| Objective:In order to observe the expression of apoptosis factor in different tissuewhen wistar rats were thirty-second weeks, neonatal wistar rats were induced by a single intraperitoneal injection of streptozotocin(STZ) to establish an animal model of insulin resistant; using different methods to treat the rats and observe the concentration of apoptosis factor in insulin resistance and treating groups, investigating the relationship of insulin resistance and apoptosis factors.Methods:1. Animal models and group divisionsThe insulin resistant model of neonatal Wistar rats was conducted by a single intraperitoneal injection of STZ at a dose of 90 mg/kg. The state of insulin resistance was assesed by measuring the fasting serum glucose level at 8 weeks after the administration of STZ. Rats with a plasma glucose level 2 hours after meals above 11.0mmol/L or fasting serum glucose level above 7.Ommol/L were selected for the experiment. The animal models were divided into 4 groups as below:normal controls (NC); insulin resistant groups (IR); dimethybiguanide groups (MET); the treatment of tetramethylpyrazine and aminouguanidine group (TMP+AG).2. The measurement of FPG and FINS:The concentrations of fasting blood glucose (FPG) and fasting insulin (FINS) were measured on the eighth and thirty-second weeks. According to the equation, insulin resistance index (IRI) was determined by the multiplication of fasting blood glucose concentration and fasting insulin concentration, which was divided into 22.5.3. The pathological changes The histological sections of kidney were dyed by hematoxylin and eosin stain to observe the changes of morphology.4. Immunohistochemical staining to assess the expression of caspase-3 and FAS in kidney,liver,brain.5. Western-blot test to detect the expression of caspase-3, FAS and P53.6. Reverse transcription-poly-mersechain reaction:to detect the concentration of mRNA with caspase-3, FAS, FAS-L and P53 in kidney, liver and brain respectively.Results:1. Result of FPG and FINS:On the eighth week treating groups had a higher FPG,FINS and IRI than NC group(P<0.01).On the thirty-second week(after administration 24weeks) MET group and TMP+AG group had a lower FPG and FINS than IR group(P<0.01), there is no statistic significance between MET,TMP+AG group with NC groups with regard to FPG, but FINS and IRI was still higher than NC group. With respect to IRI,IR group was obviously higher than NC group(P<0.01), MET and TMP+AG group was distinctly lower than IR group(P<0.01), in addition TMP+AG group was lower than MET group(P<0.05).2. Pathological results:HE staining gave the evidence that in IR group intercapillary cells were a little hyperplasia in partially; karyopycnosis was disintegrated;cytoplasm was condensed; acidophilia was added; some tissues disappeared and broke into pieces, but there was no inflammation, nephric tubule was intact and basement membrane was left outline. IR group, the liver cells showed nuclear condensation or fragmentation, eosinophils increased, loosely connected with the surrounding cells. Neuronal cells in brain tissue of IR group can be seen stained shrinkage, cell volume reduction.3. Immunohistochemical test:caspase-3 was expressed in liver, kidney and brain in each group. It was mostly in plasm, fuscescent granulations concentrated in proximal and distal nephric tubule, especially in distal nephric tubule. Nothing was in glomcrulus. There were a little positive cells in NC group, also the colour was shallow, the number in IR group was obviously added; compared to IR group, MET and TMP+AG group was lower. FAS was relatively more in liver and brain than in kidney. 4. Western-blot:the express tendency of caspase-3 was similar to Immunohistochemical test, just like below:NC group was relatively lower than IR, MET, TMP+AG group (P<0.05). MET and TMP+AG group was significantly less than IR group (P<0.05), moreover TMP+AG group was obviously less than MET group (P<0.05). expression of Fas in Liver tissue:Compared with the NC group, IR group and the MET group was significantly higher (P<0.05); after the drugs intervention compared with the IR group, MET group and TMP+AG group were reduced (P<0.05), but MET group did not change (P> 0.05),5. Semi-determination of RT-PCR:The concentration of mRNA coincides with Immunohistochemical test. FAS-L mRNA was presented in liver,brain and kidney, especially abundant in liver tissue:Among the comparisons between four groups, the expression of NC group was less, IR,MET,TMP+AG groups were higher significantly (P<0.01). MET and TMP+AG group was significantly less than IR group (P<0.01), moreover TMP+AG group was obviously less than MET group (P<0.05). P53 mRNA was existed in liver, brain and kidney, but the content was less in brain, the changes tendency was basically like capase-3 mRNA and P53 mRNA.Conclusions:1. The insulin resistance model of neonatal wistar rats were successfully induced by a single intraperitoneal injection of STZ. The level of FPG,FINS and IRI were significantly increasing as the time. Meanwhile in the animal models, the expression of various apoptosis factors was obviously changed, indicating the degree of insulin resistance was correlated with different apoptosis factors.2. The therapy of diformin and combined treatment of tetramethylpyrazine and aminoguanidine had the ability of reducing insulin resistance meanwhile decreasing the expression of casapase-3 and FAS, furthermore confirming the degree of insulin resistance was related with caspase-3 and FAS. In MET and TMP+AG groups the changes of insulin resistance was different from the variety of apoptosis factors, the diversification of TMP+AG group was greater, which indicated combined treatment of tetramethylpyrazine and aminoguanidine had a more influence to apoptosis factors. The exact mechanism needed further vestigation.3. The results show that caspase-3, FAS-L and P53 transcription and translation were significantly increased in a variety of organs and tissues of insulin resistant rats. The application of drug intervention of metformin, aminoguanidine and TMP reduced the expression of caspase-3, FAS-L and P53 and alleviated insulin resistance. Showed the formation of insulin resistance and the degree of expression of pro-apoptotic factors are related. |
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