| Background and Objective:The prevalence of Type 2 Diabetic Osteoporosis(T2DOP)is on the rise,making it a critical complication of type 2 diabetes.Hence,it is crucial to discover efficient approaches for managing type 2 diabetic osteoporosis and preserving skeletal well-being in individuals living with type 2 diabetes.Traditional Chinese medicine has conducted numerous studies on type 2 diabetic osteoporosis in the past few years,resulting in significant advances in understanding its causes and treatment strategies.Traditional Chinese medicine theory suggests that an excessive intake of food containing high levels of fat can potentially cause the insufficiency of the spleen,leading to a dysfunction of transportation of essence of water and food,eventually leading to the depletion of qi and blood.Without the nourishing of blood,the production of kidney essence is affected.The deficiency of native endowment and overstrain also lead to the deficiency of kidney essence.The lack of kidney essence resulting in the abnormality in bone growth and function,contributing to the onset of osteoporosis.The core pathogenesis of this disease is the deficiency of spleen and kidney,and invigorating the spleen and kidney and nourishing the bone marrow is the key approach to effectively treat osteoporosis in individuals with type 2 diabetes.Based on the aforementioned treatments,our research team discovered the Zuogui Pill(ZGP),a well-known chinese medicine prescription,developed by Zhang Jiebin,a physician during the Ming Dynasty.This implies that Zuogui Pill could potentially be a successful treatment for osteoporosis in individuals with diabetes.Our research team has previously discovered that Eldecalcitol(ED-71),a new vitamin D analogue,effectively treats osteoporosis in individuals with diabetes.It has been discovered in Traditional Chinese Medicine that ED71 not only enhances the effect of Zuogui Pill by tonifying essence,but also strengthens the spleen.The use of this approach enhances the therapeutic outcome of Zuogui Pill.From the standpoint of a comprehensive approach to healthcare that combines Chinese and Western medicine,the amalgamation of these two practices yields more favorable therapeutic outcomes for individuals with type 2 diabetes and osteoporosis.Hence,by considering the promotion of spleen function and kidney support,this research establishes and validates a treatment approach for type 2 diabetic osteoporosis.The study examines how the combined use of Zuogui Pill(ZGP)and Eldecalcitol(ED-71),integrating traditional and Western medicine,affects the treatment of type 2 diabetic osteoporosis.Abnormal rise of intracellular Reactive oxygen species(ROS)caused by hyperglycemic environment will lead to Oxidative stress(OS),and ROS inhibits the activity of osteoblasts and promotes the increase of osteoclast activity,resulting in the imbalance of bone formation and bone resorption.ROS has been listed as an independent risk factor for osteoporosis.Intracellular ROS levels are regulated by various factors,among which the PI3K/AKT signaling pathway is involved in the regulation of oxidative stress levels.Therefore,this study took oxidative stress and PI3K/AKT signaling pathway as the starting point to explore its mechanism,providing a modern scientific basis for the treatment of type 2 diabetic osteoporosis from the perspective of integrated Chinese and Western medicine.Research methods:1.Zuogui Pill combined with ED-71 can improve Induced type 2 diabetic osteoporosis.In order to assess the impact of ZGP combined with ED-71 on induced type 2 diabetic osteoporosis,a mouse model of induced type 2 diabetes mellitus was developed by exposing the mice to a high-fat diet and administering streptozotocin(STZ)through intraperitoneal injection.Based on the various approaches in drug administration,the study was divided into the control group,the model group,the ZGP group,the ED-71 group,and the ZGP+ED-71 group.The treatment lasted for 6 weeks,and the fasting blood glucose levels of the mice were monitored weekly.The changes of serum calcium and phosphate levels were evaluated by biochemical analysis.The hepatotoxicity of the drugs was evaluated by hematoxylin-eosin(HE)staining.Bone mass,bone histological parameters and new bone formation were evaluated by HE staining and Masson staining.The status of osteoblasts and osteoclasts was evaluated by immunohistochemical staining and tartrate resistant acid phosphatase(TRAP)staining.2.Zuogui Pill combined with ED-71 can improve spontaneous type 2 diabetic osteoporosisIn order to assess the impact of ZGP combined with ED-71 on spontaneous type 2 diabetic osteoporosis,db/db mice were used as a preclinical model.Based on the various approaches in drug administration,the study was divided into the control group,the model group,the ZGP group,the ED-71 group,and the ZGP+ED-71 group.The treatment lasted for 6 weeks,and the fasting blood glucose levels of the mice were monitored weekly.The changes of serum calcium and phosphate levels were evaluated by biochemical analysis.The hepatotoxicity of the drugs was evaluated by HE staining.Bone mass,bone histological parameters and new bone formation were evaluated by HE staining and Masson staining.The status of osteoblasts and osteoclasts was evaluated by immunohistochemical staining and TRAP staining.3.The mechanism of Zuogui Pill combined with ED-71 to improve type 2 diabetic osteoporosisMC3T3-E1 cells were cultured in vitro to induce osteogenic differentiation.35 mmol/L glucose solution was used to create a hyperglycemic environment.The impact of drugs on the development of MC3T3-E1 cells in a high-sugar environment was evaluated by real-time polymerase chain reaction(RT-qPCR),western blot,immunofluorescence,alkaline phosphatase(ALP)staining and Alizarin red(AR)staining.The levels of reactive oxygen species and related enzymes were detected by DCFH-DA fluorescent staining and western blot.The PI3K/AKT signaling pathway was detected by western blot and immunofluorescence.PI3K inhibitor LY294002 and AKT inhibitor ARQ092 were used to obstruct the PI3K/AKT signaling pathway and verify the important role of PI3K/AKT signaling pathway in Zuogui Pill combined with ED-71 in improving type 2 diabetic osteoporosis.Experimental results:1.Zuogui Pill combined with ED-71 of improved blood glucose level and bone mass in induced T2DOP miceThe blood sugar levels of mice with induced type 2 diabetes were considerably higher compared to those in the control group,resulting in significantly lower bone mass.ZGP and ED-71 administration resulted in a decrease in blood glucose levels in mice,with a greater impact observed when the two treatments were combined compared to using either treatment alone.ZGP and ED-71 demonstrate substantial enhancements on the skeletal structure of mice with induced type 2 diabetes,such as increased bone mass,heightened bone trabeculae count,volume,and width,as well as facilitation of new bone formation.This predominant effect is particularly notable in the group that received a combined treatment of ZGP and ED-71.After administering a combination of ZGP and ED-71,we observed an increase in the number of ALP and RUNX2 positive osteoblasts,as well as an enhanced expression of COL-1,and the decrease of TRAP positive osteoclasts and the expression of MMP9 in the epiphysis of the femoral shaft according to the histological staining results.2.Zuogui Pill combined with ED-71 improved blood glucose level and bone mass in spontaneous type 2 diabetes miceCompared to the wild-type mice,the db/db mice exhibited a substantial increase in blood glucose levels and a significant decrease in bone mass.The administration of ZGP and ED-71 to mice with the db/db genotype leads to a decrease in their blood glucose levels,with a greater impact observed when the two treatments were combined compared to using either treatment alone.ZGP and ED-71 were found to have a positive effect on bone density,structure,and formation in db/db mice,particularly when used together in the ZGP+ED-71 group.After administering a combination of ZGP and ED-71,we observed an increase in the number of ALP and RUNX2 positive osteoblasts,as well as an enhanced expression of COL-1 in the epiphysis of the femoral shaft,and the decrease of TRAP positive osteoclasts and the expression of MMP9 are observed according to the histological staining results.3.Zuogui Pill combined with ED-71 inhibits oxidative stress by activating PI3K-AKT signaling pathway,thus promoting osteoblast differentiationUpon initiating an osteogenic induction in MC3T3-E1 cells,exposure to a high glucose environment resulted in a significant decline in the cellular expression of RUNX2 and OCN proteins.The combined treatment of ZGP and ED-71 significantly enhanced the levels of ALP,RUNX2,and OCN proteins and mRNA in osteoblasts exposed to high glucose,promoting their differentiation and mineralization.Research has shown that when cells are exposed to high levels of glucose,it can lead to an increase in the production of reactive oxygen species(ROS)within the cells.At the same time,this high-glucose environment can also downregulate the expression of the antioxidant enzymes known as MnSOD and Catalase.The combined administration of ZGP and ED-71 showed a noteworthy decrease in cellular oxidative stress levels triggered by elevated glucose levels.Upon conducting additional analysis,it was uncovered that the combination of ZGP and ED-71 upregulated the levels of PI3K and AKT in osteoblasts.Reversal of oxidative stress inhibition and enhancement of osteogenic differentiation by ZGP and ED-71 were achieved when the PI3K-AKT signaling pathway was blocked using LY294002 or ARQ092.Conclusion:1.Zuogui Pill combined with ED-71 synergistically play the role of invigorating the spleen and kidney and nourishing the bone marrow in the theory of TCM,and have the therapeutic effect on T2DOP.2.Zuogui Pill combined with ED-71 significantly reduced blood glucose levels in induced and spontaneous type 2 diabetic osteoporosis animals.3.Zuogui Pill combined with ED-71 significantly improved the bone mass loss of induced and spontaneous type 2 diabetic osteoporosis animals by promoting the activity of osteoblasts and inhibiting the activity of osteoclasts.4.Zuogui Pill combined with ED-71 inhibited the level of oxidative stress to promote osteogenic differentiation of MC3T3-E1 cells by activating PI3K-AKT signaling pathway. |
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