Protective effect and mechanism of allicin on renal injury in rats with membranous nephropathy

Objective  To explore the effect of allicin on renal injury in rats with membranous nephropathy （MN） and elucidate its potential mechanism.

Methods  A total of 65 male Wistar rats were assigned into two groups of normal （n = 10） and modeling （n = 55）. Modeling group received an injection of cationized bovine serum albumin （C-BSA） via tail vein for MN modeling. And 50 modeling rats were randomized into five groups of model, low-dose allicin （5 mg/kg）, medium-dose allicin （10 mg/kg）, high-dose allicin （20 mg/kg） and nenazepril hydrochloride （10 mg/kg） （n = 10 each）. After once daily continuous dosing for 4 weeks, 24 h urinary total protein （24 h UTP）, blood urea nitrogen （BUN） and serum creatinine （Scr） were detected by spectrophotometry; serum levels of total protein （TP） and albumin （Alb） by biochemistry; serum levels of interleukin-1β （IL-1β）, IL-6/10 and tumor necrosis factor-alpha （TNF-α） by enzyme-linked immunosorbent assay （ELISA）. Renal histopathology was observed after stains of hematoxylin eosin （HE） and periodic acid-Schiff （PAS） and immunoglobulin G （IgG） immunofluorescence. Apoptosis of renal cells was observed through TdT-mediated dUTP nick-end labeling （TUNEL） stain. The expressions of Toll-like receptor 4 （TLR4）, nuclear factor-κB p65 （NF-κB p65）, p-NF-κB p65, B lymphoblastoma 2 （Bcl-2）, Bcl-2-associated X protein （Bax）, caspase-3 and cleaved caspase-3 in renal tissue were detected by Western blot.

Results  24 h UTP （45.07±6.19）mg, BUN （46.45±5.22）mmol/L, Scr （24.90±2.81）μmol/L were significantly higher in model group than 24 h UTP （6.41±0.82）mg, BUN （28.93±3.17）mmol/L and Scr （5.74±0.76）μmol/L in normal control group. The difference was statistically significant （P＜0.05）; TP （37.25±4.09）g/L and Alb （18.62±2.03）g/L were significantly lower in model group than TP （59.38±7.24）g/L and Alb （30.74±2.60）g/L in normal control group. The difference was statistically significant （P＜0.05）. Serum levels of IL-1β （638.14±69.22）ng/L, IL-6 （115.08±13.61）ng/L and TNF-α （341.75±40.93）ng/L were significantly higher in model group than IL-1β （157.42±18.06）ng/L, IL-6 （32.61±3.74）ng/L and TNF-α （60.28±6.51）ng/L in normal control group. Serum level of IL-10 was significantly lower in model group than that in normal control group （24.90±3.01） vs （75.14±9.76）ng/L. The difference was statistically significant （P＜0.05）. The pathological changes of renal tissue in model group included greater glomerular volume, mesangial hyperplasia, basement membrane thickening, vacuolar degeneration and necrosis of renal tubule cells, infiltration of inflammatory cells in renal interstitium and diffuse deposition of capillary loop IgG. Apoptotic index was statistically higher in model group than that in normal control group （63.90±8.74）% vs （4.58±0.52）%. The difference was statistically significant （P＜0.05）. Expressions of TLR4 （0.34±0.06） and Bax （0.27±0.05） and ratios of p-NF-κB p65/NF-κB p65 （0.93±0.18） and cleaved caspase-3/caspase-3 （0.67±0.09） were statistically higher in model group than those of TLR4 （0.03±0.01） and Bax （0.03±0.01） and those of p-NF-κB p65/NF-κB p65 （0.09±0.03） and cleaved caspase-3/caspase-3 （0.15±0.03） in normal control group. The differences were statistically significant （P＜0.05）. Expression of Bcl-2 was statistically lower in model group than that in normal control group （0.08±0.02） vs （0.37±0.06）. The difference was statistically significant （P＜0.05）. 24 h UTP （25.72±3.49）, （13.35±1.60）, （23.11±3.57）mg, BUN （39.28±4.38）, （34.17±3.62）, （40.35±4.81）mmol/L and Scr （14.61±1.85）, （9.05±1.12）, （12.72±1.50）μmol/L were statistically lower in medium/high-dose allicin and benazepril hydrochloride groups than 24 h UTP （45.07±6.19）, BUN （46.45±5.22） and Scr （24.90±2.81）μmol/L in model group. TP （46.39±5.02）, （53.47±6.58）, （45.63±5.17）g/L and Alb （23.38±2.91）, （27.50±3.04）, （24.08±2.86）g/L were statistically higher than TP （37.25±4.09）g/L and Alb （18.62±2.03）g/L in model group. The differences were statistically significant （P＜0.05）. IL-1β （349.18±44.06）, （201.33±26.07）, （317.92±38.64）ng/L, IL-6 （73.83±9.17）, （50.90±5.25）, （82.76±9.83）ng/L and TNF-α （228.14±25.60）, （101.57±12.94）, （193.82±23.74）ng/L were significantly lower than IL-1β （638.14±69.22）, IL-6 （115.08±13.61） and TNF-α （341.75±40.93）ng/L in model group. IL-10 was significantly higher than that in model group （49.26±6.35）, （62.35±7.71）, （55.60±6.15） vs （24.90±3.01）ng/L. The difference was statistically significant （P＜0.05）. Renal histopathological changes and apoptosis improved markedly and apoptotic index was significantly lower than that in model group （63.90±8.74）%, （63.90±8.74）%, （63.90±8.74）% vs （63.90±8.74）% . The difference was statistically significant （P＜0.05）. Expressions of TLR4 （0.24±0.04）, （0.09±0.02）, （0.15±0.03） and Bax （0.14±0.02）, （0.08±0.02）, （0.16±0.03） and ratios of p-NF-κB p65/NF-κB p65 （0.45±0.07）, （0.21±0.04）, （0.31±0.05） and cleaved caspase-3/caspase-3 （0.47±0.07）, （0.20±0.04）, （0.38±0.06） were significantly lower than those of TLR4 （0.34±0.06） and Bax （0.27±0.05） and those of p-NF-κB p65/NF-κB p65 （0.93±0.18） and cleaved caspase-3/caspase-3 （0.67±0.09） in model group. Expression of Bcl-2 was significantly higher than that in model group （0.19±0.04）, （0.30±0.06）, （0.28±0.05） vs （0.08±0.02）. The difference was statistically significant （P＜0.05）. Except for BUN, low/medium/high-dose allicin group demonstrated dose-dependent regulatory effects on other detection parameters （P＜0.05）. Except for IL-10/Bcl-2, regulatory effect of high-dose allicin group was better than that of benazepril hydrochloride group （P＜0.05）.

Conclusion  Allicin may reduce renal injury and protect renal function in MN rats through suppressing TLR4/NF-κB signaling pathway of modulating inflammatory responses and apoptosis.