| Hepatic ischemia reperfusion injury (HIRI) often secondary to severe trauma, hemorrhagic shock due to liver ischemia, liver surgery required line Pringle,liver transplantation, which is a complex pathophysiological process caused by multiple factors. Current study found that endoplasmic reticulum stress (ERS) had the role of cell self-protection, moderate ERS will start cell survival pathway, but the extent of the stress response is too strong or too long, ERS will cause cells apoptosis and necrosis. The cells apoptosis mediated by ERS plays an important role in HIRI. The study confirmed that hepatic ischemia reperfusion received Salvia pretreatment could reduce calcium overload and lipid peroxide free radicals damage to play cytoprotective effects, but it regulated ERS was unknown.The experiment was observed the expressions of activating transcription factor-6a(ATF-6α) and Cysteinylasparate specific proteinase-12(Caspase-12) during the periods of hepatic ischemia-reperfusion injury and the effect of Radix Salvia Miltiorrhiae(RSM) in this process, to explore whether steady state play a protective effect by adjusting the ERS. 1. ObjectiveWe established rat liver ischemia reperfusion model, and investigated the expressions of activating transcription factor-6a(ATF-6a) and Cysteinylasparate specific proteinase-12(Caspase-12) during the periods of hepatic ischemia-reperfusion injury and the effect of radix Salvia miltiorrhiae(RSM) in this process.2. Methods(1)A total of80Wistar rats were randomly divided into4groups:normal group, sham-operation group, ischemia-reperfusion group and Radix Salvia Miltiorrhiza (RSM)-pretreated group. Then the last three groups were divided into five subgroups by different reperfusion time(Oh,3h,12h,24h,72h). Every subgroup has five rats.(2)Took samples soon after broken neck executed in normal group group. The preparation of preoperation in ischemia-reperfusion group group was the same as normal group, took the middle abdominal incision about3cm into the abdominal after intraperitoneal injection anesthesia (3%pentobarbital sodium40mg/kg), then dissociated and blocked hepatic pedicle by non-invasive artery clamp for45min, and released it. Directly took samples at Oh subgroup, but sew up the wound of rats to continue feeding in other subgroups, and took samples at the reperfusion time points (3h,12h,24h, and72h). Preoperative treatment and anesthesia methords were as the same as the ischemia-reperfusion group in sham-operation group group, dissociated but not blocked hepatic pedicle, took samples at different time points. In RSM group, tail vein was injected salvia miltiorrhiza (6ml/kg).Other steps were as the same as ischemia-reperfusion group.(3)The liver preparation was put into10%formalin. The preparation for Western blot was stored in liquid nitrogen.(4) Hematoxylin and eosin stain and immunohistochemical SABC method were operated according to the procedure of the kit. Rabbit anti-ATF-6α and Caspase-12(both working concentration was1:100) were purchased from Wuhan Boster biotechnological company.(5)The expressions of ATF-6α and Caspase-12were also detected by Western blot method:liver tissue stored in-80℃refrigerator, mixed with cell lysis buffer, protein quantitatively by the Bradford method.40μg sample mixed with5X protein sample buffer which made its final concentration of1×,95℃for5min. the protein denaturation, SDS-PAGE electrophoresis separation, transferred to the PVDF membrane, the membrane on the decoloring table with5%skim milk (0.5%TBST) closed after1h to primary antibody incubation (1:500)4℃for a night at room temperature; Use thiobarbituric acid buffer (3×5min); add second antibody (1:3000) for1h incubation at room temperature, coloration by chemiluminescence method, X-ray exposure.(6)The pathohistolgical changes were detected in light and electron microscope. The expression of ATF-6α and Caspase-12protein were investigated by immunohistochemical SABC method at the different time points of hepatic ischemia45min in rats.The results were quantitated by Image pro-plus6.0software analytical system.(7) All metrology data was disposed in statistics by SPSS13.0. Factorial design ANOVE was used to analyze the main effects and interaction of all groups. The difference of every groups was tested by LSD method. P<0.05means that there was statistical difference of experimental groups.3. Results (1)The injury of the hepatocyte was aggravated with the increase of reperfusion time in micropathological structure. The peak of the injury was at the12hours reperfusion and the injury recovered gradually at the24hours reperfusion. The injury of RSM pretreatment groups was lower than that of ischemia and reperfusion groups.(2)The expression of ATF-6a were in a low levels in normal group and sham operated group. There was no statistical difference between them (P>0.05). The expression of ATF-6α was increased after hepatic ischemia, and it was increasing with the reperfusion time.The peak of ATF-6a’s expression appeared at twelve hours reperfusion, and the expression decreased at24hours. There was significant statistical difference between ischemia reperfusion group and sham operated group (P<0.01). The expression of ATF-6a in RSM pretreatment groups was higher than ischemia reperfusion groups. There were statistical difference between them (P<0.01). The expression also decreased at24hours, but did not approached natural level until72hours.(3)There was no statistical difference of the expression of Caspase-12between normal group and sham operated group (P>0.05). The expression peak appeared atl2hours and it decreased at24hours. The compare between ischemia reperfusion group and sham operated group showed significant statistical difference (P<0.01). The expression of Caspase-12in RSM pretreatment group was lower than that in ischemia reperfusion group (P<0.01).4. ConclusionsThis experimental study results showed that:the expression of ATF-6α began to increase after reperfusion0h,3h,12h,24h and72h, maintained at the high level. The peak of ATF-6α’s expression appeared at twelve hours reperfusion, the expression decreased at24hours, but still higher than the normal control group and the sham group. The results promoted that ATF-6a as the important role of endoplasmic reticulum stress involved in regulating the ischemia and reperfusion injury during Oh-72h. ATF-6a expressed in the central vein during0h and3h after reperfusion, the reason may be due to blood oxygen partial pressure in the central vein area. Oxygen radicals and a large number of metabolites accumulated in the central vein during the period of reperfusion, this process exacerbated hepatocyte around the central vein, ATF-6a enhanced the unfolded protein response to reduce hepatocyte injury.Caspase-12is an important regulatory factor in endoplasmic reticulum stress-induced apoptosis.In this experiment, the expression of Caspase-12and ATF-6a trend was very similar in temporal and spatial, Oh-3h expressed near the central vein and portal area, reached the peak at12h, throughout the liver lobule cells at24h, expressed around the central vein at72h. These results prompted that45min ischemia and subsequent reperfusion injury in rats was a big strike, as the reperfusion injury, excessive ESR played a regulatory role of ATF-6α. The cell death program via caspase cascade activation triggered, oxygen partial pressure was lowly around the central vein, hepatocyte around the central vein proned to cell death-The results suggested that the protective effect of Salvia was closely related to alleviate excessive ERS reaction.The mechanism may be as follows:it provided a certain amount of energy substrates for liver cells through the microcirculation and antioxidant effects, promoted normal metabolic activity, reduced acidic metabolites, peroxides, and a lot of the unfolded protein accumulation, reducd excessive endoplasmic reticulum stress, Salvia maintained a steady state balance of the endoplasmic reticulum stress. |
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