Simiao pill inhibits epithelial mesenchymal transition in a mouse model of chronic hyperuricemic nephropathy by inhibiting NLRP3 inflammasome activation

Abstract

Background: Simiao pill module (SMM), a traditional Chinese medicine formula, has been widely used to treat gout and gouty arthritis. The goal of this study was to investigate the effects of SMM on epithelial-mesenchymal transition (EMT) and activation of NLR family pyrin domain containing 3 (NLRP3) inflammasome in a mouse model of potassium oxonate (PO)-induced chronic hyperuricemic nephropathy (HN).

Methods: Mice were randomly divided into the following four groups: control, HN model (PO), febuxostat (FEB)-treated (PO + FEB), and SMM-treated (PO + SMM) groups. Following 6 weeks of treatment, blood samples were collected and mice were sacrificed to collect kidney samples to study the biochemical parameters associated with renal function and histopathological changes associated with HN, respectively. The samples were analyzed for the expression of markers of EMT (collagen type 3, α-smooth muscle actin [α-SMA], fibronectin, vimentin and E-cadherin) and activation of NLRP3 inflammasome (NLRP3, apoptosis-associated speck-like protein [ASC], caspase-1, interleukin [IL]-1β, and IL-18).

Results: Our results showed that hyperuricemia, impaired kidney function, and renal pathological characteristics induced by PO treatment were improved following treatment with SMM and FEB. Additionally, treatment with SMM and FEB decreased the expression of vimentin, collagen 3, fibronectin, and α-SMA, and increased the expression of E-cadherin. Moreover, NLRP3 inflammasome activation, as assessed by the increased expression of NLRP3, ASC, and caspase-1, and secretion of IL-1β and IL-18, was inhibited by treatment with SMM and FEB.

Conclusion: These results suggest that SMM inhibited EMT and NLRP3 inflammasome activation in chronic HN mice, and the beneficial effect of SMM was compared with a standard drug, FEB.

Keywords: Chronic hyperuricemic nephropathy; Epithelial-mesenchymal transition; NLRP3 inflammasome; Simiao pill.

Fig. 1

Effects of Simiao pill module (SMM) on a body weight (BW), b kidney weight (KW) and kidney index (KW/BW), c liver xanthine oxidase (XOD) activity, d serum uric acid (UA), e blood urea nitrogen (BUN), and f serum creatinine (Scr) in chronic hyperuricemic nephropathy (HN mice (n = 8). SMM improved a the loss of BW, and b increase in KW and kidney index induced by potassium oxonate (PO) injection. c SMM inhibited the activity of liver XOD. SMM decreased c UA levels and e and f ameliorated renal function as assessed by e BUN and f Scr.*p < 0.05, **p < 0.005, ***P < 0.001, ****P < 0.0001

Fig. 2

Effects of SMM on histopathological changes in chronic HN mice. Representative images of a Periodic acid–Schiff (400×) and b Masson trichrome staining (400×) to assess kidney histopathology. SMM decreased (c) histological severity scores and SMM improved histopathological changes in the kidneys in the chronic HN model (d) renal fibrosis in chronic HN mice (n = 8). **p < 0.005, ***P < 0.001, ****P < 0.0001

Fig. 3

Western blot analysis for the expression of E-cadherin, vimentin, collagen 3, fibronectin and α-SMA in renal tissues. a Representative immunoblots of protein expression in the control, PO, PO + FEB, and PO + SMM groups. Densitometry analysis for western blot results of b E-cadherin, c vimentin, d collagen 3, e fibronectin, and f α-SMA protein (n = 3). ****P < 0.0001

Fig. 4

Representative images of immunofluorescence staining (400×) for the expression of fibronectin and α-SMA in renal tissues. SMM decreased the expression of a fibronectin and b α-SMA in PO-induced chronic HN mice

Fig. 5

Relative gene expression levels of a NLR family pyrin domain containing 3 (NLRP3), b apoptosis-associated speck-like (ASC), c caspase-1, and d IL-1β in the different groups (n = 8). **p < 0.005, ***P < 0.001, ****P < 0.0001

Fig. 6

Western blot analysis for the expression of NLRP3, ASC and caspase-1 in renal tissues. a Representative immunoblots of protein expression in the control, PO, PO + FEB, and PO + SMM groups. Densitometry analysis for western blot results of b NLRP3, c ASC, and d caspase-1 (n = 3). ****P < 0.0001

Fig. 7

The secretion levels of IL-1β and IL-18 in the control, PO, PO + FEB, and PO + SMM groups. The serum levels of a IL-1β and b IL-18 in the different groups (n = 8). The protein levels of c IL-1β and d IL-18 in kidney tissues lysates (n = 8). e Representative images for immunohistochemical staining (400×) for the expression of IL-1β and TGFβ1 in renal tissues. f Relative IL-1β staining area in renal tissues (n = 3). **p < 0.005, ***P < 0.001, ****P < 0.0001