Insulin Signaling And Osteoporosis: The Investigation Of Pathogenesis And Chinese Medical Treatment

Type 2 diabetes and osteoporosis are two common diseases in the elderly. The incidence of these two diseases is increasing in recent years, which affects people’s quality of life. Various domestic and international research showed that type 1 diabetes mellitus can actually cause osteoporosis, which means type 1 diabetes is a high risk factor for osteoporosis, and can thus lead to pathologic fracture. The reason may be related to the absolute lack of insulin secretion caused by carbohydrate, lipid and protein metabolism abnormalities, which can lead to the calcium and phosphorus imbalance, thereby reducing the amount of bone. The patients with type 2 diabetes can have higher, lower or normal bone mass. The common pathogenesis of type 2 diabetes and osteoporosis is still unknown. Recent studies have found that the insulin signaling pathway may be associated with the pathogenesis of type 2 diabetes and osteoporosis.Insulin signaling pathway include: insulin receptor substrate(IRS) phosphorylation, phosphatidylinositol 3-kinase(PI3K) activation, Akt(protein kinase B, PKB) activation, molecular target of rapamycin(m TOR) activation and ribosomal protein S6 kinase(p70 S6 kinase, p70 S6 k) activation. A series of signaling cascades is initiated by insulin binding to its receptors. Initially, insulin binding induces receptor autophosphorylation, followed by the activation of receptor tyrosine kinases, and resulting in the tyrosine phosphorylation of insulin receptor substrates(IRSs). Activated insulin receptors(IR) phosphorylate IRS1 and IRS2 on tyrosine residues, enabling the recruitment of the p85-p110 Ptd Ins 3-kinase to the plasma membrane. This, in turn, generates phosphatidylinositol 3, 4, 5-trisphosphate(Ptd Ins(3, 4, 5) P3), which recruits 3-phosphoinositide-dependent protein kinases-1 and-2(PDK1 and PDK2). PDK-1 and-2 phosphorylate and activate Akt1 and Akt2, which in turn promote GLUT4 translocation to the plasma membrane and further glucose uptake.Recent studies have found that the insulin signaling pathway is not only related to glucose metabolism, but also involved in bone metabolism. PI3 K involved in the differentiation of osteoblast and osteoclast, and plays an important role in bone formation and bone reconstruction. It has found that the activation of PI3 K signaling pathway is necessary to the differentiation of MC3T3-E1 and block the activation will inhibits the differentiation of osteoblasts. Akt is one of major downstream molecule in PI3 K signaling pathway. Aktl expression is dominant in osteoblasts and osteoclasts. Related study showed that Akt1 gene knockout mice have decreased bone mass and bone resorption activity. The secondary ossification formation center delayed in Akt1 gene knockout mice. Osteoblast differentiation index(such as ALP, I collagen, osteocalcin, etc.) decreased in Akt1-/- wild-type mice. In addition, Akt regulates the differentiation of mesenchymal cells into osteoblasts with bone morphogenetic protein 2(BMP2).Osteoporosis is not only related to body weight and total body fat, but also to bone marrow fat. One theory explaining the inverse association between marrow fat and bone is that an increase in the differentiation of MSCs to adipocytes will lead to a decrease in the differentiation of MSCs to osteoblasts, as adipocytes and osteoblasts share the same progenitor, the bone marrow mesenchymal stem cells(MSCs). The differentiation of MSCs into either fat or bone is influenced by hormones, adipokines, PPARγ, as well as mechanical stimuli. Mechanisms that promote the adipogenic fate of MSCs actively suppress intracellular osteogenic signals.The increasing prevalence of osteoporosis has contributed to a considerable financial burden, both to the individual suffering from osteoporotic vertebral compression fractures(OVCFs) and society as a whole. Over the past decade, pharmaceutical companies have developed a wide collection of drugs designed to offer targeted therapeutic effects on the improvement of bone quality. These drugs have been developed based on the current understanding of bone tissue metabolism, either to stimulate the anabolic side(e.g. teriparatide and strontium products) or suppress the catabolic side(e.g. bisphosphonates). Although the available therapeutic drugs created for the treatment of osteoporosis have been shown to be effective, they introduce many negative side effects to the patients who use them. Chinese herbal medicine can combines with standard therapeutic drug for patients with OVCF. Part I: The expression of PI3 K, Akt1, Akt2 and NFκB in liver, kidney and skeletal muscel of type 2 diabetic Osteoporosis rat modelObjective: The aim of this study was to explore whether insulin signaling downstream proteins participate in the pathogenesis of type 2 diabetes mellitus and osteoporosis.Methods: 100 healthy female Wistar rats, 2.5 to 3 months old, were randomly divided into four groups after 1 week adaptability fed: normal control group(NS, N=24), ovariectomized group(NOVX, N=26), type 2 diabetes group(DS, N=24), type 2 diabetic ovariectomized group(DOVX, N=24). The T2 DM rats model was made by feeding them with high-sugar-fat feed stuff combined with low-dose STZ intraperitoneal injection, and ovariectomied to make the osteoporosis rats model. At 12 weeks after ovariectomy, rats were executed, the liver, kidney and right quadriceps muscle tissue were separated after anesthesia. Paraffin sections were produced respectively. Total RNA and protein were extrated from the different tissues. immunohistochemistry(IHS) and western blot were used to detecte the protein expression of PI3 K, Akt1,Akt2 and NFκB, while the RT-PCR was used to detecte m RNA expression of PI3 K, Akt1,Akt2 and NFκB in different groups. SPSS13.0 statistic software was used to process the experimental data. All experimental data was presented as mean±standard deviation. Variance Analysis was used to check differences between groups, P<0.05 was considered statistically significant.Results: The first part: Immunohistochemistry was used to observe the expression of PI3 K, Akt1, Akt2, NFκB in rat liver, kidney and skeletal muscle. All of these proteins expressed in liver cytoplasm, renal tubular epithelial cells or skeletal muscle cell cytoplasm, respectively. The immunohistochemistry results showed that compared with the NS group, the expression of PI3 K, Akt1 and Akt2 in NOVX, DS and DOVX groups were decreased, and the lowest level was observed in DOVX group(P <0.05). The expression of NFκB in NOVX, DS and DOVX groups were increased, and the highest level was observed in DOVX group(P <0.05). The result of liver RT-PCR showed that both the lowest level of PI3 K, Akt1, Akt2 and the highest level of NFκB were observed in DOVX group(P <0.05). There is no significant difference between DS group and DOVX group in the expression of Akt2(P> 0.05), There is no significant difference among NOVX group, DS group and DOVX group in the expression of NFκB(P> 0.05). The result of kidney RT-PCR showed that both the lowest level of PI3 K, Akt1, Akt2 and the highest level of NFκB were observed in DOVX group(P <0.05). There is no significant difference between DS group and DOVX group in the expression of PI3K(P> 0.05), There is no significant difference among NOVX group, DS group and DOVX group in the expression of Akt2(P> 0.05). The result of skeletal muscle RT-PCR showed that both the lowest level of PI3 K, Akt1, Akt2 and the highest level of NFκB were observed in DOVX group(P <0.05). There is no significant difference among NOVX group, DS group and DOVX group in the expression of PI3 K, Akt1 and NFκB(P> 0.05). The result of liver Western blot showed that the lowest level of PI3 K, Akt1 and Akt2 were observed in DOVX group(P <0.05). There is no significant difference between NOVX group and DS group in the expression of PI3K(P> 0.05). The higest level of NFκB was observed in DOVX group(P <0.05). There is no significant difference between NOVX group and DS group in the expression of NFκB(P> 0.05). The result of kidney Western blot showed that both the lowest level of PI3 K, Akt1, Akt2 and the highest level of NFκB were observed in DOVX group(P <0.05). There is no significant difference between NOVX group and DS group in the expression of PI3 K and NFκB(P> 0.05). There is no significant difference between DS group and DOVX group in the expression 10 o f Akt2(P> 0.05). The result of skeletal muscle Western blot showed that both the lowest level of PI3 K, Akt1, Akt2 and the highest level of NFκB were observed in DOVX group(P <0.05). There is no significant difference between NOVX group and DS group in the expression of Akt1 and NFκB(P> 0.05).Conclusion: The inhibition of the inslin signaling expressions of PI3 K, Akt, NFκB in kidney pathway maybe related to the pathogenesis of Type 2 diabetes with osteroposis. Part II: The relationship between bone marrow adipose Tissue and cortical bone in postmenoposal osteoporosisObjective:To explore wheather an inverse relationship between bone marrow adipose tissue and cortical bone presents in postmenoposal woemen.Methods: The second part: Collected 30 cases of female patients with type 2 diabetes who received treatment at the second department of endocrinology during January 2012 to December 2012. Right femoral bone marrow adipose tissue area(BMA), right femoral cortical bone area(CBA), right femoral subcutaneous adipose tissue(SAT), and right femoral skeletal muscle were accessed by magnetic resonance imaging. Slice Omatic image analysis software was used for data analysis. SPSS13.0 statistical analysis software was used for statistical analysis, descriptive statistics presented as mean ± standard deviation. Pearson’s correlation coefficient were calculated among CBA, BMA, weight, BMI percentage, age, SAT, and skeletal muscle. Multiple linear regression was used to calculated related data. P<0.05 was considered statistically significant.Results: The second part: Pearson correlation coefficients showed a positive association among right femur CBA, right femur BMA, weight, BMI percentile, age, right femur SAT, and right femur skeletal muscle(r=0.081,r=-0.215,r=0.175,r=-0.252,r=0.317;r=0.385,r=0.153,r=-0.298,r=-0.087,r=0.650;P <0.05, respectively).Conclusion: There is a competitive relationship exists between bone and marrow fat in cortical bone and is consistent with a similar finding in 11 cancellous bone in previous studies. Future research is needed to clarify the role of marrow fat in childhood fractures that are related to cortical bone quality. Part III: Systematic review of traditional Chinese medicine in the treatment of osteoporotic vertebral compression fracturesObjective: This study was designed to perform a systematic review of the clinical effectiveness of traditional Chinese medicine in the treatment of osteoporotic vertebral compression fractures.Methods: We performed online searches of the published literature using three electronic databases(the China National Knowledge Infrastructure, the Chinese Scientific and Technical Journals database(VIP) and Wanfang data; the time period ranged from January 2013 to December 2013. The keywords we chose to search were “Chinese herb AND herbal AND osteoporotic vertebral compression fracture”. We also used any combination of the following keywords: a) compression fracture, vertebra fracture, lumbar vertebrae fracture, OR thoracic vertebrae fracture; and b) traditional Chinese medicine OR traditional Chinese drug. We limited our selection to randomized controlled trials. All satistical analyses were performed using Rev Man 5.1 software.Results: Eleven studies were identified for this meta-analysis, representing 953 patients. Our results indicated that the bone mineral density(BMD) of patients receiving the Chinese herb treatment was higher than the BMD of patients receiving a placebo. Furthermore, our results suggested that the efficiency rate was higher still among patients who incorporated Chinese herbs into their treatment, as compared to patients who were treated with a placebo; sensitivity analyses confirmed this finding.Conclusion: Chinese herbs substantially increased BMD of spine compared to placebo or anti-osteoporotic drugs as indicated in the current clinical reports on osteoporotic vertebral compression fractures treatment.Given the increasing prevalence of financial burden of osteoporosis, Chinese herbs could provide a less invasive, cost-efficient alternative approach to increasing the BMD of patients with osteoporotic vertebral compression fractures.