Study On The Molecular Mechanism Of Inhibiting The Classical Wnt Signaling Pathway To Promote The Development Of Diabetic Neuropathy

Diabetes is currently the most important metabolic disease in the world,which seriously affects human life and health.Diabetic peripheral neuropathy is one of the most common chronic complications of diabetes.It can cause repeated lower limb infections,ulcers,non-traumatic amputations,etc.Which has a high disability rate,and seriously reduces the quality of life of patients,so the prevention and treatment of DPN has important clinical significance.Recent studies have shown that the classical Wnt signaling pathway is closely related to the nervous system and plays an important role in the formation of neural tube,dorsal root ganglia and midbrain development.Abnormal Wnt signaling pathways also play a broad role in the development of metabolic diseases.In addition,Wnt signaling pathway is also involved in the regulation of adipocyte differentiation,pancreatic development,beta cell differentiation,insulin biosynthesis,insulin secretion and signal transduction,which play an important role in the occurrence,development and complications of T2 DM.Therefore,the relationship between Wnt signaling pathway and DPN is studied,and the mechanism of action has important research value.Part ? Determination of changes in the expression of key genes of Wnt signaling pathway in patients with DPN ObjectiveThe peripheral blood of normal population,with out DPN diabetic patients and diabetic patients with DPN was used as the research object to explore the differential expression of key molecules of Wnt pathway in three groups,and to analyze the correlation between the expression level and clinical indicators of diabetes.Methods1.From Dec 2017 to May 2018,according to the Diabetes Clinical Diagnostic Criteria and DPN Diagnostic Criteria,the fasting peripheral blood of normal population,with out DPN diabetic patients and diabetic patients with DPN were collected from the Henan Provincial People's Hospital.Peripheral blood EDTAK2 was anticoagulated and stored at-80 °C until use.At the same time,clinical biochemical test data of three groups of people and medical records of T2 DM patients were collected.2.Using the Trizol method to extract total blood RNA and determine its purity and content,reverse transcription reaction to obtain cDNA of RNA.3.Using real-time PCR and the forward and reverse primers of the key molecules ?-catenin,c-myc,cyclinD1 and DKK1,and amplifying the internal reference gene ?-actin as a control to analyze the key molecules of Wnt pathway.Differences in expression in the three groups of people.4.Using the clinical blood biochemical data of the three groups of people to analyze the correlation between the key molecule expression level of Wnt pathway and HbA1 c.ResultsDifferential expression of key molecules in the Wnt pathway in three groups: Fluorescence quantitative PCR showed that ?-catenin,c-myc,and cyclinD1 were decreased in T2 DM and DPN patients,while DKK1 was elevated in T2 DM and DPN patients,and Even worse in patients with DPN.Further analysis showed that the levels of ?-catenin,c-myc and cyclinD1 were negatively correlated with HbA1 c in T2 DM population,and DKK1 level was positively correlated with HbA1 c level in T2 DM population.ConclusionThe expression levels of key genes ?-catenin,c-myc and cyclinD1 in Wnt pathway were decreased in patients with T2 DM and DPN,while the negative regulatory molecule DKK1 was abnormally highly expressed in DPN patients,and was significantly correlated with HbA1 c level,suggesting Wnt A reduction in signal path function may promote the occurrence of DPN.Part ? Effect of inhibiting Wnt signaling pathway in vivo on the development of DPN ObjectiveBy constructing a T2 DM mouse model,mice were intragastrically administered with Wnt pathway inhibitor C59 to observe the effect of inhibition of Wnt signaling pathway on the development of DPN.Methods1.Establishment of T2 DM mouse model: SPF grade 4 week old C57B6 mice,weighing 13-15 g,adaptive feeding for 1 week,randomized digital table method to select 6 normal diet as the basic control group,namely Chow-NC group The remaining mice,fed a high-fat diet with 60% fat content for four weeks,were fasted for 14 h,and were injected with streptozotocin(STZ)solution at 40 mg/(kg·d)for 5 consecutive days.After injecting STZ,10% sucrose water was consumed for 3 days.After 10 days,blood glucose was measured at the tip of the tail,and 3 random blood glucose >16.9 mmol/L was considered as successful modeling.2.Grouping of mice: T2 DM mice were divided into 3 groups according to the random number table.One group was intragastrically administered with normal saline as a control,which was T2DM-NC group;group 1 was intragastrically administered Wnt pathway inhibitor C59 10 mg/(kg·d)was injected every 2 days,that is,T2DM-C59 group,and high-fat feeding continued until the end of the experiment.3.Observe the mouse color,mental state,daily activities,drinking water and diet.The weights of the 4th,8th,14 th,17th,19 th and 21 th week of the four groups of mice were recorded.After fasting for 12 hours,the tail veins were taken to take blood to detect the fasting blood glucose of the mice.4.Mouse hind limb pain test: fixed detection of the right hind limb of the mouse,the stimulation intensity was 35%,the stimulation times were 3 times,each time interval 30 minutes,the mouse paw withdrawal time was recorded.Test every four weeks until the end of the experiment.5.Detection of exercise capacity: The number of shocks of the mice was recorded at 17 m/min for 30 minutes,and was tested every four weeks until the end of the experiment.6.Experimental animal materials: At the end of the experiment,the mice were fasted for 12 h,and after 7% chloral hydrate anesthesia,the mice eyeballs were bled,and the disposable negative pressure blood collection tube EDTAK2 was used for anticoagulation,at 4 ° C,5000 r / min,After centrifugation for 20 minutes,the supernatant was obtained,dispensed in an EP tube,and stored at-80 ° C until use.The mouse was placed on the operating table in the supine position.After the skin was disinfected,the skin of the mouse was cut in the anterior lateral part of the femur,fully exposed,and the muscles of the leg of the mouse were taken and placed in a frozen tube for rapid freezing of nitrogen for molecular biological detection.7.Determination of serum serum insulin,total cholesterol,total glycerol,high density lipoprotein,low density lipoprotein,total bile acid concentration.Results1.The main clinical features of type 2 diabetes such as insulin resistance,hyperglycemia and dyslipidemia in model mice.Intraperitoneal injection of STZ resulted in damage to islet cells,so the weight of the high-fat group was slightly lower than that of the normal control group,and the weight gain of the mice in the high-fat group was slow,and the blood glucose was elevated,which was consistent with the performance of diabetes,indicating that the T2 DM mouse model was successfully modeled.2.After inhibiting the endogenous Wnt signaling pathway,the fasting serum insulin levels,TG,C-THO and LDL were higher in mice,and the blood glucose and lipid metabolism disorders in mice were more serious,and insulin resistance was more significant.3.Liver oil red O staining results showed that: After inhibiting Wnt signaling pathway,the hepatic cell gap of the mouse became larger,the hepatic cord arrangement was disordered,and the red lipid droplets visible in the liver cells increased significantly,indicating that the lipid deposition in the liver was more serious.4.Through the detection of the foot tingling and exercise ability of the mice,it was found that the Wnt signaling pathway was inhibited,the response time of the mice to thermal pain stimulation was prolonged,and the exercise tolerance was significantly decreased.ConclusionInhibition of endogenous Wnt signaling pathway significantly increased aggravated blood glucose and lipid metabolism disorders in mice,and insulin resistance was more pronounced;peripheral nerves The degree of damage is significant,the nerve conduction velocity is reduced,the neurotrophic function is aggravated,and the exercise endurance ability is further reduced.Part ? Revealing the molecular mechanism of Wnt signaling pathway affecting the occurrence of DPN ObjectiveTwo groups of mouse skeletal muscle were used as research objects to explore the differential expression of key molecules of Wnt pathway in two groups of mice and the molecular mechanism of promoting the development of DPN.Methods1.The total RNA of muscle tissue was extracted by Trizol method and its purity and content were determined,and the cDNA of RNA was obtained by reverse transcription reaction.2.Using the quantitative PCR technique and the forward and reverse primers of the key molecules c-myc,cyclinD1,VDL1 and FZD7 of the Wnt pathway,and amplifying the ?-actin gene as a control,analyzing the key molecules of the Wnt pathway in two Differences in expression in the group of mice.Western blot further verified the protein expression of key molecules in the Wnt pathway.3.Using RNA transcriptome deep sequencing to detect changes in gene expression profiles of the two groups of mice.Using bioinformatics methods to predict target genes and molecular pathways affecting the development of DPN.4.The levels of IL-6 and HIF-1? in human serum were detected by double sandwich ELISA.The correlation between IL-6,HIF-1? level and HbA1 c was analyzed by clinical blood biochemical data of three groups.5.Real-Time PCR,Western blot and other techniques were used to detect changes in mitochondrial copy number and ATP content in skeletal muscle tissue,and to verify the molecular mechanism of inhibition of this pathway to promote the development of DPN.Results1.Real-Time PCR results showed that the expression of c-myc and cyclinD1 was down-regulated after Wnt pathway was inhibited(P<0.05).Western Blot results further confirmed that the ratio of phosphorylated ?-catenin to total ?-catenin was increased after inhibition of Wnt pathway(P < 0.05).This suggests that Wnt-C59 interferes with the normal conduction of the Wnt pathway by inhibiting the expression of key genes in the Wnt pathway.2.Based on the results of deep sequencing and bioinformatics analysis,determine the specific signaling pathways that inhibit the Wnt pathway to promote the development of DPN.3.Compared with the control group,IL-6 and HIF-1? were elevated in patients with T2 DM and DPN,and the decrease was more significant in patients with DPN,and the levels of IL-6 and HIF-1? in serum were correlated with those in diabetic patients.HbA1 c is significantly correlated.4.After inhibiting the Wnt pathway,IL-6 mRNA expression was increased in key molecules in the HIF-1 pathway metabolic pathway(P < 0.05);the ratio of phosphorylated STAT3 to total STAT3 was increased(P < 0.05)while HIF1? mRNA was present.The expression was also significantly elevated(P < 0.05).These results suggest that inhibition of Wnt pathway induces overexpression of IL-6 inflammatory factor and activates STAT3 protein,which promotes the expression of hypoxia-inducible factor;further down-regulates TFAM expression and mtDNA copy number(P < 0.05),inhibits mitochondrial respiratory oxidation and The generation of ATP aggravates the degree of damage to the peripheral nerves.ConclusionInhibition of Wnt pathway activates IL-6 / STAT3 / HIF-1? signaling pathway,induces increased expression of hypoxia-inducible factors,further reduces TFAM expression and mtDNA copy number,inhibits mitochondrial respiratory oxidation and ATP production,and promotes DPN The occurrence and development.