| Objective:In recent years, more the patients of type 2 diabetes develop to osteoporosis. Duing to the bone loss secrecy, these patients often come to hospital and kwon they have been osteoporosis because of bone fracture. It influences seriouly people's life and the quality of life. So, it is much vital for treatment of T2DM with osteoporosis to research the pathogenesis of T2DM with osteoporosis. In this study, we established type 2 diabetes osteoporosis rat model, measured the skeletal muscle gene expression involved in insulin signaling pathway in Type 2 diabetic osteoporosis rats and discussed the relationship of the pathogenesis of these two diseases.Methods:100 Wistar rats were randomly divided into four groups after 1 week feeding: normal control group(NC,N=24), ovariectomy group(NOVX,N=26) , type 2 diabetes control group(DC,N=24), type 2 diabetic rats under ovariectomy group (DOVX,N=24). NCand NOVX groups were fed with normal feedstuff while DC and DOVX groups were fed with high-sugar-fat feedstuff mixed with 20% sugar,2.5% cholesterin, 15% cooked lard) for 8 weeks. T2DM were made by intraperitoneal injection of STZ after 12 hours fasting. At the end of 9th weeks, rats with fasting blood glucose (FBG)≥7.8mmol/L and with insulin resistance were considered as T2DM.Rats chose for NOVX and DOVX group were ovariectomied under anesthesia 1 week later after T2DM model were done, and sham group only cut some fat tissue. We randomly selected 5 rats in each group and measured body weight, FBG and fasting insulin level (FINS) and bone mineral density (BMD) of lumbar 3-6 at 0 week,4 weeks,8weeks and 12 weeks after the operation. At 4 weeks,8 weeks,12 weeks after ovariectomy, five rats was randomly executed, the quadriceps femoris of which was separated after anesthesia. The expression of IGF-1, IRS-1,IRS-2 at protein level was detected by IHS (immunohistochemistry) while the expression of IGF-1,IRS-1,IRS-2 at RNA level was detected by RT-PCR. The experimental data was presented as mean±standard deviation. Variance Analysis were used to check differences between groups by SPSS13.0,P<0.05 was considered statistically significant.Results:1 The building of Type 2 diabetes osteoporosis rat model : The comparison of the FBG of DC group and DOVX group is prominent higher than NC group and NOVX group(P<0.05), and the level of INS of DC group and DOVX group is higher than NC group and NOVX group(P<0.05)too , so we can make sure that the Type 2 diabetes rat model is established . The comparison of BMD of NOVX group and DOVX group after ovariectomy 4 week,8 week and 12 week is lower than NC group and DC group . This Indicates the osteoporosis and type 2 diabetes osteoporosis rat model is established .2 Body weight of rats in each group: There were no statistically significant difference between the 4 groups weight after ovariectomy(0 week). At 4, 8, 12 weeks after ovariectomy, the body weight of NOVX group is significantly higher than other 3 groups, DS group is significantly lower than other 3 groups(P<0.05), and there is no statistically significant difference between the weight of NS group and DOVX group.3 The comparison of FBG,INSand estrogen in each group3.1 Fasting plasma glucose(FBG): After T2DM model was established, FBG of DS group and DOVX group is significant higher than NSgroup and NOVX group, after ovariectomy 4, 8, 12 week, the FBG of DS group and DOVX group is lower than earlier, but is also higher than NS group and NOVX group, the difference was statistically significant (P<0.05).3.2 Insulin level(INS): The INS of diabetic group (DS group and DOVX group) rats is significantly higher than non-diabetic group (NS group and NOVX group) rats at each period (after ovariectomy 0, 4, 8, 12 week). 3.3 The estrogen level: at the beginning after ovariectomy, the estrogen level of diabetic groups (DS and DOVX group) is significantly lower than non-diabetic groups ( NS and NOVX group ) , but there is no difference between NS group and NOVX group , DS group and DOVX group ( P>0.05 ) ; at 4 and 8 weeks after ovariectomy , the estrogen level of NOVX group is lower than NS group, and DOVX group is the lowest ( P<0.05 ) ; at 12 weeks after ovariectomy , the estrogen in NS group is the highest and DOVX group is the lowest .4 The pathology of the skeletal muscle: Under microscope ,we can see the skeletal muscle of non-diabetic groups shows stronger , muscle silk closely-arranged , while the shape of the nuclear is normal , and the number is appropriate . In NOVX group , there are more gaps between muscle silk , the number of the nuclear is fewer than NS group . The liver of diabetic groups shows distortion of hepatic lobules, obvious hepatic steatosis. With the progression of the experiment, steatosis of the hepatocyte becoming more serious, some of which look like signet ring cell, especially in DOVX group .5 Immunohistochemistry results: IGF-1, IRS-1and IRS-2 expression is located in the cytoplasm of the skeletal muscle cell . The chino granules in the cytoplasm of the skeletal muscle cell indicate they are masculine .5.1 The expression of IGF-1:at 4 week after ovariectomy,the expression of IGF-1 in NC group and DC group is significantly higher than NOVX and DOVX groups ( P<0.05 ), and there is no difference between NC group and DC group and between NOVX and DOVX groups ( P>0.05 );at 8 week after ovariectomy,the expression of IGF-1 in NC group and NOVX group is significantly higher than DC and DOVX groups ( P<0.05 ) , but there is no different between NC group and NOVX group and between DC group and DOVX group ( P>0.05) ; at 12 week after ovariectomy ,the expression of IGF-1 in NC group and NOVX group is significantly higher than DC and DOVX groups ( P<0.05) , and the expression of IGF-1 in DC group is higher than DOVX group (P<0.05) .5.2 The expression of IRS-1 : at 4 week after ovariectomy,the expression of IRS-1 in NC is much higher than DC group, and NOVX group is higher than DOVX group ; at 8 week after ovariectomy , the expression of IRS-1 in NC group and NOVX group is significantly higher than DC and DOVX groups (P<0.05) , but there is no different between NC group and NOVX group and between DC group and DOVX group (P>0.05) ; at 12 week after ovariectomy , the expression of IRS-1 in NC group and NOVX group is significantly higher than DC and DOVX groups (P<0.05) , but there is no different between NC group and NOVX group and between DC group and DOVX group (P>0.05) .5.3 The expression of IRS-2 : at 4 week after ovariectomy,the expression of IRS-2 in NOVX is prominent higher than NC, DC and DOVX groups (P<0.05) , but there is no differt between DC group and DOVX group ; at 8 week after ovariectomy , the expression of IRS-1 in NOVX group is much higher than other groups , but there is no different between DC group and DOVX group (P>0.05) ; at 12 week after ovariectomy , the expression of IRS-2 in NC group is evident higher than NOVX group and DOVX group (P<0.05) , The expression of IRS-2 in DC group is much higher than DOVX group .6 RT-PCR results :6.1 The expression of IGF-1 : at 4 week after ovariectomy,the expression of IGF-1 in NC group and DC group is significantly higher than NOVX and DOVX groups (P<0.05) , and the expression of IGF-1 in DC group is lower than NC group (P<0.05) , while there is no different between NOVX group and DOVX group (P>0.05) ; at 8 week after ovariectomy , the expression of IGF-1 in NC group and NOVX group is significantly higher than DC and DOVX groups (P<0.05) , and NOVX group is much lower than NC group (P<0.05) , while there is no different between DC group and DOVX group (P>0.05) ; at 12 week after ovariectomy , the expression of IGF-1 in NC group is much higher than other three groups (P<0.05) , while DOVX group is much lower than DC group (P<0.05) , DC group is much lower than NOVX group (P<0.05) .6.2 The expression of IRS-1 : at 4 week after ovariectomy,the expression of IRS-1 in NC group is much higher than other three groups , while DOVX group is much lower than DC group (P<0.05) , DC group is much lower than NOVX group (P<0.05) ; at 8 week after ovariectomy , the expression of IRS-1 in NC group is much higher than other three groups (P<0.05) , while DC group is much lower than DOVX group (P<0.05) , DOVX group is much lower than NOVX group (P<0.05) ; at 12 week after ovariectomy , The expression of IRS-1 in NC group is much higher than other three groups (P<0.05) , while DC group is much lower than DOVX group (P<0.05) , DOVX group is much lower than NOVX group (P<0.05) .6.3 The expression of IRS-2 : at 4 week after ovariectomy ,the expression of IRS-2 in NC group is much higher than other three groups (P<0.05) , while DOVX group is much lower than DC group (P<0.05) , and DC group is much lower than NOVX group (P<0.05) ; at 8 week after ovariectomy , the expression of IRS-2 in NOVX group is much higher than other three groups (P<0.05) , while DOVX group is much lower than DC group (P<0.05) , DC group is much lower than NC group (P<0.05) ; at 12 week after ovariectomy , the expression of IRS-2 in NC group is much higher than other three groups (P<0.05) , while DOVX group is much lower than NOVX group and DC group (P<0.05) , but there is no different between NOVX group and DC group (P>0.05) .Conclusion:1 The expressions of IGF-1, IRS-1 and IRS-2 in the skeletal muscle of NOVX group is lower than that of NC group .2 The expressions of IGF-1, IRS-1 and IRS-2 in the skeletal muscle of DC group is lower than that of NC group .3 The expressions of IGF-1, IRS-1 and IRS-2 in the skeletal muscle of DOVX group is lower than that of NC, NOVX and DC groups .4 It may be relevant that the pathogenesis of Type 2 diabetes with osteroposis to the inhibition of the expressions of IGF-1, IRS-1 and IRS-2 in the skeletal muscle . |
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