Relation Of Free Fatty Acid And Peripheral Neuropathy Of Type 2 Diabetes And Mechanism Study

A significant increase in type 2 diabetes mellitus can be demonstrated from all epidemiological studies.Diatebic neuropathy (DN) is one of the most frequent and troublesome complications of diabetes mellitus. In the U.K.Prospective Diabetes Study(UKPDS),over 11% of patients had diabetic neuropathy in the time of diagnosis of type 2 diabetes.At 12 years,thers were obverous diabetic neuropathy in 71% of men patients and 51% of women patients. It is the major reason for morbidity and mortality among diabetic patients.Diabetic peripheral neuropathy(DPN) is the most common chronic complication of diabetes,accourting for substantial morbidity and mortality and resulting in huge health care costs.There are varied clinical presentations of DPN with involvement of proximal or distal peripheral sensory and motor nerves,as well as autonomic nerves.Although there has been significant progress in the understanding of the clinical aspects of these conditions, it is difficult to answer the pathogenic mechanism of diabetic neuropathies.In the past few years,animal and clinic studies reveled that pathogenesis of diabetic neuropathy has multifactorial causes, such as:non-enzymatic glycation of proteins ,oxidative stress, the polyol pathway, immune mechanisms, neurotrophic growth factor ,genetic susceptibility.In the recent years studies,it is found that dyslipidaemia and inflammation are important in the devolpment of insulin resistant,diabetes and complations of diabetes.In 2002,Mcgarry belive that dysregulation of fatty acid metabolism is the etiology of type 2 diabetes,and there is increasing evidence that low-grade inflammation is closely involved in the pathogenesis of type 2 diabetes and associated complications.But the relationship between dyslipidaemia,inflammation and diabetic peripheral neuropathy is unkown。In our study,the first and second part are clinic research。By logistic regression analysis, to study the risk factors in the patients with type 2 diabetic peripheral neuropathy and relation between FFA,TNF-α,IL-6 and diabetic peripheral neuropathy.In the third and forth part of reaserch, Female Wistar rats were fed with high-fat diet to induce insulin resistance.Hyperglycemia was developed by intraperitoned injection in these rats with 30mg/kg streptozotocin(STZ)after 8 weeks.The rats were accepted as diabetic in the light of their blood glucose exceeded 7.0mmol/L after 1 month of STZ injection.The serum FFA is high in these rats and continue to feed with the high-fat diets.Then, the model rats were divided into two groups: diabetic peripheral neuropathy group(DPN group) and treatment group(the rats were fed with fenofibrate 30mg/kg.d)(DPNFgroup).We explore the possible relation between FFA,inflammation and diabetic peripheral neuropathy,and the effect of treatment with fenofibrate. To providing a new study direction of improving diabetic peripheral neuropathy. The paper contains four parts below:Part one: Risk factors for diabetic peripheral neuropathy in type 2 diabetes mellitus patientsObjective: To study the risk factors in the patients with type 2 diabetic peripheral neuropathy.Methods: 547 type 2 diabetic patients were tested for the nerve conduction velocities. The patients were divided into diabetic peripheral neuropathy group and non-diabetic peripheral neuropathy group. Blood pressure, triglycerides (TG), total cholesterol (TC), HDL-C, LDL-C, body mass index (BMI), glycosylated hemoglobin (HbA1c), insulin, C peptide and urinary albumin/creatinine (A/C) were measured for all patients. All the risk factors were included in the statistical analysis.Results:Duration of diabetes, age, fasting plasma glucose,2-hour plasma glucose, TG, TC, urinary A/C and HbA1c in diabetic peripheral neuropathy group were significantly higher than that in non-diabetic peripheral neuropathy group(p<0.05 or p<0.01). The level of fasting insulin and plasma C peptide ,2-hour insulin and plasma C peptide were significantly lower than that in non-diabetic peripheral neuropathy group(p<0.05 or p<0.01).Conclusions: By logistic regression analysis, it was found that the significant risk factors of diabetic peripheral neuropathy in type 2 diabetic patients were age, duration of diabetes,HbA1c, urinary A/C, fasting and 2-hour glucose,C peptide and insulin.Part two: The relation between FFA,TNF-α,IL-6 and diabetic peripheral neuropathy in type 2 diabetes mellitus patientsObjective: 92 type 2 diabetic patients were divided into diabetic peripheral neuropathy group and non-diabetic peripheral neuropathy group. Serum FFA,TNF-α,IL-6 were measured for all patients and analysis the relationship between FFA,TNF-α,IL-6 and diabetic peripheral neuropathy.Methods: According to the 1997 ADA criteria for DM diagnosis and the nerve conduction velocities, 92 type 2 diabetic patients were divided into diabetic peripheral neuropathy group (DPN group) and non-diabetic peripheral neuropathy group(NDPN group). Blood pressure, triglycerides (TG), total cholesterol (TC), HDL-C, LDL-C, body mass index (BMI), glycosylated hemoglobin (HbA1c), insulin, C peptide and urinary albumin/creatinine (A/C) ,serum FFA ,TNF-αand IL-6 were measured for all patients. All the risk factors were included in the statistical analysis.Results: 1,Compared with NC group, the DBP,BMI,SBP,FPG,HbAlc,Fins,TG,LDL-C were higher in the DPN group and NDPN group(p<0.05 or p<0.01); the HDL-c was lower in the DPN group and NDPN group( p<0.01)。2,Compared with NDPN group, duration,SBP, DBP,FPG,HbAlc,Fins,TG,LDL-C were higher in the DPN group(P<0.05P<0.01).3,Compared with NC group,serum FFA TNF-αand IL-6 were higher in the DPN group and NDPN group(p<0.05 or p<0.01).4,Compared with NDPN group,serum FFA TNF-αand IL-6 were higher in the DPN group (p<0.05).5,By logistic regression analysis,with or without diabetic peripheral neuropathy as a dependent variable and with SBP,DBP,HbAlc,urinary A/C,Fins,FBG,TG,LDL-C,TNF-α,FFA,IL-6 as independent variable,finally duration,urinary A/C,HbAlc,HoMA-IR,TNF-α,FFA,IL-6 entered the equations.6,Serun TNF-αand IL-6 had a positive correlation with serum FFA.Conclusions: The level of serum FFA,TNF-αand IL-6 of DPN group were significantly increased Compared with NC group and NDPN group. By logistic regression analysis suggests that serum FFA,TNF-αand IL-6 are the risk factors of diabetic peripheral neuropathy. Serum FFA have positive correlation with serum.TNF-α,IL-6,Part three: Relation between serun FFA and diabetic peripheral neuropathy in type 2 diabetes rats and fenofibrate treatment.Objective: To set up rat model of type 2 diabetes peripheral neuropathy and observe the correlation of serum FFA and peripheral neuropathyMethods: Male Wistar rats weighed 300g were divided into normal control control (NC) group (n=10), high-fat diet (HF) group (n=30).The rats in control group were fed with a regular low fatty acids diet containing 10.3% fat, 24.2% protein, and 65.5% carbohydrate as percentage of total calories. The rats in high-fat diet group were fed regular diets mixed with 30% lard, containing 59.8% fat, 20.1% protein and 20.1% carbohydrate as a percentage of total calories. The rats in high-fat diet group were injected by intraperitoned with 30mg/kg streptozotocin(STZ)after 8 weeks.The rats were accepted as diabetic in the light of their fasting blood glucose exceeded 7.0mmol/L after 4 weeks of STZ injection.Total 23 rats'fasting blood glucose exceeded 7.0mmol/L ,and were randomly devided into diabetic peripheral neuropathy (DPN) group (N=11)and fenofibrate treatment (DPNF) group (N=12). The twe groups rats were continue to feed with the high-fat diets.The rats in DPNF group were given with fenofibrate 30mg/kg.d through intragastric administration once a day.The body weights were determined every two weeks for 32 weeks. The electrophysiology was examined every four weeks.The blood sample was collected by cardiac puncture after rats were anesthetized with diethyl ether for the biochemical analysis.At the end of 32 week, the rats were killed with phenobarbital sodium, and sciatic nerve were taken out freeze-clamped with copper clamps precooled in liquid N2 and were stored in -70℃refrigerator.Results: 1,After 8 weeks of fed with high-fat diet,compared with NC group, the bodyweight,FBG,FINS,TC,TG,FFA were higher in HF group(P<0.05 or P<0.01). After 4 weeks of STZ injection, compared with NC group,the body weight and Fins were sinple(P>0.05) ,and FBG,TC,TG,FFA were higher(P<0.05或P<0.01)in the HF group.2,At the end of 32 weeks, compered with DPN group , BG,TG,FFA were lower in DPNF group(P<0.05或P<0.01).The FBG and Fins were lower than DPNF group,but not signifecient(P>0.05).3,After 4 weeks of STZ injection, tibial nerve conduction velocity,latency period,withdrawa mechanical threshold and withdrawal thermal latency were not signifecient(P>0.05).At the end of 32 weeks, compered with NC group, tibial nerve conduction velocity lower,latency period longer,withdrawa mechanical threshold and withdrawal thermal latency higher in DPN group(P<0.05或P<0.01). Compered with DPN group, tibial nerve conduction velocity faster,latency period shorter,withdrawa mechanical threshold and withdrawal thermal latency lower in DPNF group(P<0.05或P<0.01).4,With electron microscope, lamina medullares of myelin sheath was chorisis, neurite was atresia in DPN group. But pathological changes were abatement in DPNF group.5,Serum FFA has a positive correlation with latency period and has a negative correlation with tibial nerve conduction velocity.Conclusions: 1,After fed with high-fat diet after 8 weeks,insulin resistance developed in rats。These rats were intraperitoned injected with a low doze of STZ. After 4 weeks of STZ injection,fasting blood glucose and serm insuin was higher than NC group,so the rat model of type 2 diabetes mellitus was set up .The serum FFA was higher in the rat model. These rats continued to fed with high-fat diet,and diabetic peripheral neuropathy developed at 32th week. 2,Correlation analyses suggested that serum FFA associated with tibial nerve conduction velocity,latency period shorter,withdrawa mechanical threshold and withdrawal thermal latency lower。3,After treated with fenofibrate,serum FFA lowered and pathology damage of sciatic nerve improved,so that indicated serum FFA has a correlation with diabetic peripheral neuropathy.Part four: The expression of NF-κB,TNF-α,IL-6 in sciatic nerve in rats of type 2 diabetic peripheral neuropathy and fenofibrate treatment.Objective: To observe the expression of NF-κB,TNF-α,IL-6 in sciatic nerve in rats of type 2 diabetic peripheral neuropathy and fenofibrate treatment. To analyze the correlation of NF-κB,TNF-α,IL-6 and diabetic peripheral neuropathy.Methods: Animal grouping and the samples acquirement were the same as part one. The expression at protein and mRNA level of NF-κB,TNF-α,IL-6 were measured by Western-blot method and PCR method respectively.Results: 1,At the end of 32th week, compared with NC group, the expression of protein and mRNA level of NF-κB,TNF-α,IL-6 in sciatic nerve were higher in the DPN group rats(P<0.01); Compared with DPN group, after fenofibrate treatment ,the expression of protein and mRNA level of NF-κB,TNF-α,IL-6 in sciatic nerve were lower in DPNF group (P<0.05). 2,Correlation analyses suggested that the expression of protein and mRNA level of TNF-α,IL-6 have a positive correlation with NF-κB.And the expression of NF-κB has a positive correlation with serum FFA.Conclusions: 1,The expression of protein and mRNA level of NF-κB,TNF-α,IL-6 in sciatic nerve in DPN group were higher and after fenofibrate treatment the expression of protein and mRNA level of NF-κB,TNF-α,IL-6 lower, and pathology damage of sciatic nerve improved.That indicated inflammation mybe one of the mechanisms of diabetic peripheral neuropathy.2,Correlation analyses suggested, the expression of protein and mRNA level of NF-κB,TNF-α,IL-6 in sciatic nerve associated with serum FFA,...