Radix Rehmannia Glutinosa inhibits the development of renal fibrosis by regulating miR-122-5p/PKM axis

Abstract

Objective: It is acknowledged that Radix Rehmanniae Praeparata (RR) can regulate hormone metabolism, reduce blood glucose, resist aging, help to sedate patients and promote diuresis. The study aims to investigate the mechanism of how RR influences the development of renal fibrosis by regulating the miR-122-5p/PKM axis.

Methods: Unilateral ureteral obstruction (UUO) was applied to induce renal fibrosis in mice in vivo, and human tubular epithelial HK2 cells treated by transforming growth factor-β (TGF-β1) were used to induce renal fibrosis in vitro. Interleukin 6 (IL-6) and tumor necrosis factor-α (TNF-α) in mouse serum were detected by Enzyme-linked immunosorbent assay (ELISA); fibronectin (FN) and type I collagen (Col-I) in renal tissue were detected by Western blotting; serum creatinine (Cr) and blood urea nitrogen (BUN) were analyzed by kits. Hematoxylin-eosin (HE) staining and Masson staining were utilized to assess the degree of pathological damage and fibrosis. Cell viability and apoptosis in the in vitro model were detected by MTT and Flow cytometry. Dual-luciferase reporter assay was performed to determine intermolecular targeting relationships.

Results: RR could inhibit IL-6 and TNF-α levels, decrease the levels of FN and Col-I and improve the renal function indexes (serum Cr and BUN) in UUO mice (all P<0.05). In addition, RR was able to promote the up-regulation of miR-122-5p expression in UUO mice in vivo (P<0.05). MiR-122-5p expression was down-regulated and PKM expression was up-regulated in HK2 cells treated with TGF-β1 (all P<0.05). RR inhibited renal fibrosis progression by regulating the miR-122-5p/PKM axis. Inhibition of miR-122-5p or overexpression of PKM could promote apoptosis of TGF-β1-treated HK2 cells, inhibit their viability, aggravate fibrosis, and attenuate the protective effect of RR on the cells. The protective effect of RR promoted by overexpression of miR-122-5p was partially counteracted by PKM.

Conclusion: RR can inhibit renal fibrosis progression by regulating the miR-122-5p/PKM axis.

Keywords: PKM; Radix Rehmannia Glutinosa; miR-122-5p; renal fibrosis.

Figure 1

RR could improve renal function and inhibit renal pathological changes in mice. A. HE staining (The arrows indicated injured cells and tubular basement membrane) (400×). B. Masson staining (The blue part indicated by the arrow indicated tissue collagen fiber deposition; 400×). C. HE staining quantitative results. D. Masson staining quantitative results. E. Cr level detection. F. BUN level detection. n=10, *P<0.05.

Figure 2

RR had the anti-inflammatory and anti-renal fibrosis effects. A. Detection of IL-6 and TNF-α levels. B. Detection of mRNA levels of FN and Col-I. C, D. Detection of protein levels of FN and Col-I. n=10, *P<0.05.

Figure 3

RR could regulate miR-122-5p with PKM. A. Heatmap of differentially expressed miRNAs in the dataset of GSE162794 (Sham:UUO). B. Volcano map of up- and down-regulated miRNAs (Sham:UUO). C. Expression level of miR-122-5p in GSE162794 dataset samples. D, E. Detection of expression levels of miR-122-5p in mouse renal tissue versus HK2 cells. n=10, *P<0.05.

Figure 4

Down-regulation of miR-122-5p could promote the progression of injury in in vitro models and eliminate the protective effect of RR in vitro. A. Detection of transfection efficiency of inh-miR-122-5p (n=3). B. Detection of IL-6 and TNF-α levels (n=3). C. Detection of mRNA levels of FN and Col-I (n=3). D, E. Detection of protein levels of FN and Col-I (n=3). F. MTT assay of cell viability (n=3). G. Flow cytometry assay of apoptosis (n=3). *P<0.05.

Figure 5

A targeting relationship was proved between miR-122-5p and PKM. A. Intersection results of target genes from TargetScan and StarBase websites. B. Pathway annotation analysis results of target genes. C. Protein interaction analysis between potential target genes and risk genes. D. Results of dual-luciferase reporter assay (n=3). *P<0.05.

Figure 6

Evaluation of PKM expression. A, B. Detection of PKM expression levels in mouse renal tissues (n=10) and HK2 cells (n=3). C. Correlation analysis results between miR-122-5p and PKM (n=3). D. Effect of miR-122-5p mimic on PKM expression. E. A target relationship was proved between PKM and RR (genes related to RR components on the left and protein interaction analysis between PKM and genes related to RR components on the right). *P<0.05.

Figure 7

Up-regulation of PKM promoted the progress of injury in in vitro models and eliminated the protective effect of RR in vitro. A. Detection of IL-6 and TNF-α levels (n=3). B. Detection of mRNA levels of FN and Col-I (n=3). C, D. Detection of protein levels of FN and Col-I (n=3). E. MTT assay of cell viability (n=3). F. Flow cytometry assay of apoptosis (n=3). *P<0.05.

Figure 8

Overexpression of miR-122-5p could inhibit the progression of renal fibrosis in the in vitro model, which was partially counteracted by PKM. A. IL-6 and TNF-α levels (n=3). B. mRNA levels of FN and Col-I (n=3). C, D. Protein levels of FN and Col-I (n=3). E. Cell viability measured by MTT (n=3). F. Apoptosis measured by Flow cytometry (n=3). *P<0.05.

Figure 9

The mechanism by which Rehmannia Glutinosa regulates miR-122-5p/PKM axis to inhibit the progression of renal fibrosis.