The Effect Of Angelica Sinensis Extract On SHR Blood Pressure And Target Organs And Molecular Mechanism Exploration

Purpose1.To confirm the hypotensive effect of angelica sinensis alcohol extract on SHR and the functions of myocardial remodeling and endothelial improving,and evaluate the safety.2.To explore the mechanism of angelica sinensis alcohol extract intervening SHR and protecting target organs via Th17/Treg Equilibrium.Method1.Cochrane,Web of science,PubMed,Embase,CNKI,Wanfang data and CBM were systematically searched.Researches about angelica or compounds containing angelica treating hypertension or its complications were included.EXCEL 2013 was used to integrate and process the data,and the result is showed intuitively with the bubble diagram.2.The organic acids of angelica sinensis were prepared by 70%ethanol hot reflux method and spray drying method.High performance liquid chromatography method was used to determine the content of ferulic acid in angelica sinensis alcohol extract,and the quality of angelica sinensis alcohol extract was controlled by ferulic acid content.3.The SHR model was established and randomly divided into six groups:blank group?WKY?,model group,benazepril group,angelica sinensis alcohol extract high,middle and low dose group.In the blank group,intragastric administration was applied with equal volume of saline,and the positive control group with benazepril tablets 10mg/D,once a day for 8weeks.The physiological function and blood pressure of the rats were monitored.The SHR heart cardiac morphology and function were evaluated by color Doppler,HE staining and Masson staining.The contents of NF-??,VCAM-1,ET-1,ICAM-1 and eNOS in serum were detected with ELISA,Western blot and RT-PCR methods.And the expression of RAAS system and oxidative stress components were monitored based on protein gene level.The safety of angelica extract was evaluated by liver and kidney functions.4.Based on the network pharmacology,the signal pathway of Angelica in the treatment of hypertension and target organ protection was screened.5.The SHR model was established and randomly divided into blank group?WKY?,model group,benazepril group and angelica sinensis alcohol extract high-dose group.The serum inflammatory indexes IL-6,CRP and Th17,Treg expression factors IL-17,IL-23,IL-10 and TGF?1 were detected by ELISA.The peripheral blood Th17 and Treg cells were monitored by flow cytometry and the Th17/Treg ratio was calculated.The expression of transcription factors Fox P3 and ROR?t in SHR kidney tissue were detected by qPCR.Western Blot was used to detect the expression of p38,p-p38,FoxO1,p-foxo1,SGK1 and IL-23R in SHR kidney tissue.Result1.Evidence MappingRCT studies included were analyzed,total effectiveness outcome indicators accounted for 69.4%,the effective rate is statistically different from that of the control group;the traditional Chinese medicine used in the research intervention measures is divided into 8categories,such as supplementing qi and activating blood,supplementing qi and nourishing blood,supplementing qi,nourishing yin and activating blood,most of which are activating blood and dissolving stasis formula.In the literature included,the treatment group and the control group have statistical differences in aspects of blood pressure reduction,target organ protection,adverse reactions,etc.2.Preparation and quality control of angelica sinensis alcohol extractThe alcohol extract of Angelica is brown powder.The HPLC conditions were C18column,mobile phase consisted of acetonitrile-0.1%phosphoric acid solution.Gradient elution time:0-30 min,A:95%-5%?V/V?,B:5%-95%?V/V?;detection wave length was 316 nm;the method had strong specificity and precision,and the recovery rate could meet the requirements of in vitro analysis.The average content of ferulic acid in angelica extract was?151.525±0.002?%??g/g?.3.Evaluation index and safety of Angelica sinensis alcohol extract on SHRGeneral physiology:compared with the model group,the rats in benazepril group and angelica medium and high dose alcohol extract group were active,responsive,sleep less,body hair luster,bow back performance less,weight increased;no obvious diarrhea symptoms were found in each group during the experiment.After 2,4,6 and 8 weeks of administration,the systolic and diastolic pressures of benazepril group and Angelica high-dose group were lower than those of the model group?P<0.01?.Morphology,structure and function of the heart:?1?Color Doppler ultrasound display:compared with the blank group,the LVEDD and LVESD of the heart in the model group decreased,IVSTD and LVPWTD increased significantly,EF,FS and E/A decreased?P<0.05?.Compared with the model group,in benazepril group,LVEDD and LVESD increased,EF,FS and E/A increased,IVSTD and LVPWTD increased,while in high dose angelica alcohol extract,LVEDd,LVESD,EF,FS and E/A increased significantly?P<0.05?.?2?HE staining showed that,compared with the blank group,SHR in the model group had increased cardiomyocyte volume,disordered myocardial fiber shape,deepened nuclear staining,increased intercellular space with obvious tissue collagen deposition and inflammatory cell infiltration;compared with the model group,SHR in the benazepril group and the high-dose Angelica alcohol extract group,there was no obvious infiltration of inflammatory cells and collagen deposition in the intercellular matrix.?3?Masson staining showed that,compared with the blank group,a large number of collagen fibers,widened intercellular space,loss of fusiform structure,and a large number of collagen deposition around blood vessels were found in the myocardial tissue space of the model group;compared with the model group,the myocardial fiber space and collagen deposition around blood vessels were significantly reduced in the high-dose benazepril and angelica alcohol extract group,and the fusiform of myocardial cells were significantly reduced.The structure is clearly visible.Vascular endothelial function and RAAS system index:?1?compared with the blank group,the expression of VCAM-1,ET-1,ICAM-1 protein in the model group increased,gene expression was significantly increased,the expression of eNOS protein in the oxidative stress factor decreased,and gene expression was significantly decreased?P<0.01?;compared with the model group,VCAM-1,ET-1 and ICAM-1 protein in the benazepril group and the high dose Angelica alcohol extract group Protein expression decreased,gene expression decreased,eNOS protein expression increased,gene expression increased?P<0.01?.?2?Compared with the blank group,AT₁-R protein expression increased,gene expression increased,AT₂-R protein expression decreased and gene expression decreased in the model group?P<0.05?;compared with the model group,AT₁-R protein expression decreased,gene expression decreased,AT₂-R protein expression increased and gene expression increased in the benazepril group and Angelica alcohol extract group?P<0.05?.Safety evaluation:compared with the blank group,the serum biochemical indexes ALT,AST,BUN and CREA of the experimental groups were all at the same level,without statistical difference.4.Study on the mechanism of Angelica in the treatment of hypertension by network pharmacologyIn the treatment of hypertension with angelica,it is mainly involved in biomembrane synthesis,brain gut axis development,intracellular calcium signal transduction,smooth muscle contraction,stimulation of cAMP synthesis,lipopolysaccharide response,G protein coupled signal transduction and other biological processes;MAPK/SGK1/FoxO1/RoR?t/IL-23R signal pathway may play a role in reducing SHR model blood pressure and target organ protection by regulating the balance of Th17/Treg.5.Mechanism of angelica sinensis alcohol extract on blood pressure and target organ protection in SHRRenal histopathology:compared with the blank group,the glomerular fibrosis of SHR model group was obvious,the epithelial cells of renal tubules were swollen,the lumen was narrowed,the capillaries around renal tubules were compressed,the morphology was irregular,the stroma was swollen,and the inflammatory cells were infiltrated;compared with the model group,the glomerular fibrosis of rats in benazepril group and high dose angelica alcohol extract group was reduced,and the shape of renal tubules was regular,and the inflammatory infiltration decreased.Serum inflammation index:?1?compared with the model group,the expression of IL-6 and CRP in benazepril group and Angelica alcohol extract high dose group decreased?P<0.05?.?2?compared with the blank group,the percentage of Th17 cells increased,the percentage of Treg cells decreased and the ratio of Th17/Treg cells increased in the model group;compared with the model group,the percentage of Th17 cells decreased,the percentage of Treg cells increased and the ratio of Th17/Treg cells decreased in the benazepril group and the high-dose angelica alcohol extract group?P<0.05?.?3?compared with the model group,the levels of IL-17 and IL-23 in benazepril group and angelica high-dose alcohol extract group were significantly lower?P<0.05?.?4?compared with the model group,the levels of IL-10 and TGF?1 in benazepril group and angelica high-dose alcohol extract group were significantly lower?P<0.05?.Expression of ROR?t and Fox P3:?1?compared with the blank group,the expression of ROR?t in the model group decreased?P<0.05?;the expression of ROR?t in the benazepril group and the high-dose angelica alcohol extract group was lower than that in the model group?P<0.05?.?2?compared with the blank group,the expression of Fox P3 in the model group was higher?P<0.05?;the expression of FoxP3 in the benazepril group and the angelica alcohol extract high dose group was higher than that in the model group?P<0.05?.Expression of p38/MAPK pathway related factors:compared with the blank group,the expression levels of p-p38,SGK1,p-FoxO1 and IL-23R in the renal tissue of SHR in the model group increased?P<0.05?;compared with the model group,the expression levels of p-p38,SGK1,p-FoxO1 and IL-23R in the renal tissue of rats in the benazepril group and the high dose angelica alcohol extract group decreased?P<0.05?.Conclusion1.Current research status has revealed that angelica is effective in reduction of MAP and target organ protection and the adverse effects rate is low,but the clinical efficacy need to be proved by further researches.2.Angelica sinensis alcohol extract can improve the general situation of SHR,reduce the blood pressure level of SHR model to a certain extent,reverse the myocardial remodeling,improve the endothelial function of SHR,and has good safety.3.The imbalance of Th17/Treg is the key factor of hypertension in SHR model.The high-dose angelica sinensis alcohol extract group can reduce the blood pressure and target organ protection of SHR model by regulating the balance of Th17/Treg mediated by p38MAPK/SGK1/FoxO1/RoR?t/IL-23R.