The Expression Of GRP78/94Induced By Pretreatment Of Radix Salvia Miltiorrhiza In Hepatic Ischemia Reperfusion Rats

Hepatic ischemia reperfusion injury is an important factor that impaire liver functions during liver surgery and transplantation. The mechanism and treatment of this injury have been generally investigated.Endoplasmic reticulum (ER) is an important organelle in eukaryotic cells. It is mainly involved in protein synthesis, modification and folding; steroids, cholesterol and other lipid biosynthesis and calcium ion storage. ER is one of the most important sources of other membranous organelles in the cells. It occupies the central position in the membranous systems. But it is very sensitive, and the endoplasmic reticulum ischemia-reperfusion injury may cause dysfunction of energy metabolism.Free radicals in cells increase and endoplasmic reticulum calcium overload, all of these can be stimulus of endoplasmic reticulum dysfunction, namely the endoplasmic reticulum stress (ERS). In recent years, the effect of glucose regulated proteins (GRPs) in ischemia-reperfusion injury has come into notice.GRPs is a kind of endoplasmic reticulum molecular chaperon, which is produced by endoplasmic reticulum stress. GRP78is also known as an immunoglobulin heavy chain binding protein (Bip), which is a core of unfolded protein response (UPR). GRP78is regarded as the Signature molecule of ERS and UPR. GRP94is the most GRPs in endoplasmic reticulum, it can combine with unfolded polypeptide chain, prevent protein gathered together, assist protein folding, stretch, assembly and transport, inhibit the secretion of protein folding error.Radix salvia miltiorrhiza (RSM) is a Chinese herbal medicine, which has an effect of activating blood circulationand dissipating blood stasis. It can prevent the calcium overload in liver cells during HIRI, reduce the injury of oxygen free radicals and lipid peroxidation, protect the function of cell membrane, block the cross effect of Fas and perforin, coordinate with bcl-2expression. In the past, the protecting mechanism of RSM is considered to be associated with its improvement of microcirculation, resistance of oxygen free radicals, calcium and retardation. But there are no reports talkingabout the relationship between RSM and GRPs. The main purpose of this study is to realize the expressions of GRP78and GRP94during HIRI and its significance, explore the influences of RSM on the expressions of GRP78and GRP94, further understand the molecular mechanism of RSM for liver protectionand provide more powerful evidences of using RSM to prevent HIRI.Chapter One:The expressions of GRP78and GRP94during different hepatic ischemia process in rats1. ObjectiveTo investigate the expression of GRP78, GRP94, Caspase-12and p-Aktl during different periods of hepatic ischemia in rats.2. Methods(1) Fifty-five Wistar rats were randomly divided into three groups:Normal group (n=5), sham operation group (n=25) and ischemia reperfusion group (n=25). The last two groups were divided into five subgroups by different hepatic ischemia times (0,3,12,24,72h). There were five Wistar rats in each group. And then we built the rat model of ischemia injury.(2) Preoperative fasting for12h, free drinking water. After abdominal anaesthesia, skin preparation and disinfection, we cut the normal group into abdomen and take liver tissue for assay. The control group was anatomically dissected portal vein; the ischemia-reperfusion group was placed artery clamp forceps in the hepatoduodenal ligament telecentric side for45min, and collected liver tissue in different reflow time (0h,3h,12h,24h, and72h). Half of the liver tissues were fixed in10%formalin. Enclose the left liver tissues with aluminium foil paper, and put them into liquid nitrogen container.(3) HE and immunohistochemical staining was undertaken according to the illustrationg of the kit The concentration of rabbit anti rat GRP78polyclonal antibody, rabbit anti rat GRP94polyclonal antibody, and rabbit anti rat Caspase-12polyclonal antibody is1:100. These antibodys were purchased from Santa Cruz.(4) The pathohistolgical change was detected under microscope. The expression of GRP78, GRP94and Caspase-12protein was investigated by immunohistochemical staingingduring different periods of hepatic ischemia reperfusion. The results were quantitated by Image pro-plus6.0software analytical system.(5) The expression of p-Aktlwas investigated by Western blot during different periods of hepatic ischemia reperfusion. Rabbit anti rat p-Aktl polyclonal antibody was purchased from Santa Cruz company.(6) All of the data were expressed as mean±standard deviation (x±s). SPSS13.0statistical software was used for data analysis. Multiple groups of samples were compared with ANOVA (LSD and Games-Howell were used for analysis of variance. P<0.05was considered statistically significent.3. Results(1) The pathological morphology:The structure of hepatic lobule was kept integrity in normal group and sham operation group. Liver cell was showed as polyhedron shape, and cell nucleus is in the centerin, which is big and round. In ischemia reperfusion group, liver cell was light to moderate swollen, after reperfusion Oh, hepatic sinus became narrow; reperfusion3h later, the swollen liver cell was obviously, the cytoplasm was puffing. Some liver cell appears to be acidophilic; after12h of reperfusion, the swollen and puffed liver cells diffused; after24h of reperfusion, the injury was the heaviest. Some of the hepatic lobule became deformation, and some of them appeared to be flake necrosis; after72h of reperfusion, the tide of hepatic sinus became narrow and reduced, some of the hepatic lobule reconstructed.(2) The expression of GRP78:Tan-yellow staining in the cytoplasm was considered positive expressioncell. The expressions of GRP78were weak in normal group and sham operated group. In the corresponding time point, the expression of GRP78in ischemia reperfusion group was obviously higher than that of normal group and sham operation group(p<0.01). The expression of GRP78began to elevate at0-3h after ischemia-reperfusion, the peak time was12h (p<0.01). The expression was still in a relatively peak value at24h, higher than normal group and sham operation group about one order of magnitude, higher than the reperfusion0h about2times. GRP78expression was also higher than normal group and sham operation group at72h. (3) The expression of GRP94:The expressions of GRP94in normal group and sham operation group were weak. At the same time point, the expression of GRP94in ischemia reperfusion group was obviously higher than in sham operation group (p<0.01). The expression of GRP94began to rise at0～3h after ischemia-reperfusion, and the peak time was12h (p<0.01). The expression was still in a relatively peak value at24h, higher than the normal group and sham operation group. At the time point of72h, GRP94expression was higher than that of normal group and sham operation group, which was the same level with ischemia-reperfusion group.(4) The expression of Caspase-12:Tan-yellow staining in the cytoplasm was considered positive-expression. The expressions of Caspase-12during different periods of time in ischemia reperfusion group were obviously higher than in the same time point of normal group and sham operation group. In the ischemia reperfusion group, Caspase-12had a strong expression at Oh, then continued to rise at3h (p<0.01), and reached its peak at12h (p<0.01), then began to reduce at24h, but is still far higher than that of normal group and sham operation group. At the time point of72h, Caspase-12expression was the same with ischemia-reperfusion group, but higher than normal group and sham operation group (p<0.01).(5) The expression of p-Akt1:The expression of normal group and sham operation group were weak. In ischemia reperfusion group, the expression was significantly higher than that of the other two groups. It started to express at Oh, peaked at12h, reduced but still in a higher level during24h-72h.4. ConclusionHepatic ischemia-reperfusion injury in rats can be triggered by the activation of Caspase cascade in endoplasmic reticulum; GRP78and GRP94may elevate sugar by endoplasmic reticulum reduction and dysplasia and play a role in cell protection. p-Aktl probably raise important signal molecules of GRP78expression through the PI3K/Akt signaling pathway.Chapter Two:The expression of GRP78/94induced by pretreatment of radix salvia miltiorrhiza in hepatic ischemia reperfusion rats1. ObjectiveWe aim to build rat model of hepatic ischemia and reperfusion injury and to investigate the expression of GRP78, GRP94, Caspase-12and p-Aktl during different periods of hepatic ischemia in rats. The protective effect of radix salvia miltionrrhiza (RSM) pretreatment was also investigated.2. Method(1) Eighty Wistar rats were randomly divided into four groups:normal group, sham operation group, ischemia and reperfusion group, RSM pretreatment group. Then the groups were divided into five subgroups by different reperfusion time (Oh,3h,12h,24h, and72h). Every group has five rats.(2) Preoperative fasting for12h, free drinking water. After abdominal anaesthesia, skin preparation and disinfection, we cut the normal group into abdomen and take liver tissue for assay. The control group was anatomically dissected portal vein; the ischemia-reperfusion group was placed artery clamp forceps in the hepatoduodenal ligament telecentric side for45min, and collected liver tissue in different reflow time (0h,3h,12h,24h,72h). Salvia miltiorrhiza pretreatment groups:salvia miltiorrhiza injection (0.6g/kg) and normal saline to40ml/kg, preoperative tail vein into, anesthesia and surgical methods with ischemia reperfusion group. Half of the liver tissues were fixed in10%formalin. Enclose the left liver tissues with aluminium foil paper, and put them into liquid nitrogen container(3) HE and immunohistochemical staining was undertaken according to the illustrationg of the kit.The concentration of rabbit anti rat GRP78polyclonal antibody, rabbit anti rat GRP94polyclonal antibody, and rabbit anti rat Caspase-12polyclonal antibody is1:100. These antibodys were purchased from Santa Cruz.(4) The pathohistolgical change was detected under microscope. The expression of GRP78, GRP94and Caspase-12protein was investigated by immunohistochemical staingingduring different periods of hepatic ischemia reperfusion. The results were quantitated by Image pro-plus6.0software analytical system.(5) The expression of p-Aktlwas investigated by Western blot during different periods of hepatic ischemia reperfusion. Rabbit anti rat p-Aktl polyclonal antibody was purchased from Santa Cruz company.(6) All of the data were expressed as mean±standard deviation (x±s). SPSS13.0statistical software was used for data analysis. Multiple groups of samples were compared with ANOVA (LSD and Games-Howell were used for analysis of variance. P<0.05was considered statistically significant.3. Results(1) The pathological morphology:The structure of hepatic lobule was kept integrity in normal group and sham operation group. Liver cellwas showed as polyhedron shape, cell nucleus was in the centerin, which was big and round. In ischemia reperfusion group, liver cell was light to moderate swollen, after reperfusion Oh, hepatic sinus became narrow; reperfusion3h later, the swollen liver cell was obviously, the cytoplasm was puffing.some liver cell appears to be acidophilic; after 12h of reperfusion, the swollen and puffed liver cells diffused; after24h of reperfusion, the injury was the most heavy. Some of the hepatic lobule became deformation, and some of them appeared to be flake necrosis; after72h of reperfusion, the tide of hepatic sinus became narrow and reduced, some of the hepatic lobule reconstructed. In the same time, the injury was lighter in RSM group than in ischemia reperfusion group.(2) The expression of GRP78:Tan-yellow staining in the cytoplasm was considered positive expressioncell. The expressions of GRP78were weak in normal group and sham operation group in the corresponding time point, the expression of GRP78in ischemia reperfusion group was obviously higher than that of normal group and sham operation group(p<0.01). The expression of GRP78began to elevate at0-3h after ischemia-reperfusion, the peak time was12h (p<0.01). The expression was still in a relatively peak value at24h, higher than normal group and sham operation group about one order of magnitude, higher than the reperfusion0h about2times. GRP78expression was also higher than normal group and sham operation group at72h. In RSM group, the expression of GRP78was weaker than ischemia reperfusion group at the corresponding reperfusion times (p<0.01).(3) The expression of GRP94:The expressions of GRP94in normal group and sham operation group were weak. At the same time point, the expression of GRP94in ischemia reperfusion group was obviously higher than in sham operated group (p<0.01). The expression of GRP94began to rise at0-3h after ischemia-reperfusion, and the peak time is12h (p<0.01).The expression was still in a relatively peak value at24h, higher than the normal group and sham operation group. At the time point of72h, GRP94expression was higher than that of normal group and sham operation group, which was the same level with ischemia-reperfusion group. In RSM group, the expression of GRP94was weaker than ischemia reperfusion group at the corresponding reperfusion times (p<0.01).(4) The expression of Caspase-12:Tan-yellow staining in the cytoplasm was considered positiveexpression. The expressions of Caspase-12during different periods of time in ischemia reperfusion group are obviously higher than in the same time point of normal group and sham operation group. In the ischemia reperfusion group, Caspase-12had a strong expression at Oh, then continued to rise at3h (p<0.01), and reached its peak at12h(p<0.01),then began to reduce at24h, but was still far higher than that of normal group and sham operation group. At the time point of72h, Caspase-12expression was the same with ischemia-reperfusion group, but higher than normal group and sham operation group(p<0.01). In RSM group, the expression of Caspase12was weaker than ischemia reperfusion group at the corresponding reperfusion times (p<0.01).(5) The expression of p-Aktl:The expression of normal group and sham operation group were weak. In ischemia reperfusion group, the expression was significantly higher than that of the other two groups. It started to express at Oh, peaked at12h, reduced but still in a higher level during24h-72h. In RSM group, the expression of p-Aktl was stronger than ischemia reperfusion group at the corresponding reperfusion times.4. ConclusionsRSM may adjust the expressions of GRP78and GRP94, which played a protective effect during hepatic ischemia reperfusion in rat, throuth reducing the burden of endoplasmic reticulum and promoting gluconeogenic pathway.