| Part 1 The protective effect of sappanone A on cisplatin-induced kidney damage in miceObjective:Animal experiment was used in this study to investigate whether SA could protect the renal injury induced by CP or not.And to explore the possible mechanism by detecting the level of oxidative stress,release of inflammatory mediators and cell apoptosis which will provide an important basis for clinical using of SA on improving the CP-induced renal injury.Methods:Balb/c mice were randomly divided into five groups:the control group(Control),CP group(20 mg/kg),CP+SA low-dose group(10 mg/kg),CP+SA middle-dose group(20 mg/kg)and CP+SA high-dose group(40 mg/kg).Serum levels of BUN and Cr content were detected;HE and observation of renal histopathological changes were used to evaluate the injury of renal tissue.The apoptosis of kidney tissue was detected by the TUNEL;qRT-PCR was used to detect the mRNA of TNF-α,IL-1β,Nrf2 and HO-1 and the expression of TNF-α,IL-1β,HO-1 and p-p65,p65,IκB,p-IκB and Nrf2 proteins in kidney tissue of mice were respectively detected by ELISA or western blot.Result:1 The results of BUN and Cr assay showed that BUN and Cr levels in CP group were significantly increased compared with control group(P<0.01),while serum BUN and Cr levels in CP+ SA groups were significantly lower than that in CP group in dose-dependent manner(P<0.05).2 Renal histopathological observation showed that renal tissue injury was more sever in CP group,showing renal tubular dilation,tubular necrosisand shedding a large number of cells,a large number of cell debris accumulation within the lumen.However,the kidney tissue in SA+CP group showed a slight injury,renal proximal tubule epithelial cells became flattened in addition,the degree of partial deletion tubule cells was relatively light.The results of TUNEL showed that CP increased the ratio of apoptosis,while SA could decrease the apoptosis rate induced by CP in renal tissue.3 Compared with control group,SOD activity significantly decreased in CP group(P<0.05);when administered in combination with cisplatin and sappanone A,SOD activity was restored in a dose-dependent manner.4 MDA level was significantly higher(P<0.05)in CP group than that in control group;when administered in combination with cisplatin and sappanone A,the MDA was significantly reduced in a dose-dependent manner;5 The results of western blot showed that the p-p65 and p-IκB expression was significantly higher in CP group than that in control group(P<0.05);when administered in combination with cisplatin and sappanone A,NF-κB activity in kidney tissue was significantly reduced in a dose-dependent manner.6 Fluorescence quantitative PCR results showed that compared with normal control group,TNF-α and IL-1β mRNA in renal tissue of CP group was significantly higher(P<0.05),whereas the expressions of Nrf2 and HO-1mRNA were significantly reduced(P<0.05).When administered in combination with cisplatin and sappanone A,TNF-α and IL-1β mRNA decreased significantly(P<0.05),while Nrf2 and HO-1 mRNA expressions were significantly increased(P<0.05);7 The results of western blot and ELISA showed that TNF-α and IL-1βprotein expression was significantly higher(P<0.05),while Nrf2 and HO-1protein expression was significantly decreased in CP group(P<0.05)compared to the control group.When administered in combination with cisplatin and sappanone A,TNF-α and IL-1β protein was significantly decreased(P<0.05);while Nrf2 expression and HO-1 protein wassignificantly increased(P<0.05)in dose-dependent manner.Summary:1 SA could improve the renal injury induced by CP,which might be correlated with inflammation or/and oxidative stress inhibitory and decrease of tubule cell apoptosis.2 SA protected the renal tubule cell induced by CP partly through inhibiting the activation of NF-κB signal pathway,reducing the inflammation and renal tubule cell apoptosis.Part 2 The protective effect of sappanone A on the HK-2 cells induced by cisplatinObjective: Cell culture was used in this study to investigate the mechanism of SA on CP-induced HK-2 cells injury by determining the cell apoptosis,release of TNF-α and IL-1β and pNF-κBp65 expression.Methods: HK-2 cells were cultured in DMEM/F12 medium supplemented with 10% fetal bovine serum and random divided into five groups:control group(DMSO group),CP group(5 μM);5 μM CP+10 μM SA group;5 μM CP +20 μM SA group and 5 μM CP+30 μM SA group.MTT was used to detect the proliferation;FCM was used to detect the apoptosis of HK-2 cells;The level of MDA and ·OH in HK-2 were detected using colorimetry;ELISA was used the determine the content of TNF-α,IL-1β in the supernatant of HK-2 cells and western blot was used to measure the expressions of p-p65,p65,IκB,p-IκB and Nrf2 proteins.Result:1 MTT showed that the proliferation level of HK-2 significantly decreased in CP group,while SA could increase the proliferation level of HK-2 in dose-dependent manner induced by CP.2 FCM showed that the ratio of apoptosis significantly increased in CP group compared with control group,while SA decreased the cell apoptosis induced by CP in dose-dependent manner.3 ELISA results showed that TNF-α and IL-1β protein levels were significantly higher in CP group than that in control group(P<0.05).Whenadministered in combination with cisplatin and sappanone A,TNF-α and IL-1β protein levels were significantly decreased(P<0.05)compared with CP group in dose-dependent manner.4 MDA and ·OH protein levels were significantly higher in CP group than that in control group(P<0.05).When administered in combination with cisplatin and sappanone A,MDA and ·OH protein levels were significantly decreased(P<0.05)compared with CP group in dose-dependent manner.4 The expression of p-p65 and p-IκB protein increased and IκB protein expression decreased in CP group compared with control group.While the p-p65 and p-IκB down-regulated in SA+CP group in dose-dependent manner.The results of western blot showed that Nrf2 nucleoprotein expression was significantly decreased in CP group(P<0.05)compared with that of control group When administered in combination with cisplatin and sappanone A,Nrf2 expression was significantly increased(P<0.05)in dose-dependent manner.Summary:SA protected the renal tubule cell partly through inhibiting the activation of NF-κB signal pathway,increasing the expression of Nrf2 nucleoprotein,reducing the inflammation and renal tubule cell apoptosis.Part 3 The precise role of Nrf2 signal pathway in SA anti-cispl-、 atin-induced HK-2 cell injuryObjective: To determine the precise role of Nrf2 signal pathway in SA anti-cisplatin-induced HK-2 cell injury by measuring the effect of Nrf2 knockdown on cell apoptosis,oxidative stress,activity of NF-κB and release of IL-1β in the HK-2 cells.Methods: The experimental cells were divided into 8 groups:normal cells group;normal HK-2 plus CP,normal HK-2 plus SA,normal HK-2 plus CP and SA,Nrf2 siRNA HK-2 group,Nrf2 siRNA HK-2 plus CP group,Nrf2 siRNA HK-2 plus SA group,Nrf2 siRNA HK-2 plus CP and SA group.The level of MDA,·OH and SOD in HK-2 were detected using colorimetry.Cell viability of each group was detected by MTT assay,apoptosis was detectedby TUNEL and flow cytometry;Nrf2,HO-1 and p-p65 protein expression was detected by western blot method;ELISA was used to detect the content of IL-1β in supernatant.Result:1 Flow cytometry and TUNLE results showed that the apoptosis rate of HK-2 cell in CP group increased and SA decreased the ration of apoptosis induced by CP.However,knockdown of Nrf2,SA could not improve the apoptotic level and the ratio of apoptosis increased.2 The expression of MDA and ·OH increased in CP group compared with control group;while MDA and ·OH significantly decreased in SA+CP group compared with CP group.However,the MDA and ·OH significantly increased in SA+CP+siRNA group compared with SA+CP group.3 The expression of HO-1 in HK-2 cells transfected with Nrf2 siRNA was significantly lower than that in CP +SA group.4 CP up-regulated the expression of p-p65 protein,and SA decreased the p-p65 expression incduced by CP.However,the p-p65 protein expression in CP+SA+Nrf2 siRNA was significantly up-regulated compared with CP+SA group.5 The result of ELISA showed that knockdown of Nrf2 expression could increase the content of IL-1β in supument of HK-2 of CP+SA group.Sumary:1 Knockdown of Nrf2 in HK-2 cells could upregulate the apoptosis rate,reversed cell damage of SA anti-CP-induced..2 Knockdown of Nrf2 in HK-2 cells significantly decreased the expression of HO-1 protein in SA+CP group.3 Knockdown of Nrf2 in HK-2 cells significantly reversed p-p65 expression induced by SA+CP,and upregulated the release of IL-1βConclusions:1 SA could improve the renal tissue injury induced by CP,which might correlated with improving inflammation and/or oxidative stress,and thereby decreased the cell apoptosis.2 SA protected the renal tissue injury induced by CP partly through upregulating the expression of Nrf2,increasing the expression of HO,thereby inhibiting oxidative stress.3 SA reversed the apoptosis of the renal tubular epithelial cells induced by CP,which might correlate with inhibiting the activation of NF-κB and decreasing the release of inflammatory mediator. |
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