Traditional Mongolian medicine (HHQG) attenuates CCl4-induced acute liver injury through inhibiting monocyte/macrophage infiltration via the p-P38/p-JNK pathway

Abstract

Ethnopharmacological relevance: Honghua Qinggan 13 Flavor Pills (HHQG), whose Mongolian name is Guri Gumu-13, is a traditional Mongolian medicine, that was stated in the "Diagnosis and Treatment of Ming Medical Code". The HHQG has been included in the Mongolian Medicine Division of the Ministry of Health Drug Standards (1998 edition). Based on our clinical expertise, HHQG demonstrated satisfactory therapeutic effects in hepatitis and liver failure. However, the pharmacological effects and potential mechanisms of HHQG have not been investigated.

Aim of the study: In this study, we combined network pharmacology, transcriptomics, and molecular biology to detect the underlying mechanism for the effect of HHQG on acute liver injury in mice.

Materials and methods: Network pharmacology was used to explore the pathways involved in the protective effect HHQG in acute liver injury. This effect was further verified by injecting carbon tetrachloride (CCl₄; 10 mL/kg, i.p.) to induce acute liver injury in mice. Serum markers of liver injury, morphology, histology, and monocyte/macrophage infiltration in the liver tissue were investigated. Transcriptomics further defined the HHQG targets. Transwell analysis was performed to confirm that HHQG inhibited monocyte/macrophage RAW.264.7 infiltration. qPCR and Western blot were performed to explore the mechanism of action of HHQG.

Results: Network pharmacology showed that HHQG exerted anti-oxidative and anti-inflammatory effects and promoted metabolic effects against acute liver injury. Pretreatment of mice with HHQG significantly maintained their body weight and decreased serum tumor necrosis factor-alpha (TNF-α) levels induced by CCl₄ treatment in vivo. Histopathological examination further confirmed that HHQG protected the liver cells from CCl₄-induced damage. Importantly, HHQG significantly inhibited CCl₄-induced monocyte/macrophage infiltration. Transcriptomic analysis revealed that HHQG significantly reduced the expression of chemokines and cell adhesion molecules. We determined that HHQG significantly downregulated the expression of the key chemokine (monocyte chemokine protein-1, CCL2) at the gene and protein levels. Further research showed that HHQG inhibited chemokine production in hepatocytes by inhibiting the p-P38 and p-JNK pathways, thereby reducing monocyte/macrophage infiltration.

Conclusions: These combined data showed that HHQG alleviated acute liver injury in mice, and further verified that HHQG exerted protective effects by inhibiting the production of CCL2 and reducing the infiltration of monocyte/macrophage by inhibiting the p-P38 and p-JNK pathways.

Keywords: Acute liver injury; CCL2; HHQG; Monocyte/macrophage; Traditional Mongolian medicine.