The effect and mechanism of freeze-dried powder of Poecilobdella manillensis on improving inflammatory injury of rat glomerular mesangial cells through TXNIP / NLRP3 pathway

Abstract

Objective: Diabetic kidney disease (DKD) is a common complication of diabetes mellitus. The pathophysiological changes in platelet function and the hypercoagulable state associated with DKD are closely linked to inflammatory processes. Poecilobdella manillensis (PM), a type of leech known for its anticoagulant and antithrombotic properties, has the potential to modulate the inflammatory response in DKD. This study aims to investigate the effect of freeze-dried powder of PM on improving inflammatory injury in rat glomerular mesangial cells and to explore its underlying mechanism.

Methods: Lipopolysaccharide (LPS) stimulated HBZY-1 rat mesangial cells to establish an in vitro DKD inflammation model. After the intervention with the water extract of freeze-dried powder of PM (FDPM), cell viability, NO content, and the levels of inflammatory factors such as IL-1β, IL-18, and TNF-α were assessed. Finally, utilizing transcriptomics technology, RT-qPCR, and Western blot methods, the mechanism by which FDPM improves inflammatory injury in rat glomerular mesangial cells was explored and preliminarily validated.

Results: FDPM effectively enhances cell viability and inhibits the production of NO and related inflammatory factors. Transcriptomic analysis suggests that FDPM may exert these effects by regulating the TXNIP/NLRP3 signaling pathway. The mRNA and protein expressions of TXNIP, NLRP3, and MCP-1 in the model cells were reversed by FDPM.

Conclusion: FDPM may improve the micro-inflammatory state of DKD and slow the progression of the disease by regulating the TXNIP/NLRP3 signaling pathway. This study provides a scientific basis for the clinical application of PM DKD treatment.

Keywords: Diabetic nephropathy; NLRP3 inflammasome; NLRP3 signaling pathway; Poecilobdella manillensis freeze-dried powder; Rat mesangial cells; TXNIP; Transcriptomics.

Fig. 1

The in vitro modeling conditions and the safe dose range of FDPM. (A) Cell inhibition rate after 12 h of intervention with different concentrations of LPS; (B) Cell inhibition rate after 24 h of intervention with different concentrations of LPS; (C) Screening of the safe dose range of FDPM. ∗∗∗P < 0.001, compared with the normal control group, n = 6.

Fig. 2

Effects of FDPM intervention on LPS-induced HBZY-1 cell injury and inflammatory response. (A) Effect of FDPM on cell viability following LPS intervention (n = 6); (B) Effect of FDPM on NO content following LPS intervention (n = 6); (C) Effect of FDPM on IL-1β levels in cell supernatant following LPS intervention (n = 3); (D) Effect of FDPM on IL-18 levels in cell supernatant following LPS intervention (n = 3); (E) Effect of FDPM on TNF-α levels in cell supernatant following LPS intervention (n = 3). ∗∗∗P < 0.001, compared with the normal control group; ^###P < 0.001, compared with the model group.

Fig. 3

(A) Venn diagram of genes expressed differently; (B) Volcanic diagram of difference of expression in model group and normal control group; (C) Volcanic diagram of difference of expression in FDPM-H administration group and model group.

Fig. 4

(A) GO function secondary classification annotation of IDEGs; (B) IDEGs were enriched in the first 20 items of biological processes (BP); (C) IDEGs were enriched in the top 20 items of cellular components (CC); (D) IDEGs were enriched in the first 20 items of molecular functions (MF).

Fig. 5

KEGG enrichment analysis of IDEGs.

Fig. 6

(A) KEGG pathway enrichment chord diagram comparing the model group with the normal control group; (B) KEGG pathway enrichment chord diagram comparing the FDPM-H administration group with the model group. On the right side, the pathway information related to significant enrichment of the differential genes is displayed. On the left side, the pathway genes are sorted by log2FC in descending order.

Fig. 7

Effects of FDPM on TXNIP/NLRP pathway in LPS-induced cellular inflammatory injury. (A) Relative expression of TXNIP mRNA in each group; (B) Relative expression of NLRP3 mRNA in each group; (C) Relative expression of MCP-1 mRNA in each group; (D) Western blot bands of TXNIP, NLRP3, and MCP-1 proteins; (E) Relative content of TXNIP protein in each group; (F) Relative content of NLRP3 protein in each group; (G) Relative content of MCP-1 protein in each group. ∗P < 0.05, ∗∗∗P < 0.001, compared with the normal control group; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001, compared with the model group, n = 3. The original Western blot bands are shown in Figs. S3(A–D)-S5 (A–D) in the supplemental file.