Yiqi Huoxue Tongluo formula-containing serum attenuates high glucose-induced injury in microglia via MAPK pathways: Network pharmacology-based analysis and biological validation

Introduction

The Yiqi Huoxue Tongluo formula (YQHX), containing Astragalus mongholicus Bunge, Angelica sinensis (Oliv.) Diels, Rehmannia glutinosa Libosch., Pueraria lobata (Willd.) Ohwi, Corydalis yanhusuo W.T.Wang, Spatholobus suberectus Dunn, Clematis chinensis Osbeck, is a proprietary formula of the First Affiliated Hospital of Anhui University of Chinese Medicine, and can attenuate diabetic neuropathy pain (DNP). This study aimed to explore the mechanisms underlying YQHX-mediated DNP alleviation. Additionally, the effects of YQHX on BV2 microglia injured by high glucose via the mitogen-activated protein kinase (MAPK) signaling pathways were investigated.

Methods

Liquid chromatography tandem mass spectroscopy (LC-MS/MS) was performed to identify the chemical compounds in YQHX. The potential mechanism of YQHX for treating DNP was explored through network pharmacology analysis. The established glucose-induced BV2 cell injury model was treated with varying concentrations of YQHX-containing serum. The effects of YQHX-containing serum and MAPK pathway inhibitors on microglial cells were assessed by Cell Counting Kit-8 and enzyme-linked immunosorbent assays. The messenger RNA (mRNA) and protein levels of microglial marker OX42 were measured. Additionally, phosphorylated-p38, -c-Jun-terminal kinase, and -extracellular signal-regulated kinase and downstream nuclear factor-κB/p65 and phosphorylated-activating transcription factor 2 (ATF2) proteins were detected by western blotting.

Results

In total, 503 chemical components were identified in YQHX by LC-MS/MS analysis. Network pharmacology results showed 203 active ingredients and 363 overlapping targets, respectively, as potential therapeutic candidates for YQHX-mediated DNP treatment. Additionally, the Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis yielded a total of 105 signal pathways. Notably, YQHX considerably improved the cell viability of BV2 cells (P < 0.01), remarkably reduced the production of proinflammatory factors (P < 0.01), significantly increased the production of anti-inflammatory factors (P < 0.01), and markedly inhibited the mRNA and protein levels of OX42 (P < 0.01). Furthermore, YQHX treatment inhibited MAPK activation and ATF2 expression.

Conclusions

YQHX may protect against high-glucose-induced BV2 microglial cell injury by inhibiting the activation of MAPKs and downstream ATF2, thus, reducing microglial activation and proinflammatory cytokine release. These results provide a foundational theoretical basis for exploring the further mechanism of YQHX in treating DNP, and they provide a novel target for the application of traditional Chinese medicine.