| Objective: To investigate the Protective effect and mechanism of chlorogenic acid(CGA)from Eucommia ulmoides(EU)on hepatic ischemia-reperfusion injury(HIRI)from in vivo and in vitro levels.Methods: 1.In vivo experiments: In vivo level study of the protective mechanism of CGA from EU against HIRI: 24 rats were randomly divided into sham-operated group(Sham),ischemia-reperfusion + CGA group(I/R+CGA)and ischemia-reperfusion group(I/R).20 m L/kg.d of CGA from EU was given by gavage at 100 mg/kg.d in the I/R+CGA group.After10 d of continuous pretreatment,the I/R+CGA and I/R groups were established as HIRI models in rats(ischemia for 1 h and reperfused for 4 h).After modeling,the liver tissue was taken to detect the following indexes:(1)HE staining to observe the histopathological damage of liver in each group,and the difference of histopathological score between groups were analyzed.(2)ROS fluorescence probe(DHE)was used to detect the expression of ROS in liver tissues during HIRI in rats.(3)Effect of CGA from EU on sterile inflammatory response in rat HIRI:(1)Western-blot to detect the expression level of IRF-1 and immunoprecipitation(IP)combined with Western-blot to detect the level of acetylated HMGB1(Ac-HMGB1)in liver tissue.(2)RT-q PCR and Western-blot were used to detect HMGB1,RAGE,TLR-4 m RNA and protein levels,respectively.(3)Western-blot was performed to detect the expression levels of TLR-4/NF-κB signaling pathway-related proteins: My D88,P65,P-p65,P-IκB-α,IκB-α,IL-1β,TNF-α.(4)The effect of CGA from EU on HIRI-induced apoptosis in liver tissue of rats:(1)TUNEL staining was used to detect apoptosis in liver tissue of each group.(2)Western-blot was used to detect the expression levels of mitochondrial apoptosis pathway-related proteins: BCL-2,Bax,Cyt-c,Cleaved-caspase9,Cleaved-caspase3,ENDOG and AIF.2.In vitro experiments:(1)To further clarify the protective mechanism of CGA from EU on HIRI aseptic inflammatory response: normal human hepatocyte line HL-7702(L02)was cultured and passaged,and TLR-4 overexpressing L02 cell was constructed.The effect of CGA from EU on the viability of L02 cells was examined by CCK8,and low,medium and high dose CGA were selected to culture with TLR-4 overexpressing L02 cells according to the CCK8 results,and the optimal CGA concentration was obtained for the experiment.The cells were divided into control group(NC),TLR-4 overexpression group(OE)and OE+CGA group.The OE+CGA group was pretreated with CGA for 48 h,and the expression levels of TLR-4/NF-κB signaling pathway proteins: TLR-4,My D88,P65,P-p65,P-IκB-α,IκB-α were detected by Western-blot.(2)To further clarify the protective mechanism of CGA from EU on HIRI-induced apoptosis:(1)Cells were divided into NC group,hypoxia/reoxygenation group(H/R)and H/R+CGA low,medium and high dose groups,the latter were given low,medium and high dose CGA from EU and cultured with L02 cells for 48 h.H/R models were established in both H/R group and CGA pretreatment group(hypoxia for 6 h and 12 h of reoxygenation).Cells were collected to detect the expression of Cleaved-caspase3 in each group,and the optimal dose of CGA from EU was determined for subsequent experiments.(2)The cells were divided into NC group,H/R group and H/R+CGA group,and the cell treatment and hypoxia-reoxygenation model were established as before.Cells were collected to detect the following indicators:(A)The ROS production level of each group was detected by ROS fluorescence probe(DCFH-DA)and flow cytometry.(B)Mito-Tracker Red CMXRos and Annexin V-FITC fluorescence probes were used to determine the H/Rinduced mitochondrial damage and apoptosis in L02 cells.(C)Western-blot was used to detect the effects of CGA from EU on the expression levels of mitochondrial apoptosis pathway related proteins: BCL-2,Bax,Cyt-c,Cleaved-caspase9,Cleaved-caspase3,ENDOG and AIF.Results: 1.In vivo experiments:(1)The liver histopathology score in the I/R+CGA group was significantly lower than that in the I/R group(P<0.017).(2)The fluorescence intensity of ROS was significantly reduced in the I/R+CGA group compared with the I/R group(P<0.01).(3)The effects of CGA from EU on the HIRI-induced aseptic inflammatory response in rats:(1)the expression levels of IRF-1 and Ac-HMGB1 were significantly decreased in the I/R+CGA group compared with the I/R group(P<0.01);(2)The expression of HMGB1,TLR-4,RAGE m RNA and protein were significantly decreased in the I/R+CGA group compared with the I/R group(P<0.01).(3)My D88,P65,P-p65,P-IκB-α,IL-1β,TNF-α protein expression levels were significantly lower in the I/R+CGA group compared with the I/R group(P<0.01),while IκB-αprotein expression levels were significantly higher than those in the I/R group(P<0.01).(4)Effect of CGA from EU on HIRI-induced hepatocyte apoptosis in rats:(1)TUNEL staining results showed that apoptotic cells were significantly reduced in the I/R+CGA group compared with the I/R group(P<0.01).(2)Compared with the I/R group,the expression of BCL-2 was significantly increased in the I/R+CGA group(P<0.05),while the expression of Bax,Cyt-c,Cleaved-caspase9,Cleaved-caspase3,ENDOG and AIF were significantly decreased(P<0.05).2.In vitro experiments:(1)The protective mechanism of CGA from EU on HIRI aseptic inflammatory response: TLR-4 overexpressing L02 cell was identified and successfully constructed;CCK8 assay results showed that CGA(12.5 ～ 200 μM)had no toxic effect on L02cells(P<0.05),and 200 μM CGA reduced TLR-4 protein expression level most significantly(P<0.01).Compared with the OE group,the expression levels of TLR-4,My D88,P65,P-p65 and P-IκB-α protein were significantly decreased(P<0.05),while the expression levels of IκB-α protein were significantly increased(P<0.05)in the OE+CGA group.(2)Protective mechanism of CGA from EU against HIRI-induced apoptosis:(1)Cleaved-caspase3 protein levels were significantly decreased in the H/R+CGA medium and high dose groups compared with the H/R group(P<0.05),with the high dose group(200 μM)being the most significant(P<0.01),and this dose was the optimal dose.(2)The ROS fluorescence level was significantly decreased in the H/R+CGA group compared with the H/R group(P<0.01).(3)Compared with the H/R group,the mitochondrial fluorescence intensity was enhanced and apoptosis was reduced in the H/R+CGA group(P<0.01).(4)Compared with the H/R group,the expression of BCL-2 was significantly increased(P<0.05);the expression of Bax,Cyt-c,Cleaved-caspase9,Cleaved-caspase3,ENDOG and AIF proteins were significantly decreased(P<0.05).Conclusion: The current study suggests that : CGA from EU exerts a protective effect against HIRI by reducing the production of ROS during HIRI,inhibiting the HMGB1/TLR-4/NF-κB signaling pathway-mediated sterile inflammatory response during HIRI,and attenuating HIRIinduced mitochondrial damage. |
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