| Objective: To explore the effect and mechanism of the neuritin over-expression in bone marrow on peripheral neuropathy in the model of type2 diabetic mice(dbdb).Methods 1.Established model of transgenic mice with over expression of neuritin in bone marrow,two pairs of transgenic mice(neuritinloxp/+_lyz-Cre/+and misty/+)were propagated,to obtain the type2 diabetic mice with high expression of Neuritin in bone marrow.The experiment was divided into 8 groups:(1)lean db/m mouse as control group(2)type 2 diabetic mouse(TD2)(3)the metformin + type 2 diabetic mouse(TD2+MET)(4)the type 2 diabetic mouse with over expression of neuritin in bone marrow(A/Lyz-Cre+TD2)(5)the metformin +the type 2diabetic mouse with over expression of neuritin in bone marrow(A/Lyz-Cre+TD2+MET)(6)the lean db/m mouse with over expression of neuritin in bone marrow(A/Lyz-Cre+db/m).Mouse were administered intragastrically at age tenth weeks,the administration lasted for 8 weeks,body weight measured each week,every two weeks to monitor blood glucose.2.After 8-week treatment,nerve conduction velocity were carried out in each group of mice,Monitoring the concentration of TC,TG,HDL-C and LDL-C in the plasma of mice,the SDF-1 levels in bone marrow supernatant,in plasma,insciatic nerve supernatant were detected by ELISA in each group of mice.3.Tissue section of femur were stained by HE and oil red O for observing the pathological damage and fat accumulation.Immunofluorescence method is used for detecting the nerve density(TH),the microvessel density(CD31),the expression of neuritin in bone marrow in each group of mice.4.The pathologic structure of sciatic nerve was observed using a transmission electron microscope in each group of mice.Immunofluorescence method is used for detecting the neuron regeneration(GAP43),nerve fiber density(NF200),neuromyelination(GFAP),Schwann cell proliferation(S100+Brdu)in sciatic nerve in each group of mice.5.The primary BMSCs were provided from bone marrow for cell migration experiments.Detecting the effect of SDF-1 alpha,AMD3100(CXCR4 inhibitor),Ly294002(Pakt inhibitor)on the migration of BMSC.Observe the changes in BMSC migration in type 2 diabetic mouse and the type 2 diabetic mouse with over expression of neuritin in bone marrow.Immunofluorescence method is used for detecting the expression of CXCR4 and SDF-1 alpha in bone marrow,and the expression of Pakt in the sciatic nerve.Result: 1.MNCV and SNCV in TD2 mice were significantly lower than those in db/m mice(P < 0.05),MNCV and SNCV in treatment group(A/Lyz-Cre+TD2,A/Lyz-Cre+TD2+MET and TD2+MET mice)were significantly improved(P < 0.05).2.During 8-week treatment,the speed of rising of blood glucose concentration is slowed down in the treatment group,and after 8-week treatment,their FBG was significantly lower in TD2mice(P<0.05).The body weight of the type 2 diabetic model in mice with over expression of neuritin in bone marrow(A/Lyz-Cre+TD2 mice,A/Lyz-Cre+TD2+MET mice)was reduced significantly(P<0.05),the body weight of TD2+MET mice was not significantly changed compared with TD2 mice.The concentration of TC and LDL-C in serum in treatment group was lower than it in TD2 mice,but the difference was not statistically significant(P>0.05),and the concentration of TG was lower than it in TD2 mice,and the difference was significant(P<0.05).For HDL-C,the concentration in type 2diabetic model in mice with over expression of neuritin in bone marrow was significantly lower in TD2 mice(P<0.05),the concentration in TD2+MET mice was lower than it in TD2 mice,but the difference was not statistically significant(P>0.05).3.The femoral HE staining showed that: the cell density was significantly improved in treatment group,and lower than the db/m mice and A/Lyz-Cre+db/m mice,compared to TD2 mice(P<0.05).Oil red O staining showed that the positive rate in treatment group was not different with that of TD2 mice(P>0.05),and they are all lower than that of db/m mice and A/Lyz-Cre+db/m mice(P<0.05).4.Immunofluorescence staining showed that:compared to db/m mice,A/Lyz-Cre +db/m mice,the positive rate of TH and CD31 positive area in bone marrow was significantly lower in TD2 mice(PCD31<0.001,PTH<0.05),while the expression were significantly improved in the treatment group compared with the TD2 mice(PCD31<0.05,PTH<0.05).5.Transmission electron microscope showed mitochondrial crista disappeared,mitochondrial membrane swelling,axonal membrane and myelin membrane separation,and disappearance of myelin lamellar structure.And the pathological symptoms were slightly relieved in TD2+MET mice,the pathological symptoms were significantly improved in A/Lyz-Cre+TD2 mice,A/Lyz-Cre+TD2+MET mice.Immunofluorescence staining showed that the expression of GAP43 in the sciatic nerve in TD2 mice and TD2+MET mice was significantly lower than that of A/Lyz-Cre +db/m mice,A/Lyz-Cre+TD2mice,and A/Lyz-Cre+TD2+MET mice(P<0.01).The proliferation rate of Schwann cell in sciatic nerve was decreased significantly in TD2 mice compared with db/m mice,A/Lyz-Cre+db/m mice(P<0.05),the proliferation rate was significantly improved in A/Lyz-Cre+TD2 mice and A/Lyz-Cre+TD2+MET mice(P<0.01).The distribution area of GFAP in sciatic nerve of TD2 mice and TD2+MET mice was much lower than that of db/m mice(P<0.05),And the distribution area in A/Lyz-Cre+TD2 mice and A/Lyz-Cre+TD2+MET mice was significantly higher than that of TD2 mice(P<0.01).There was no significant difference in the expression of NF200 in sciatic nerve.6.The result of BMSC migration in vitro showed that SDF-1alpha can indeed enhance the migration of BMSC,and it migrate along the direction of the SDF-1 alpha concentration gradient,When the CXCR4inhibitor(AMD3100)and the Pakt inhibitor(Ly94002)were treated,the mobility of BMSC was significantly reduced.The BMSC migration rate in TD2 mice was significantly lower than that in db/m mice,and the neuritin in bonemarrow contributes to the migration of BMSC.ELISA results showed:concentration of SDF-1 alpha in sciatic nerve was significantly increased in TD2 mice,and the gradient distribution rate of concentration of SDF-1 alpha from bone marrow to sciatic nerve is more obvious in model mice with the over expression of neuritin in bone marrow(A/Lyz-Cre+TD2 mice,A/Lyz-Cre+TD2+MET mice).immunofluorescence staining of slices of femur with SDF-1 alpha +CXCR4: in type 2 diabetes(including type 2 diabetes model with over expression of neuritin),SDF-1alpha and CXCR4 were co-localized.Immunofluorescence staining with GFAP+Pakt in sciatic nerve sections: in type2 diabetes mice,the damage of the myelin of sciatic nerve induce activation of Pakt and the high expression of neuritin in bone marrow would promote the activation of Pakt.Conclusion: 1.Neuritin in bone marrow nerve can repair neuropathy in bone marrow of type 2 diabetes and improve bone marrow function.2.Neuritin in bone marrow nerve can improve the migration ability of bone marrow mesenchymal stem cells.3.Neuritin in bone marrow nerve can repair the peripheral neuropathy in type 2 diabetes by activating PI3K/Akt pathway through SDF-1 alpha /CXCR4 axis.It is presumed that bone marrow mesenchymal stem cells play an important role in the repairing process. |
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