RESUMO
Objective To investigate the effect and mechanism of atorvastatin (ATV) on the inflammatory response of human renal tubular epithelial cells (HK-2 cells) induced by calcium oxalate crystals.Methods HK-2 cells were divided into control group (normal medium),ATV group (after 3 h pretreatment with 40 μmol/L ATV,replaced with normal medium),calcium oxalate crystal stimulation group (4 mmol/L calcium oxalate crystal) and ATV treatment group (after 3 h pretreatment with 40 μmol/L ATV,replaced with 4 mmol/L calcium oxalate crystals).After 12 h,the cells were collected,and the expression levels of NLRP3 and Cleaved caspase-1 were detected by immunohistochemical staining and Western blotting.The expression level of NF-κB was detected by immunofluorescence and Western blotting.The cell culture supernatant was collected to detecte the concentrations of interleukin-1 β (IL-1β) and intedeukin-18 (IL-18) by enzyme linked immunosorbent assay (ELISA).Results Western blot analysis showed that the relative expression of NLRP3 (0.125 ±0.013 vs.0.135 ±0.007) and Cleaved caspase-1 (0.090 ±0.014 vs.0.095±0.006) was decreased in the ATV group compared with the control group,but the difference was not statistically significant (P ＞ 0.05).The relative expression of NLRP3 (0.315 ±0.021 vs.0.135 ± 0.007,P ＜ 0.001) and Cleaved caspase-1 (0.235 ± 0.008 vs.0.095 ± 0.006,P ＜0.001) was significantly increased in the calcium oxalate crystal stimulation group compared with the control group.While the relative expression of NLRP3 (0.245 ±0.007 vs.0.315 ±0.021,P ＜0.05) and Cleaved caspase-1 (0.170 ±0.017 vs.0.235 ±0.008,P ＜0.05) in the ATV treatment group was significantly lower than that in the calcium oxalate crystal stimulation group.The results of immunohistochemical staining showed that the expression trends of NLRP3 and Cleaved caspase-1 in each group were consistent with those obtained by Western blotting.The ELISA results showed that the concentration of inflammatory factors IL-1 β [(162.00±21.21)pg/ml vs.(183.50±7.78) pg/ml,P＞0.05] and IL-18 [(176.50±24.12)pg/ml vs.(182.50 ± 20.51) pg/ml,P ＞ 0.05] in the ATV group was lower than that in the control group,but the difference were not statistically significant (P ＞ 0.05).The concentrations of IL-1β [(850.50 ± 48.79)pg/ml vs.(183.50 ± 7.78) pg/ml,P ＜ 0.001] and IL-18 [(526.00 ± 39.61) pg/ml vs.(182.50 ±20.51)pg/ml,P ＜0.001] were significantly increased in the cell culture medium of the calcium oxalate crystal stimulation group compared with the control group,while the concentrations of IL-1 β [(452.50 ±36.06)pg/ml vs.(850.50±48.79) pg/ml,P＜0.01] and IL-18 [(403.50 ±23.33)pg/ml vs.(526.00 ±39.61)pg/ml,P ＜0.05] was significantly reduced in the cell culture medium of the ATV treatment group compared with the calcium oxalate crystal stimulation group.Western blot analysis showed that the relative expression of NF-κB (0.105 ±0.021 vs.0.100 ±0.014) in the ATV group was decreased compared with the control group,but the difference was not statistically significant (P ＞ 0.05).The relative expression of NF-κB (0.295 ±0.035 vs.0.100 ±0.014,P ＜0.001) in the calcium oxalate crystal stimulation group was significantly increased compared with the control group.While the relative expression of NF-κB (0.160 ± 0.012 vs.0.295 ± 0.035,P ＜ 0.05) in the ATV treatment group was significantly lower than that in the calcium oxalate crystal stimulation group.The expression of NF-κB by immunofluorescence staining was consistent with the results of Western blotting.Conclusions Calcium oxalate crystals can induce the inflammatory response of HK-2 cells,while ATV can exert anti-inflammatory effects by inhibiting the activation of NLRP3 inflammasome and decreasing the secretion of inflammatory factors IL-1β,IL-18 and the expression of NF-κB.
RESUMO
Objective@#To investigate the effect and mechanism of atorvastatin (ATV) on the inflammatory response of human renal tubular epithelial cells (HK-2 cells) induced by calcium oxalate crystals.@*Methods@#HK-2 cells were divided into control group (normal medium), ATV group (after 3 h pretreatment with 40 μmol/L ATV, replaced with normal medium), calcium oxalate crystal stimulation group (4 mmol/L calcium oxalate crystal) and ATV treatment group (after 3 h pretreatment with 40 μmol/L ATV, replaced with 4 mmol/L calcium oxalate crystals). After 12 h, the cells were collected, and the expression levels of NLRP3 and Cleaved caspase-1 were detected by immunohistochemical staining and Western blotting. The expression level of NF-κB was detected by immunofluorescence and Western blotting. The cell culture supernatant was collected to detecte the concentrations of interleukin-1β (IL-1β) and interleukin-18 (IL-18) by enzyme linked immunosorbent assay (ELISA).@*Results@#Western blot analysis showed that the relative expression of NLRP3 (0.125±0.013 vs. 0.135±0.007) and Cleaved caspase-1 (0.090±0.014 vs. 0.095±0.006) was decreased in the ATV group compared with the control group, but the difference was not statistically significant (P>0.05). The relative expression of NLRP3 (0.315±0.021 vs. 0.135±0.007, P<0.001) and Cleaved caspase-1 (0.235±0.008 vs. 0.095±0.006, P<0.001) was significantly increased in the calcium oxalate crystal stimulation group compared with the control group. While the relative expression of NLRP3 (0.245±0.007 vs. 0.315±0.021, P<0.05) and Cleaved caspase-1 (0.170±0.017 vs. 0.235±0.008, P<0.05) in the ATV treatment group was significantly lower than that in the calcium oxalate crystal stimulation group. The results of immunohistochemical staining showed that the expression trends of NLRP3 and Cleaved caspase-1 in each group were consistent with those obtained by Western blotting. The ELISA results showed that the concentration of inflammatory factors IL-1β [(162.00±21.21)pg/ml vs. (183.50±7.78) pg/ml, P>0.05] and IL-18 [(176.50±24.12)pg/ml vs.(182.50±20.51)pg/ml, P>0.05] in the ATV group was lower than that in the control group, but the difference were not statistically significant (P>0.05). The concentrations of IL-1β[(850.50±48.79)pg/ml vs. (183.50±7.78)pg/ml, P<0.001] and IL-18 [(526.00±39.61)pg/ml vs. (182.50±20.51)pg/ml, P<0.001] were significantly increased in the cell culture medium of the calcium oxalate crystal stimulation group compared with the control group, while the concentrations of IL-1β [(452.50±36.06)pg/ml vs. (850.50±48.79) pg/ml, P<0.01] and IL-18 [(403.50±23.33)pg/ml vs. (526.00±39.61)pg/ml, P<0.05] was significantly reduced in the cell culture medium of the ATV treatment group compared with the calcium oxalate crystal stimulation group. Western blot analysis showed that the relative expression of NF-κB (0.105±0.021 vs. 0.100±0.014) in the ATV group was decreased compared with the control group, but the difference was not statistically significant (P>0.05). The relative expression of NF-κB (0.295±0.035 vs. 0.100±0.014, P<0.001) in the calcium oxalate crystal stimulation group was significantly increased compared with the control group. While the relative expression of NF-κB (0.160±0.012 vs. 0.295±0.035, P<0.05) in the ATV treatment group was significantly lower than that in the calcium oxalate crystal stimulation group. The expression of NF-κB by immunofluorescence staining was consistent with the results of Western blotting.@*Conclusions@#Calcium oxalate crystals can induce the inflammatory response of HK-2 cells, while ATV can exert anti-inflammatory effects by inhibiting the activation of NLRP3 inflammasome and decreasing the secretion of inflammatory factors IL-1β, IL-18 and the expression of NF-κB.