2.1 β-sitosterol improves renal function and albuminuria in model rats
Within 8w after administration, compared with the normal control group (NC group), the renal function of the model group changed significantly, as shown in figure 1, ALB increased obviously (figure 1A, P < 0.01), CREA (figure 1C, P < 0.005) and BUN (figure 1D, P < 0.05) decreased significantly, these results demonstrate that a model of acute nephritis was successfully established. Meanwhile, figures 1 show that the ALB (figure 1A), CREA (figure 1C) and BUN (figure 1D) in the β-sitosterol group improved significantly compared with those in the model group. Specifically, 44.47±3.35g/L, 74.03±4.31 μmol/L and 6.88±1.17 mmol/L in high dose group (HD group) , 40.32±3.22g/L, 83.35±3.38 μmol/L and 8.19±1.09 mmol/L in low dose group(LD group). It is worth noting that the therapeutic effect of LD group is better than that of HD group. TP (figure 1B) was no significant difference among these four groups.
2.2 β-sitosterol improves renal pathology in model rats
After HE staining, There was abnormal changes among 4 groups under microscope. As shown in the figure2A, the glomerulus in the NC group has clear structure, normal size, and the arrangement of renal tubular epithelial cells is regular and orderly. In the AC group, the glomerular volume increased significantly, some renal tubular epithelial cells were vacuolar degeneration, and a small amount of inflammatory cell infiltration was observed; After β-sitosterol intervention, compared with the AC group, the glomerular volume increased, the degree of renal tubular vacuolar degeneration decreased and the inflammatory cell infiltration decreased in each dose group.
The results of Masson staining as shown in the figure 2B, the collagen fibers of glomerulus, renal tubules and interstitium in the NC group were stained normally. Compared with the NC group, the glomerular basement membrane in the AC group was thickened, the blue staining of collagen fibers was significantly increased, vacuolar degeneration was observed in some renal tubules, and the blue staining of collagen fibers in renal interstitium was increased. After β-sitosterol intervention, compared with the AC group, the thickening of glomerular basement membrane in each dose group decreased in varying degrees, and the blue staining of glomerular basement membrane and renal interstitial collagen fibers decreased.
Next, TGF in tissues was detected by immunohistochemistry, as shown in figure 2C, brown staining particles can be seen in renal tubular epithelial cells of the normal control group, indicating the expression of TGF. Compared with the normal NC group, only a small amount of brown staining particles can be seen in renal tubular epithelial cells of the AC group, indicating that the expression of TGF is significantly weakened. After β-sitosterol intervention, compared with the AC group, the brown stained granules of renal tubular epithelial cells in HD and LD groups increased in varying degrees, suggesting that the expression of TGF recovered.
2.3 β-sitosterol improves renal Inflammatory level in model rats
For detection the effects of β-sitosterol in vivo, the inflammatory factors MCP-1, iNOS and TNF-α, IL-1 β and IL-6 was detected. At week 8, MCP-1, iNOS levels in AC group were significantly higher than those in NC group (figure 3A and B, P < 0.001), IL-1 β, IL-6 and TNF-α levels were higher than those in NC group (figure 3C, D and E, P < 0.005). The levels of inflammatory factors in LD group and HD group were significantly lower than those in AC group, and those in HD group were significantly lower than those in LD group (figure 3). The difference of some indexes was statistically significant. Specifically, 3.35±0.25ng/L, 0.31±0.01 U/mg,51.25±5.42 pg/mL, 61.67±4.62 pg/mL and 39.32±4.71 pg/mL in high dose group (HD group) , 5.49±0.15ng/L, 0.35±0.04 U/mg, 68.66±8.49 pg/mL, 73.67±6.49 pg/mL and 51.88±5.93 pg/mL in in low dose group(LD group). It is worth noting that the therapeutic effect of LD group is better than that of HD group. There was no significant difference in the therapeutic effect between the high-dose group and the low-dose group, indicating that low-dose β-sitosterol can achieve the effect of reducing inflammation.
2.4 β- Sitosterol reduces the level of apoptosis
Annexin V-FITC/PI staining and flow cytometric analysis were performed to determine the degree of cell apoptosis in all groups. As shown in figure 4A and B, the apoptosis rate in AC group (Q2, 16.6%, Q3,14.8%) increased significantly compare with NC group (Q2, 2.01%, Q3,2.61%). This apoptosis caused by inflammation can be improved by β-sitosterol. The apoptosis rates in HD and LD group both decreased significantly, Specifically, Q2, 5.57%, Q3, 5.56% in HD group and Q2, 8.04%, Q3, 7.70% in HD group (figure 4C and D). It is worth noting that the therapeutic effect of HD group is better than that of LD group.
2.5 β-sitosterol enhances SIRT1-induced autophagy and inhibits NLRP3 inflammasome
We further tested whether β-sitosterol inhibits the activation of NLRP3 inflammasome through the activation of Sirt1. Western blot results showed that compared with the NC group, the expression of sirt in the AC group was significantly decreased, and the LD group and HD group could increase the expression of sirt to a certain extent in renal tissue (figure 5A). Meanwhile, we found that the expressions of NLRP3 and caspase-1 were significantly increased in the AC group, while their expressions were decreased in both the LD and HD groups(figure 5A). Quantitative analysis of blot showed significant differences between the LD and HD groups compared to the AC group (figure 5B, C and D) These findings suggest that β-sitosterol inhibits NLRP3 inflammasome by activating the expression of SIRT1-mediate autophagy.