A Network Pharmacology-based Approach To Explore The Active Ingredients And Molecular Mechanism Of Lei-gong-gen Formula Granule On A Spontaneously Hypertensive Rat Model

Objective: Lei-gong-gen formula granule(LFG) is a folk prescription derived from Zhuang nationality,the largest ethnic minority among the 56 nationalities in China.It is composed of three herbs,namely Centella asiatica(L.)Urb.,Eclipta prostrata(L.)L.,Smilax glabra Roxb.It has been widely used as health protection tea for many years to prevent hypertension.The purpose of this study is to validate the antihypertensive effect of LFG on a spontaneously hypertensive rat(SHR)model and further to identify the active ingredients of LFG,corresponding targets,and its anti-hypertension mechanisms.Methods:Firstly,a spontaneously hypertensive rat model was used to observe the effects of LFG on the blood pressure,body weight,and heart rate.The serum levels of NO,ANG-Ⅱ,and ET-1 were measured and hematoxylin-eosin staining was applied to observe the pathology of heart.Secondly,network pharmacology analysis was performed to collect the ingredients of LFG by using the database of traditional Chinese medicine(TCMSP,TCMID,BATMAN-TCM)and to predict the ingredients,their corresponding targets,and hypertension associated pathways.Then the predicted results were verified by molecular biology methods such as RT-q PCR and Western blot.Finally,the potential active ingredients were predicted by molecular docking technology,and the LFG chemical constituents were analyzing and identifying by UPLC-QTOF/MS technology.Results: Compared with model group,LFG significantly reduced blood pressure and increased serum NO content in SHR rats.LFG ameliorated the pathological changes such as cardiac hypertrophy and interstitial inflammation.Based on the results of public database searching,53 active ingredients from LFG were collected,which link to 765 potential targets.828 hypertension associated targets were retrieved.The potential targets of antihypertensive effect were found by comparing LFG genes with hypertension genes,and 12 key targets were obtained after screening.12 key genes were validated using RT-q PCR,and the results showed that LFG could upregulate the m RNA expression of NOS and SRC in thoracic aorta.Pathway enrichment results showed that PI3K-AKT signaling pathway was closely related to the expression of NOS3 and SRC.To verify this result,western blot was used to detect the key proteins(PI3K,AKT,and p-AKT)of PI3K-AKT signaling pathway.The results showed that LFG significantly increased the protein expression levels of PI3 K and phosphorylated AKT in SHR rats,suggesting that LFG may play a role through the PI3K-AKT signaling pathway.Molecular docking study further supported that p-hydroxybenzoic acid,cedar acid,shikimic acid,salicylic acid,nicotinic acid,linalool,and histidine can be well binding with NOS3,SRC,PI3 K,and AKT.These ingredients were potential ingredients for LFG antihypertensive effects.And p-hydroxybenzoic acid was confirmed to exist in LFG extract.Conclusion: LFG can reduce the blood pressure of SHR rats,which might be attributed to upregulating SRC expression level,activating of PI3K-AKT-NOS3 signaling pathway to further increase the serum NO level for promoting vasodilation.p-Hydroxybenzoic acid,cedar acid,shikimic acid,salicylic acid,nicotinic acid,linalool,and histidine might be the underlying ingredients which affect the potential targets for LFG antihypertensive effect.