributed to sandalwood and lavender;Geranylgeraniol can resist thrombosis and atherosclerosis.From the above analysis results,it can be preliminarily determined that the volatile component is one of the main medicinal substances in the CGT.2.CGT can reduce the systolic blood pressure of spontaneously hypertensive rats to a certain extent.Although the blood pressure of rats cannot reach the normal level,after long-term medication,the systolic blood pressure of SHR can be maintained at a low level,and the effect of stabilizing is obvious.CGT has better liver protection function.Comprehensive HE staining results can clearly show that CGT can significantly improve the kidney and heart damage caused by hypertension,effectively regulate the occurrence and development of hypertension,and prevent and repair the organic damage of the heart and kidney.3.CGT can effectively inhibit the excessive activation of RAAS（Renin-angiotensin-aldosterone system）,and reduce the excitability of sympathetic nerves by reducing the content of Renin,AngⅡ,and ALD.At the same time,the decrease in the content of ET-1 strengthens the expansion capacity of blood vessels and reduces the pressure around the blood vessels,thereby achieving the effect of lowering blood pressure.PCR results showed that CGT can down-regulate the expression of ACE and AT1R m RNA,and up-regulate the expression of e NOS m RNA,which also inhibits the excessive activation of RAAS through gene regulation.4.CGT can significantly reduce the area of blue collagen fibers in the rat kidney,and avoid renal fibrosis caused by the aggravation of renal injury.Both WB and PCR test results showed that CGT can activate the cell survival and proliferation pathway PI3K/AKT,and significantly up-regulate the expression of PI3K and AKT,thereby improving kidney damage and greatly slowing down the process of renal fibrosis.