Naringin ameliorates the high glucose-induced rat mesangial cell inflammatory reaction by modulating the NLRP3 Inflammasome

Abstract

Background: The Nucleotide binding and oligomerization domain-like receptorfamily pyrin domain-containing 3 (NLRP3)-inflammasome plays an important role in various diseases, including a variety of kidney diseases. Naringin exhibits anti-inflammatory and anti-oxidation effects among others, but its specific mechanisms are not clear. We investigated the expression of the NLRP3-inflammasome under high-glucose conditions, assessed the effects of naringin on that process, and further elucidated the role of naringin in the pathogenesis of diabetic kidney disease(DKD).

Methods: To assess the therapeutic potential of naringin and the mechanisms involved, we cultured rat glomerular mesangial cells and grouped them according to different glucose concentrations, different action times, different concentrations of MCC950, and different concentrations of naringin.The cell proliferation was measured by MTT assay. The expression of Interleukin-1β(IL-1β) and Interleukin18 (IL-18) in the cell supernatant were detected by ELISA. The expression and activity of NLPR3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and Caspase-1 were detected by Western Blot.

Results: The expressions of NLRP3, ASC, caspase-1, IL-1β, and IL-18 in rat glomerular mesangial cells were significantly higher in the high glucose (HG) group than in the control normal glucose (NG) group and exhibited time-dependence activity. The expression levels of NLRP3, caspase-1, IL-1β, and IL-18 in different treatment groups were significantly lower compared with the HG group after 48 h of MCC950 pre-treatment (p < 0.05). Pre-treatment with naringin produced the same results. Naringin also inhibited the proliferation of cells.

Conclusions: The NLRP3-inflammasome potentially plays a role in the process of activation and inflammation of glomerular mesangial cells as induced by high-glucose conditions. Naringin inhibited the proliferation of cells that were induced by high glucose. Further, it reduced the expression of inflammatory factors that are mediated by NLRP3 through the NLRP3-caspase-1-IL-1β/IL-18 signaling pathway, which makes naringin a potentially novel treatment for DKD disease.

Keywords: Diabetes mellitus; Diabetic kidney disease; NLRP3 inflammasome; NLRP3-caspase-1-IL-1β/IL-18 signaling pathway; Naringin.

Fig. 1

Effects of different concentrations of glucose on the proliferation of rat glomerular mesangial cells at various time points (24 h,48 h,72 h).Proliferation was measured by MTT. NG:normal control group,H1:glucose concentration of 15 mmol/L,H2:glucose concentration of 25 mmol/L,H3:glucose concentration of 30 mmol/L,H4:glucose concentration of 35 mmol/L,H5:glucose concentration of 50 mmol/L.Data are presented in mean ± SD,^□P < 0. 05 versus normal control group

Fig. 2

Effects of different concentrations of glucose on the protein expression of NLRP3.The protein was measured by western blotting.GAPDH was used as an internal loading control. NG:normal control group,H1:glucose concentration of 15 mmol/L,H2:glucose concentration of 25 mmol/L,H3:glucose concentration of 30 mmol/L,H4:glucose concentration of 35 mmol/L,H5:glucose concentration of 50 mmol/L.Data are presented in mean ± SD,^△P < 0. 05 versus normal control group

Fig. 3

a, b, c Expression of NLRP3, ASC and Caspase-1 in NG,HM,HG group at various time points (24 h,48 h,72 h).The protein was measured by western blotting.GAPDH was used as an internal loading control. NG:normal control group,HM:mannitol osmotic pressure control group,HG:high glucose group.Data are presented in mean ± SD, ^□P < 0. 05 versus normal control group

Fig. 4

a, b Expression of IL-1β and IL-18 in NG,HM,HG group at various time points (24 h,48 h,72 h).The protein levels of IL-1β and IL-18 in culture supernatants were determined by ELISA. NG:normal control group,HM:mannitol osmotic pressure control group,HG:high glucose group.Data are presented in mean ± SD, ^□P < 0. 05 versus normal control group

Fig. 5

a, b Effects of varying MCC950 concentrations (0,1,10,50 and 100 μmol/L) on NLRP3 and Caspase-1 expression within the high glucose group. The protein was measured by western blotting.GAPDH was used as an internal loading control. NG:normal control group,HG:high glucose group.Data are presented in mean ± SD, ^□P < 0. 05 versus normal control group, ^△P < 0. 05 versus high glucose group

Fig. 6

a, b Effects of varying MCC950 concentrations (0,1,10,50 and 100 μmol/L) on IL-1β and IL-18 expression within the high glucose group. The protein levels of IL-1β and IL-18 in culture supernatants were determined by ELISA. NG:normal control group,HG:high glucose group.Data are presented in mean ± SD, ^□P < 0. 05 versus normal control group, ^△P < 0. 05 versus high glucose group

Fig. 7

a, b Effects of (a) different naringin concentrations (0,0.01,0.1, 1.00,10.00 and 100umol/L),(b) different naringin concentrations (0,5,10,20,40 and 80 umol/L) on the activity of normal glucose and proliferation of high glucose in rat glomerular mesangial cell.Proliferation was measured by MTT. NG:normal control group,HG:high glucose group,DMSO:DMSO content of 1% group.Data are presented in mean ± SD, ^□P < 0. 05 versus normal control group, ^△P < 0. 05 versus high glucose group

Fig. 8

a, b Effects of different naringin concentrations (0,5,10,20,40 and 80 umol/L) on the expression of NLRP3 and Caspase-1 within the high glucose group. The protein was measured by western blotting.GAPDH was used as an internal loading control. NG:normal control group,HG:high glucose group.Data are presented in mean ± SD, ^□P < 0. 05 versus normal control group, ^△P < 0. 05 versus high glucose group

Fig. 9

a, b Effects of different naringin concentrations (0,5,10,20,40 and 80 umol/L) on the expression of IL-1β and IL-18 within the high glucose group.The protein levels of IL-1β and IL-18 in culture supernatants were determined by ELISA. NG:normal control group,HG:high glucose group.Data are presented in mean ± SD, ^□P < 0. 05 versus normal control group, ^△P < 0. 05 versus high glucose group

Fig. 10

a, b Effects of naringin (40umol/L) and MCC-950 (100umol/L) on the expression of NLRP3 and Caspase-1 within the high glucose group. The protein was measured by western blotting.GAPDH was used as an internal loading control. NG:normal control group,HG:high glucose group,Nar:naringin 40umol/L + HG,MCC950:MCC-950 100umol/L + HG.Data are presented in mean ± SD, ^□P < 0. 05 versus normal control group, ^△P < 0. 05 versus high glucose group

Fig. 11

a, b Effects of naringin (40umol/L) and MCC-950 (100umol/L) on the expression of IL-1β and IL-18 within the high glucose group.The protein levels of IL-1β and IL-18 in culture supernatants were determined by ELISA. NG:normal control group,HG:high glucose group,Nar:naringin 40umol/L + HG,MCC950:MCC-950 100umol/L + HG.Data are presented in mean ± SD, ^□P < 0. 05 versus normal control group, ^△P < 0. 05 versus high glucose group