Lei-gong-gen formula granule attenuates hyperlipidemia in rats via cGMP-PKG signaling pathway

Ethnopharmacological relevance: 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 cardiovascular and cerebrovascular diseases such as hyperlipidemia and hypertension.

Aim of the study: This study validated the lipid-lowering effect of LFG in a hyperlipidemia rat model. Then we employed network pharmacology and molecular biological approach to identify the active ingredients of LFG, corresponding targets, and its anti-hyperlipidemia mechanisms.

Materials and methods: Hyperlipidemia rat model was established by feeding male Sprague-Dawley rats with high-fat diet for two weeks. LFG (two doses of 10 and 20 g/kg) was administered orally to hyperlipidemia rat model for 4 weeks, twice per day. Serum levels of total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C) were monitored in rats pre and post-treatment. Hematoxylin-eosin staining was applied to observe the pathology and lipid accumulation of liver. We then performed network pharmacology analysis to predict the ingredients, their associated targets, and hyperlipidemia associated targets. Pathway analysis with significant genes was carried out using KEGG pathway. These genes and proteins intersectioned between compound targets and hyperlipidemia targets were further verified with samples from hyperlipidemia rats treated with LFG using Real-time RT-PCR and Western Blot.

Results: LFG attenuated hyperlipidemia in rat model, and this was characterized with decreased serum levels of TC, LDL-C, liver wet weight, and liver index. LFG alleviated the hepatic steatosis in hyperlipidemia rats. Network pharmacology analysis identified 53 bioactive ingredients from LFG formula (three herbs), which link to 765 potential targets. 53 hyperlipidemia associated genes were retrieved from public databases. There were 10 common genes between ingredients-targets and hyperlipidemia associated genes, which linked to 20 bioactive ingredients. Among these 10 genes, 3 of them were validated to be involved in LFG's anti-hyperlipidemia effect using Real-time RT-PCR, namely ADRB2 encoding beta-2 adrenergic receptor, NOS3 encoding nitric oxide synthase 3, LDLR encoding low-density lipoprotein receptor. The cGMP-PKG signaling pathway was enriched for hyperlipidemia after pharmacology network analysis with ADRB2, NOS3, and LDLR. Interestingly, expression of cGMP-dependent protein kinase (PKG) was downregulated in hyperlipidemia rat after LFG treatment. Molecular docking study further supported that ferulic acid, histidine, p-hydroxybenzoic acid, and linalool were potential active ingredients for LFG's anti-hyperlipidemia effect. LC-MS/MS analysis confirmed that ferulic acid and p-hydroxybenzoic acid were active ingredients of LFG.

Conclusion: LFG exhibited the lipid-lowering effect, which might be attributed to downregulating ADRB2 and NOS3, and upregulating LDLR through the cGMP-PKG signaling pathway in hyperlipidemia rat. Ferulic acid and p-hydroxybenzoic acid might be the underlying active ingredients which affect the potential targets for their anti-hyperlipidemia effect.

Keywords: Centella asiatica (L.) Urb.; Eclipta prostrata (L.) L.; Hyperlipidemia; Network pharmacology; Smilax glabra Roxb..