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
Authors: Li Qiaofeng , Lan Taijin , He Songhua , Chen Weiwei , Li Xiaolan , Zhang Weiquan , Liu Ying , Zhang Qiuping , Chen Xin , Han Yaoyao , Su Zhiheng , Zhu Dan , Guo Hongwei 
Published: March 2021

Abstract
Background

Lei-gong-gen formula granule (LFG) is a folk prescription derived from Zhuang nationality, which is the largest ethnic minority among the 56 nationalities in China. It consists of three herbs, namely Eclipta prostrata (L.) L., Smilax glabra Roxb, and Centella asiatica (L.) Urb. It has been widely used as health protection tea for hundreds of years to prevent hypertension in Guangxi Zhuang Autonomous Region. The purpose of this study is to validate the antihypertensive effect of LFG on a SHR model and further to identify the active ingredients of LFG and its anti-hypertension molecular mechanism.
Methods

Firstly, a spontaneously hypertensive rat model was used to observe the effects of LFG on blood pressure, body weight, and heart rate. The serum levels of NO, ANG Ⅱ, and ET-1 were measured and H&E staining was applied to observe the pathology of the 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 active compounds, their corresponding targets, and hypertension associated pathways. Then the predicted results were verified by molecular biology experiments such as RT-qPCR and western blot. Finally, the potential active compounds were predicted by molecular docking technology, and the LFG chemical constituents were analyzed and identified by UPLC-QTOF/MS technology.
Results

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 candidate active compounds from LFG were collected, which link to 765 potential targets. 828 hypertension associated targets were retrieved. 12 overlapped targets both related to candidate active compounds from LFG and hypertension were screened and used as the potential targets of LFG on antihypertensive effect. And the 12 overlapped targets were validated using RT-qPCR, and the results showed that LFG could upregulate the mRNA expression of NOS3 and proto-oncogene tyrosine-protein kinase SRC (SRC) in the 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 the PI3K-AKT signaling pathway. The results showed that LFG significantly increased the protein expression levels of PI3K and phosphorylated AKT in SHR rats, suggesting that LFG may active PI3K-AKT signaling pathway to decrease hypertension. 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, PI3K, and AKT. UPLC-QTOF/MS analysis confirmed that p-hydroxybenzoic acid, shikimic acid, salicylic acid, and nicotinic acid existed in LFG.
Conclusion

LFG can reduce the blood pressure of SHR rats, which might be attributed to increasing the serum NO level for promoting vasodilation via upregulating SRC expression level and activating the PI3K-AKT-NOS3 signaling pathway. p-Hydroxybenzoic acid, shikimic acid, salicylic acid, and nicotinic acid might be the underlying compounds for LFG antihypertensive effect.