Berberine attenuates uric acid-induced cell injury by inhibiting NLRP3 signaling pathway in HK-2 cells

Abstract

Hyperuricemia (HUA) is a common chronic metabolic disease that can cause renal failure and even death in severe cases. Berberine (BBR) is an isoquinoline alkaloid derived from Phellodendri Cortex with strong antioxidant, anti-inflammatory, and anti-apoptotic properties. The purpose of this study was to investigate the protective effects of berberine (BBR) against uric acid (UA)-induced HK-2 cells and unravel their regulatory potential mechanisms. The CCK8 assay was carried out to detect cell viability. The expression levels of inflammatory factors interleukin-1β (IL-1β) and interleukin-18 (IL-18) and Lactate dehydrogenase (LDH) were measured using Enzyme-linked immunosorbent assays (ELISA). The expression of the apoptosis-related protein (cleaved-Caspase3, cleaved-Caspase9, BAX, BCL-2) was detected by western blot. The effects of BBR on the activities of the NOD-like receptor family pyrin domain containing 3 (NLRP3) and the expression of the downstream genes were determined by RT-PCR and western blot in HK-2 cells. From the data, BBR significantly reversed the up-regulation of inflammatory factors (IL-1β, IL-18) and LDH. Furthermore, BBR down-regulated protein expression of pro-apoptotic proteins BAX, cleaved caspase3 (cl-Caspase3), cleaved caspase9 (cl-Caspase9), and enhanced the expression of antiapoptotic protein BCL-2. Simultaneously, BBR inhibited the activated NLPR3 and reduced the mRNA levels of NLRP3, Caspase1, IL-18, and IL-1β. Also, BBR attenuated the expression of NLRP3 pathway-related proteins (NLRP3, ASC, Caspase1, cleaved-Caspase1, IL-18, IL-1β, and GSDMD). Furthermore, specific NLRP3-siRNA efficiently blocked UA-induced the level of inflammatory factors (IL-1β, IL-18) and LDH and further inhibited activated NLRP3 pathway. Collectively, our results suggested that BBR can alleviate cell injury induced by UA. The underlying unctionary mechanism may be through the NLRP3 signaling pathway.

Keywords: Berberine; Hyperuricemia; NLRP3; Uric acid.

Fig. 1

Chemical structure of BBR

Fig. 2

Cytotoxicities of BBR and UA in HK-2 cells. A and B Effects of treatment with BBR (2.5, 5, 10, 20, 40, 80, 100 μM) for 24 and 48 h on the viability of HK-2 cells. C and D Effects of treatment with UA (5, 10, 20, 40, 60 mg/dL) for 24 and 48 h on the viability of HK-2 cells. Values represent the means ± SD (n = 6). BBR, berberine; UA, uric acid. ^#P < 0.05, ^##P < 0.01 vs Con

Fig. 3

Effects of BBR on the level of inflammatory factors and LDH in HK-2 cells. A and B Effects of BBR on the level of inflammatory factors in HK-2 cells. C Effects of BBR on the level of LDH in HK-2 cells. Values represent the means ± SD (n = 6). BBR, berberine; UA, uric acid (20 mg/dL); ZY, Z-YVAD-FMK, (10 μM). ^##P < 0.01 vs Con; ^&P < 0.05, ^&&P < 0.01 vs UA

Fig. 4

Effects of BBR on apoptosis-related genes in HK-2 cells. A Western blot analyses of cl-Caspase3, cl-Caspase9, BAX, BCL-2, and β-actin. B Intensity of cl-Caspase3, cl-Caspase9, BAX, and BCL-2 relative to β-actin. Values represent the means ± SD (n = 3). BBR, berberine (20 μM); UA, uric acid (20 mg/dL); cl-Caspase3, cleaved-Caspase3; cl-Caspase9, cleaved-Caspase9. ^##P < 0.01 vs Con; ^&P < 0.05, ^&&P < 0.01 vs UA

Fig. 5

Effect of BBR on NLRP3 pathway in HK-2 cells using RT-PCR. A–D Relative mRNA expression levels of NLRP3, Caspase1, IL-18, and IL-1β. Values represent the means ± SD (n = 6). BBR, berberine (20 μM); UA, uric acid (20 mg/dL). ^#P < 0.05, ^##P < 0.01 vs Con; ^&P < 0.05, ^&&P < 0.01 vs UA

Fig. 6

Effect of BBR on NLRP3 pathway in HK-2 cells using Western blot. A Western blot analyses of NLRP3, ASC, cl-Caspase1, Caspase1, IL-18, IL-1β, GSDMD, and β-actin. B–H Intensity of NLRP3, ASC, cl-Caspase1, Caspase1, IL-18, IL-1β, and GSDMD relative to β-actin. Values represent the means ± SD (n = 3). BBR, berberine (20 μM); UA, uric acid (20 mg/dL); cl-Caspase1, cleaved-Caspase1. ^#P < 0.05, ^##P < 0.01 vs Con; ^&P < 0.05, ^&&P < 0.01 vs UA

Fig. 7

Effects of BBR on the level of inflammatory factors and LDH in HK-2 cells following NLRP3 siRNA knockdown. A and B Effects of BBR on the level of inflammatory factors following NLRP3 siRNA knockdown. C Effects of BBR on the level of LDH following NLRP3 siRNA knockdown. Values represent means ± SD (n = 6). BBR, berberine (20 μM); UA, uric acid (20 mg/dL). ^##P < 0.01 vs Con; ^&P < 0.05, ^&&P < 0.01 vs UA

Fig. 8

Effect of BBR on NLRP3 pathway in HK-2 cells following NLRP3 siRNA knockdown. A Western blot analyses of ASC, cl-Caspase1, Caspase1, IL-18, IL-1β, GSDMD, and β-actin. B–G Intensity of ASC, cl-Caspase1, Caspase1, IL-18, IL-1β, and GSDMD relative to β-actin. Values represent the means ± SD (n = 3). BBR, berberine (20 μM); UA, uric acid (20 mg/dL); cl-Caspase1, cleaved-Caspase1. ^#P < 0.05, ^##P < 0.01 vs Con; ^&P < 0.05, ^&&P < 0.01 vs UA