| Background:Previous research indicates that Radix salvia miltiorrhiza (RSM) can block calcium channel, which significantly reduces the calcium overload and lipid peroxidation damage of free radical, and prevents ischemic reperfusion injury. Eukaryotic translation initiation factor2α (eIF2α) is an important regulating factor in the process of translation, which is regulated by the PKR like endoplasmic reticulum kinase (PERK), a sensor for the unfolded protein in endoplasmic reticulum lumen. When the endoplasmic reticulum stress(ERS) occurs, PERK was activated by the unfolded protein response(UPR), eIF2α is phosphorylated and transformed into p-eIF2α, which makes an inhibition work on most mRNA translation, but it also specially activates the translation of some mRNA, and synthetizes specific proteins, to regulate the expression of target genes, thereby regulating endoplasmic reticulum environment, alleviating the ERS, and helping the endoplasmic reticulum the steady state balance, refraining from the ischemia reperfusion injury. But the continued excessive ERS will active Caspasel2, a specific protein about apoptosis, which induces apoptosis by endoplasmic reticulum pathway. Whether RSM can weake the unfolded protein response, stabilize the environment within the endoplasmic reticulum, and reduce cell apoptosis induced by endoplasmic reticulum pathway through affect the phosphorylation of eIF2a or not has not yet been confirmed. Base on the observation of RSM preconditioning on hepatic ischemia reperfusion in rats at different time of p-eIF2a and the expression of Caspasel2, we investigate the possible mechanisms of ischemia reperfusion injury in the homeostatic regulation of endoplasmic reticulum, and provide a theoretical basis for prevention and treatment of liver ischemia reperfusion injury.Materials and methods:1. Materials:80healthy male adult Wistar rats(body weight:200±20g), purchased from experimental animal and disease prevention and control center in Hubei province [license No.:SCXK (E)2008-0005]; RSM injection [ChiaTai Qingchunbao pharmaceutical, batch number:Z33020177]; internal reference P-actin (number:Sc-1616r) and rabbit anti-rat Caspasel2polycloonal antibody purchased from Santa company (item number:sc-5627), p-eIF2a rabbit anti-rat polyclonal antibody was purchased from CST company (item number:#3398); extraction of total cellular protein kit, SDS-PAGE gel preparation kit and other reagents purchased from Google biotechnology company.2. The experimental group:A total of80healthy male Wistar rats were divided into four groups:normal control group (n=5), sham group (n=25), ischemia reperfusion group (n=25) and RSM preconditioning group (n=25). The experimental group according to different reperfusion time (Oh,3h,12h,24h and72h) is divided into5subgroups, each subgroup n=5.3. Preparation of animal model:Preoperative fasting12h, free drinking water. The normal control group were executed after abdominal cut directly into the liver tissue; the experimental rats with (3%,40mg/kg) sodium pentobarbital intraperitoneal injection anesthesia, cut from upper middle incision about3cm; sham operation group anatomised hepatic portal but not clamped the hepatic pedicle; ischemia reperfusion group retracted the liver after opened abdomen, exposured and freed the hepatoduodenal ligament, clamped the hepatoduodenal ligament of distal placement with bulldog clamp and removed the artery clamp after45min, recovered blood flow, prepared the model of ischemia reperfusion injury. In sham group and ischemia-reperfusion group,40ml/kg of normal saline was injected through tail vein before ischemia; In RSM pretreatment group, liver ischemia-reperfusion model was prepared as ischemia-reperfusion group, RSM (6ml/kg) diluted to40ml/kg with normal saline was injected through tail intravenous before ischema. The experimental rats of each group in different reperfusion time of observation points were executed, cut to save the liver tissue in liquid nitrogen and formaldehyde.4. The expression of PERK, p-eIF2a and Caspasel2detected fay Western blot method:Tissue washed2-3times with cold TBS, to remove blood, cut into small pieces in the homogenizer, thoroughly homogenized on ice. Adding10times volume of tissue extraction reagent (Adding the phosphorylation of cooktail+protease inhibitor before a few minutes), and the homogenate was transferred to the1.5ml centrifuge tube. Oscillated and iced30min, repeated pipetting complete lysis of cells.12000r/min centrifuged5min, collecting the supernatant as total protein solution. Measured protein concentration with Bradford method, calculating the volume of solution containing40p.g protein is the sample volume.10%separation gel and5%spacer gel were used to twelve alkyl sulfide-polyacrylamide gel electrophoresis (SDS-PAGE), separated and transfered film (PVDF film was activated by methanol before using, transfered film according to200mA,1hour of conditions), confined with5%skim milk in room temperature30min, then500times dilution of PERK, p-eIF2a and Caspasel2antibodies were added. Incubated at room temperature for2hours, and wash three times with the degradation of TBST at room temperature on the shaking table every5min. Diluted the second antibody with3000times TBST, incubate at room temperature for45min. washing three times with0.5%oTBST at room temperature on rocking bed every5min, then ncubation with second antibody. The chemiluminescence reagents reacted with antibody, X-ray film exposuring, developing, fixing and the results were observed. The films were scaned by gel imaging and analysis system, and the optical density of the target zones were acquired from Alpha software processing system. The ratio of sample and reference were compared.5. The expression of PERK, p-eIF2a and Caspase12detected by Immunohistochemical method:All of the liver tissues were fixated, paraffin embedded, cut as serial4μm sections, and immersed sections with dimethylbenzene and graded ethanol, then3%hydrogen peroxide was used to inactivate endogenous peroxidase at room temperature10min. Fixed antigen with hot citric acid buffer solution, dropt goat serum as antigen closed solution, then placed in room temperature20min. Dropped appropriate dilution of antibody (1:100) and kept sections in4℃overnight. Washed by phosphate buffered solution (PBS)2min x3times; added biotinylated second antibody and kept in room temperature for20min; washed with PBS5minx4; dyed with DAB solution, the color reaction time was controled under microscopie. Staineded again with hematoxylin, dehydrated in graded ethanol, transparented with xylene, and sealed up with neutral gum. The position of immune complexes and strength of the immune response were observated under the high power microscope. When brown orange granule appeared in cytoplasm, the rusults were defined as positive; otherwise, the results were defined as negative. Image Pro-Plus6software was used to analyse immunohistochemical images in each group:Ten200times images were randomly selected to analyze, and the integrated optical density (IOD) of each image positive values was detected.6. Pathological observation:Rat liver specimens were fixed with10%formalin, embedded,and sliced routinely. Dyed by HE staining procedure, the pathological changes were observed under light microscope.7. The statistical method:The mean±standard deviation for all data (x±s), using SPSS13.0statistical software for data analysis, groups of samples were compared with factorial design analysis of variance (LSD for equal variances assumed and Games-Howell for equal variances not assumed), P<0.05as statistically significant difference.Results:1. Dynamic changes of PERK detected by Western blot:Normal control group and sham group had no statistical difference; the expression of Oh in ischemia reperfusion group PERK was significantly increased,3h continued to rise,12h reached the highest value, although24h was lower than the rest of time but still higher than that of normal group and sham operation group,72h was also expressed high levels of expression; change process of RSM pretreatment group PERK also increased firstly and then decreased, Oh increased, peaked at12h,24h decreased, but72h was still higher than that of normal group, sham group, ischemia reperfusion group. Among the three groups were significantly different (F=1927.24, P<0.01), and compared with each time point were significantly different (F=49.91, P<0.01).2. Dynamic changes of PERK detected by immunohistochemistry:Normal control group and sham group, no significant difference of the integral optical density of PERK protein expression between the two groups; ischemia reperfusion group, the protein expression of PERK in Oh started to increase,3h continued to rise, reached a maximum at12h,24h began to decline,72h decreased to normal level. In addition to other groups and the sham group in72h group, there were significant between times; and ischemia reperfusion group similarity, integral optical density value of PERK in RSM pretreatment group in Oh began to rise,12h reached the highest value,72h returned to normal levels, except the72h group, the rest groups and normal control group and sham group corresponding to each time point were compared its differences with statistical significance, in addition in addition to Oh group and72h group, the rest groups integral optical density value and ischemia reperfusion time points corresponding comparison also had a significant statistical difference. Compared among the experimental groups were significantly different (F=30643.55, P<0.01), and significantly different in different observation time (F=109371.6, P<0.01).3. Dynamic changes of p-eIF2a detected by Western blot:The expressions of p-eIF2a were no significant difference between normal control group and sham group (P>0.05), while in ischemia reperfusion group, reperfusion3h, significantly increased,12h peaked,24h was still in high level, and the normal control group and sham group had significant difference (P<0.01), until then perfusion72h declined to baseline levels over the same perriod (P>0.05); RSM group phosphorylation of eIF2a expression in3h, the observation time point of12h,24h were significantly higher than those in ischemia reperfusion group (P<0.01), and72h of reperfusion, was still at a relatively high level expression. Among the three groups were significantly different (F=258.59, P<0.01), and compared with each time point were significantly different (F=95.10, P<0.01).4. The dynamic changes of the p-eIF2a detected by immunohistochemistry: Immunohistochemical analysis of integral optical density of normal control group and sham group, p-eIF2a were no significant difference (P>0.05); integral optical density in ischemia reperfusion group p-eIF2a value changes as follows:Oh gradually increased,3h continued to rise,12h reached the highest value,24h slightly decreased but still in high expression levels,72h decreased to near normal group and sham group level; In RSM pretreatment group, p-eIF2a expression generally showed higher than the same period in ischemia reperfusion group level, and the control group and sham operation group were significantly higher than that of the difference in ischemia reperfusion group, variation trended and ischemia in the concern group similar still, until the72h was significantly higher than that in normal control group, sham group, ischemia reperfusion group. Comparison of the experimental groups were significantly different (F=63.52, P<0.01), and a comparison of different observation points were significantly different (F=250.07, P<0.01).5. Dynamic changes of Caspase12detected by Western blot:No statistically significant difference between the normal group and sham group (P>0.05); the expression of Caspasel2and ischemia reperfusion group at ischemia45min and reperfusion3h, significantly increased,12h peaked,24h was still at a relatively high level, although72h had decreased, but still higher than that of sham operation group and normal control group (P<0.01); at the same time, the expression of RSM pretreatment group Caspasel23h,12h,24h and72h were higher than those in normal control group and sham group (P<0.01), but significantly lower than the observation of ischemic reperfusion group (P<0.01). Among the three groups were significantly different (F=287.93, P<0.01), and compared with each time point were significantly different (F=70.91, P<0.01).6. Dynamic changes of Caspase12detected by immunohistochemistry: Normal control group integral optical density expression group and sham group Caspase12protein values were not statistically significant; integral optical density of each time point in the sham group and the value of Caspase12was also not a statistically significant difference; expression in ischemia reperfusion group Caspase12in Oh began to rise,3h continues to increase,12h reaches the maximum value,24h decreased compared with group12h, but still higher than that in normal group and sham operation group, and72h was still higher than normal levels of expression; the period of RSM pretreatment group Caspase12integral optical density value was significantly higher than that in normal group and sham group, RSM preconditioning group although exhibiting and ischemia reperfusion group increased the process of reduction, but with the same period in ischemia reperfusion group, RSM preconditioning group Caspase12integral optical density value was significantly lower than that in ischemia reperfusion group, with significant statistical difference. Compared among the experimental groups were significantly different (F=53.48, P<0.01), and were significantly different in different reperfusion time (F=189.56, P<0.01).7. Pathological observation:The normal structure of hepatic lobules in control group and sham group of basic integrity, liver cell continuous cable, and round nucleus. Compared with the same period, RSM pretreatment group damage than ischemia reperfusion group,3h showed mild cellular swelling, slightly heavier12h liver cell swelling, slight spotty necrosis of liver cell proliferation,24h expression to a certain extent based on72h spotty necrosis, morphological close to normal.Conclusions:The experimental results showed that in the early stage (hepatic ischemia-reperfusion (0h) and p-eIF2α (3h)), the expression of PERK was increased, significantly higher than the normal control group and sham group, this was a sign of liver ischemia reperfusion induced endoplasmic reticulum stress start. The expression of p-eIF2α in RSM pretreatment group from the3h～72h throughout the observation time was always higher than that of the untreated group, and the expression of caspase12over the same period was significantly lower than that of the untreated group. Our researches showed that RSM pretreatment was helpful to enhance the expressions of PERK and p-eIF2a in the period of reperfusion, and inhabited the ERS induced-apoptosis via PERK/p-eIF2a pathway, protected hepatocyte and liver tissue from damage. |
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