The Expression Of Caspase-3、Bax And Bcl-2When XNJI Was Injected In Diabetic Rats’ Abdominal Which Appear Subarachnoid Hemorrhage

IntroductionXingnaojing injection is an refined vein preparations based on traditional Chinesemedicine of Angongniuhuangwan(AGNHW). Main ingredients have musk, borneol, gardenia, turmeric, etc. which have the effectof therapy for waking up a patient from unconsciousness.removing heat to cool blood, detoxification pain, For clinical common first aid into drugs. The present study showed that XNJI can inhibit the cerebral vasospasm after subarachnoid hemorrhage. And reduce cerebral ischemic oxygen. Coma patients of awake rate increased significantly. The brain function effect is obvious. It is widely applied in salvage treatment of nervous system disease which is emergency, danger, severe patient, also usually applicationed in treatment of subarachnoid hemorrhage.Cell apoptosis (apoptosis), also known as programmed cell death, in cell apoptosis in caspase activation as a starting point, the contents of dead cells formed apoptotic body (apoptotic bodies) by a complete package function of cell membrane. And swallowed by other cells, usually without omissions of death cells contents and without inflammation, which is a natural cell death, also called cell suicide. For many animals cell apoptosis plays a very important role in body development and stability. Has proved that there are many diseases be connected with abnormal of apoptosis mechanisms. Now regards as, genetic in cells directly controlling cells apoptosis’ occurrence and development. Throughout signal transduction outside of the cells impact these gene expression or its expression the activation of the products, with the results that control cell apoptosis indirectly. there are many research about the apoptosis mechanisms, in different types, different growth stages of the cell has different apoptosis ways, but the difference is mainly manifested in the early induced apoptosis, there was difference between the molecular mechanism, and as apoptosis is starting, different early apoptotic signals are together to a common executive pathways, lead to similar characteristic morphological and biological chemical change. Caspase-3is an important one of Caspase family, and was confirmed in the path of the downstream, it is the common downstream effects part of all sorts of apoptosis pathways, it enzymolysis a series of cells protein survive required directly or indirectly, promote cell apoptosis. Bcl-2family is indispensable in neural cell apoptosis inhibit process, is a apoptosis inhibitor genes, Bcl-2proteins can be reduced concentration in cells and the nuclears, directly control the permeability of mitochondria outer membrane and regulation of apoptosis signal transduction. Bax is also important promote apoptosis genes, its induce the mechanism apoptosis is Bax homologous dimers formation, Bcl-2protein inhibiting apoptosis with form different source dimmers with Bax to achieve. Bcl-2/Bax as a balance system, a overexpression of Bax cell can accelerate apoptosis, Bcl-2overexpression just the reverse,so inferences Bcl-2/Bax rate determine the development direction cell apoptosis.With the increasing of global diabetes incidence, and subarachnoid hemorrhage patients with diabetes is appearing a growing trend. To seek feasible treatment For this kind of subarachnoid hemorrhage patients as an arduous task for medical workers. So to establish a stable of diabetes subarachnoid hemorrhage model are very important. This experiment Application chain with urea bacteria element (Streptozocin, STZ) intraperitoneal injection method to set up the diabetic rats model, then the pillow big pool two fresh autologous blood into the method to establish the diabetic rats subarachnoid hemorrhage model. To study the influence of the prognosis of diabetes to rats when they appear SAH by observe the mortality and to explore dead time of SAH diabetes rats. And then further application intervention with XNJI, observation the apoptosis of neurons in the hippocampus, To investigate the change of Caspase-3、Bax and Bcl-2expression in hippocampus when the diabetic rats appear subarachnoid hemorrhage(SAH),and the effect of xingnaojing injection(XNJI)to these apoptotic factors. To provide the theory basis for preventive treatment brain damage of diabetes patients after SAH.Part I Contrast studies of setting up subarachnoid hemorrhage rat models with which whether it has diabetes mellitusObjectiveTo study the influence of the prognosis of diabetes to rats when they appear SAH.Material and methodAnimalsRandom weight at250-300g adult male Wistar rat63. animals are provide by animal laboratory center of Shandong University. Breeding conditions for room temperature18～25℃, relative humidity environment for50%～80%, and free water diet. Feeding adaptability for1week. Healthy adult male rats are feed with high sugar and high protein for one month.MethodsRandomly divided into two groups, diabetes SAH group42only, SAH control group21only. To induce the diabetes models, the asult male wistar rats were injected intraperitoneally with streptozotocin (STZ) once, SAH models were constructed by two-time fresh autologous blood injection in the cisterna magna of diabetic rats.10%Chloral Hydrate solution intraperitoneal injection of400mg/kg. After the success of anesthesia, preservedskin of neck back and groin area, take prone to a rat fixed in stereo controller, antisepsis of neck back surgery area skin, open skin along the Central Line, blunt dissection subcutaneous tissue and muscle layer until to fully revealed pillow membrane. Then fixed rat in rats fixed frame bytaking the supine position. Disinfect the right groin area skin, open skin along the groin, blunt dissection subcutaneous tissue and the artery until the artery fully exposed. Set aside by extraction fresh blood about0.4mL.. Ligation femoral artery in the nearly heart quickly. Puncture the pillow membrane,pumping out cerebrospinal fluid0.1mL carefully, put autologous fresh blood about0.3mL into the cisterna magna slowly, limited within2min. And then suture the nuchal region and groin area step by step. Keep head low for30min,ensure blood distributed in the basement pool. Evenly, the same way after the other side the artery blood0.3mL cisterna magna injected after48h.ResultsDiabetes SAH rats, death and only, the mortality rate is78.57%; SAH die group4only, and mortality was18.09%. The number of deaths significantly greater than the control group SAH in0-24h、24-48h、48-72h.But there is no statistical significance in72-96h.ConclusionDiabetes can obviously increase the deadth rate after subarachnoid hemorrhage Especially for the first72h.Part ⅡThe expression of Caspase-3、Bax and Bcl-2when Xingnaojing was injected in diabetic rats’abdominal which appear subarachnoid hemorrhageObjectiveTo investigate the change of Caspase-3、Bax and Bcl-2expression in hippocampus when the diabetic rats appear subarachnoid hemorrhage(SAH),and the effect of xingnaojing injection(XNJI)to these apoptotic factors.Material and methodAnimalsRandom weight at250-300g adult male Wistar rat63, animals are provide by animal laboratory center of Shandong University. Breeding conditions for room temperature18～25℃, relative humidity environment for50%～80%, and free water diet. Feeding adaptability for1week. Healthy adult male rats are feed with high sugar and high protein for one month.MethodsDesign28adult male Wistar rats were divided into4groups randomly:naive group; diabetic group;diabetic rats SAH group; XNJI treatment group. To induce the diabetes models, the asult male wistar rats were injected intraperitoneally with streptozotocin (STZ) once, SAH models were constructed by two-time fresh autologous blood injection in the cisterna magna of diabetic rats.10%Chloral Hydrate solution intraperitoneal injection of400mg/kg. After the success of anesthesia, preservedskin of neck back and groin area, take prone to a rat fixed in stereo controller, antisepsis of neck back surgery area skin, open skin along the Central Line, blunt dissection subcutaneous tissue and muscle layer until to fully revealed pillow membrane. Then fixed rat in rats fixed frame bytaking the supine position. Disinfect the right groin area skin, open skin along the groin, blunt dissection subcutaneous tissue and the artery until the artery fully exposed. Set aside by extraction fresh blood about0.4mL.. Ligation femoral artery in the nearly heart quickly. Puncture the pillow membrane, pumping out cerebrospinal fluid0.1mL carefully, put autologous fresh blood about0.3mL into the cisterna magna slowly, limited within2min. And then suture the nuchal region and groin area step by step. Keep head low for30min.,ensure blood distributed in the basement pool. Evenly. the same way after the other side the artery blood0.3mL cisterna magna injected after48h. Expressions of Caspase-3mRNA. Bax mRNA, and Bcl-2mRNA were detected using Real-Time PCR. To identify these changes further. Western blot was adopted to investigate the protein changes of these three molecules after SAH.ResultsCompared with native group, Expression decreased compared with diabetic rats SAH group (P<0.05, P<0.01),while the Bcl-2mRNA(8.35±1.37vs5.99±2.69) was increased (P<0.05) compared with diabetic rats SAH group, of Caspase-3mRNA (7.80±2.33vs2.06±0.72) Bax mRNA(7.69±2.55vs2.09±0.51), and Bcl-2mRNA (5.99±2.69vs3.41±1.47) increased in vary degrees in hippocampus of diabetic rats SAH group (P<0.01、P<0.01、P<0.05). In XNJI treatment groups, Expression of Caspase-3mRNA (4.92±1.85vs7.80±2.33) and Bax mRNA(5.05±1.36vs7.69±2.55).ConclusionBy inhibiting the apoptosis and promoting antiapoptosis, XNJI has an obvious neuroprotective effect on hippocampal tissue of diabetic rats SAH.