Objective: To systematically evaluate the multidimensional therapeutic effects of Zishui Qingre Decoction (ZQD) on lupus nephritis (LN) and to elucidate its multi-target mechanism based on bioactive components in blood.
Methods: BLAB/c mice were randomly divided into six groups: blank control group, blank medication group, LN model group, low-dose ZQD group, medium-dose ZQD group, and high-dose ZQD group. Liquid chromatography-mass spectrometry (LC-MS) was used to identify the effective prototypical components in blood after ZQD administration. Urinary protein levels were detected using dipstick tests. Serum creatinine and anti-dsDNA antibody levels were measured using enzyme-linked immunosorbent assay (ELISA) to assess the effects of ZQD on renal function and autoimmune status in mice. Hematoxylin and eosin (H&E) staining of kidney and colon tissues was performed to observe the pathological changes in glomerular inflammation, crescent formation, and colonic mucosal structure and inflammatory cell infiltration before and after ZQD intervention. The effects of ZQD on gut microbiota structure and metabolites were analyzed using 16S rRNA gene sequencing and LC-MS, and potential metabolic pathways influenced by ZQD were further explored. Based on identified blood components, differentially expressed genes in LN were screened from public databases such as GEO and GENE CARDS. A protein-protein interaction (PPI) network was constructed using network pharmacology methods, and KEGG enrichment analysis was performed to determine relevant signaling pathways, thereby establishing a component-target-pathway-disease network to explore the therapeutic targets and signaling pathways of ZQD for LN. Molecular docking was used to verify the binding ability of core components to key targets. Additionally, Western blot (WB) was employed to detect the protein expression levels of PDK1, PI3K, and AKT, evaluating the impact of ZQD on key proteins in the PI3K-AKT signaling pathway in LN model mice. Quantitative real-time polymerase chain reaction (qPCR) was used to detect the mRNA expression levels of PI3K, Raf1, ERK, and Itgal, assessing the effects of ZQD on key signaling pathways and immune cell activation-related genes in LN model mice.
Results:
- By combining the analysis of blank herbs, blank animal serum, and the blank medication group, endogenous background interference was deducted, and 109 compounds in blood after ZQD administration in the LN model were ultimately identified. Based on their sources and metabolic transformation characteristics, these compounds were classified into three grades. Combined with the matching scores of secondary mass spectra, the top seven compounds with the highest matching degrees were finally selected.
- Semi-quantitative dipstick tests showed that the low-, medium-, and high-dose ZQD groups significantly reduced urinary protein levels in model mice (P < 0.05). ELISA results indicated that the recovery of serum creatinine and anti-dsDNA antibody levels in the medium-dose ZQD group was significantly better than in the low-dose group (P < 0.05) and was not statistically different from the high-dose group (P > 0.05).
- H&E staining pathology observations revealed that medium- and high-dose ZQD interventions significantly improved renal and colonic tissue damage in LN model mice. In kidney tissues, mesangial proliferation and inflammatory cell infiltration were reduced, and the proportion of crescent formation was significantly lower in the medium- and high-dose groups compared to the model group. In colonic tissues, the integrity of the colonic mucosa was markedly restored, the number of goblet cells increased significantly, and the degree of inflammatory cell infiltration in the mucosa and submucosa was markedly reduced in the medium-dose group.
- 16S rRNA gene sequencing combined with non-targeted fecal metabolomics analysis found that the abundance of pathogenic or opportunistic pathogens in the gut microbiota of LN model mice increased, including Patescibacteria, Campylobacterota, and Saccharimonadaceae. ZQD intervention effectively increased the α-diversity and F/B ratio of the gut microbiota and shifted the community structure toward that of the healthy control group. In terms of microbial composition, ZQD not only reduced the abundance of Patescibacteria and Saccharimonadaceae but also increased the abundance of short-chain fatty acid-producing bacteria such as Muribaculum and Rikenellaceae. In the model group, the levels of short-chain fatty acids (butyric acid and propionic acid) and secondary bile acids (ursodeoxycholic acid) were significantly reduced, while lipopolysaccharide and inosine levels were significantly increased. After ZQD intervention, the levels of these disrupted metabolites were significantly restored, approaching those of the blank control group. Further metabolic pathway enrichment analysis showed that differentially abundant metabolites were significantly enriched in primary bile acid biosynthesis, butyrate metabolism, and purine metabolism pathways.
- Based on the prediction of 70 class I blood prototypical components and database screening, 31 intersection targets between ZQD and LN were obtained. Network pharmacology PPI network analysis showed that ZQD mainly involved 29 protein targets in treating LN, with 24 upregulated and 5 downregulated targets. GO and KEGG analysis results indicated that key targets were significantly enriched in signal transduction structures such as cell membranes and membrane rafts, with molecular functions focused on protein kinase and receptor binding activity, primarily regulating biological processes such as cell proliferation, apoptosis, and activation. Pathway analysis further confirmed that these functions were achieved through key signaling pathways such as MAPK, PI3K-Akt, and Rap1, which are closely related to cell adhesion junctions and infection-immune-related signals, systematically revealing the potential mechanisms of ZQD's multi-target regulation of immune-inflammatory networks in treating LN. Molecular docking results showed that core blood components could stably bind to key targets, with binding energies all less than -5 kcal/mol.
- WB and qPCR results showed that the PI3K-AKT signaling pathway was significantly activated at both the transcriptional and protein levels in LN model mice compared to the control group. Key proteins PDK1, PI3K, and AKT were significantly upregulated, and the mRNA expression of key genes such as PI3K and Raf1 was also significantly increased. ZQD significantly inhibited the overactivation of the PI3K-AKT signaling pathway in LN. Compared to the model group, the protein expression levels of PDK1, PI3K, and AKT were significantly downregulated in the ZQD group, and the mRNA expression of downstream key effectors PI3K, Raf1, and AKT was also significantly decreased.
Conclusion:
- ZQD identified 109 blood compounds, and seven core components were selected through mass spectrometry matching, providing a clear pharmacological material basis.
- ZQD significantly reduced urinary protein levels, with the medium-dose group showing the best improvement in serum creatinine and anti-dsDNA antibodies.
- ZQD can significantly reduce pathological damage to kidney and colon tissues, restoring tissue structure and cell morphology.
- ZQD regulates the gut microbiota structure, increases beneficial bacteria, and reduces pathogenic bacteria, while normalizing short-chain fatty acid and bile acid metabolism, improving gut microecology and metabolic homeostasis.
- Network pharmacology analysis shows that ZQD can act on multiple targets in signaling pathways such as MAPK and PI3K-AKT, systematically regulating immune-inflammatory networks.
- ZQD can effectively inhibit the abnormal activation of the PI3K-AKT signaling pathway, providing key mechanistic evidence and clinical application potential for the treatment of LN.
Keywords: Lupus Nephritis; Zishui Qingre Decoction; Network Pharmacology; Metabolomics
