High Glucose Influence The Ability Of1,25（OH）₂D₃to Stimulate Nerve Growth Factor Secreting In Schwann Cells

BackgroudDiabetic Peripheral neuropathy is the most common and the most complicated one of chronic complications of diabetes.In our country, the incidence of type2diabetes were up to9.7%, and the patients with peripheral neuropathy were60-70%. The pain, numbness, and secondary to diabetic foot infection,amputation, resulted in the quality of life of the patients to decrease significantly and medical costs to increase significantly.1,25(OH)2D3is the active metabolite of Vitamin D3in vivo.Recent epidemiological and clinical studies have showed that Vitamin D3deficiency and diabetic peripheral neuropathy are closely related and inadequate Vitamin D3is independent risk factor of diabetic peripheral neuropathy. The latest USA Health and Nutrition Examination Survey (NHANES) statistics have showed that, insufficient Vitamin D3in81%adult patients with diabetes relates with diabetic neuropathy closely,even avoiding these potentially confounding factors,such as, the demographic factors,obesity, complications, drug treatment for neuropathy and diabetes duration. In a clinical study of210patients with diabetes, neuropathy group of25-hydroxyvitamin D3(25(OH) D3:in vivo detection index for vitamin D3active substances) levels were lower than those without neuropathy group,and25(OH) D3deficiency levels in the group with and without neuropathy were accounted for81.5%and60.4%, respectively. A prospective studies of painful diabetic neuropathy and vitamin D3had shown the relationship:vitamin D3deficiencies were present in51patients with painful diabetic neuropathy, the mean plasma25(OH) D3concentration was18ng/mL.Pain scores and25(OH) D3concentrations were negatively correlated. After supplement with adequate vitamin D3,the pain score decreased significantly. However, the mechanism of which remains unclear.Schwann cells play an important role in the peripheral nervous system, such as: the formation of myelin, supportion and nutrition, promoting the recovery of the axons and neurons and secreting neuro trophic factors (e.g.:Nerve Growth Factor) Nerve growth factor plays an important role in nerve regeneration in peripheral neuropathy.Nerve growth factor levels decrease to some extent resulting the onset of axonal regeneration failure and diabetic peripheral neuropathy. Under high glucose conditions, declining levels of nerve growth factor reduces the dorsal root ganglion neurons sprouting.As further studies in recent years on1,25(OH)2D3, the glial cells of the central nervous system-stellate cells’protective effect has been confirmed:1,25(OH)2D3inhibits inducible nitric oxide synthase enzyme expression and reduces the synthesis of nitric oxide and cell inflammatory response; Increases expression of nerve growth factor mRNA and protein in stellate cells, inducing neurotrophin protective effect; increases glutathione pool outside stellate cells, and significantly reduces LPS-mediated of nitrite, scavenging reactive oxygen species.But the studies on the fluence of1,25(OH)2D3on the peripheral nervous system, especially the relationship between1,25(OH)2D3and Schwann cells is very fewer.Although many studies have shown that1,25(OH)2D3mediated expression of nerve growth factor in various cell, but only one article had shown Schwann cells up-regulated gene expression of nerve growth factor, but did not mention their secretion levels of nerve growth factor.Although studies have shown that a derivative of vitamin D3dose-dependent increases sciatic nerve growth factor in diabetic rat and prevents nerve growth factor depletion. But almost no research concerned the relationship between high glucose and1,25(OH)2D3affect on Schwann cells.1,25(OH)2D3through the vitamin D receptor,binding retinoic acid x receptor or through signaling molecules, such as the ERK1pathway activates CYP24A1gene expression.25(OH) D3-24-hydroxylase (CYP24A1) can metabolize1,25(OH)2D3or25(OH) D3into inactive the1,24(OH)2D3or24(OH).Studies have shown CYP24A1increased expression in the kidney of diabetic animal model.Whether CYP24A1expression in Schwann cells exists, how the metabolic genes of1,25(OH)2D3will change in high glucose and whether that involves in the relationship between high glucose and1,25(OH)2D3and affects on NGF secretion by Schwann cells, will be the focus of our study.To investigate relationship between diabetic neuropathy and1,25(OH)2D3and whether high glucose affects on1,25(OH)2D3metabolism, we treat RSC96rat schwann cells as a model to explore influence of the high glucose and1,25(OH)2D3on nerve growth factor secreting by Schwann cells, and the interaction relationship between the two.Chapter1Effects of high glucose and1,25(OH)2D3on secretion levels of nerve growth factor by Schwann cellsObjective:To study the effect by high glucose and1,25(OH)2D3on secretion levels of nerve growth factor by RSC96Schwann cells.Methods:1)Effect of high glucose on secretion of NGF by Schwann cells:The Schwann cells were cultured in5.6mmol/L glucose (5.6G),17mmol/L glucose (17G) and17mmol/L mannitol (17M) DMEM culture medium for96hours.Experiments were divided into three groups:①5.6G group②17G group③17M group.Enzyme-linked immunosorbent assay (elisa) detected supernatant for levels of nerve growth factor and then detected absorbance OD values obtained from microplate readerfor. Each experiment set three wells.The experiment was performed twice.2)Effect of1,25(OH)2D3on secretion of NGF by Schwann cells:1,25(OH)2D3(D310-7mol/1) and solute ethanol treated Schwann cells respectively in5.6mmol/L glucose DMEM medium for48hours.Experiments were divided into three experimental groups:①5.6G group②D3+5.6G group③ethano1+5.6G group.Enzyme-linked immunosorbent assay (elisa) detected supernatant for levels of nerve growth factor and then detected absorbance OD values obtained from microplate reader. Each experiment set three wells.The experiment was performed twice.3)Effect of high glucose on secretion of NGF by Schwann cells treated with1,25(OH)2D3:the Schwann cells were cultured in5.6mmol/L glucose (5.6G),17mmol/L glucose (17G) and17mmol/L mannitol (17M) DMEM culture medium for48hours,combination with1,25(OH)2D3for additional48huours.Experiments were divided into three groups:①5.6G group②17G group③17M group.Results:1)To give5.6mmol/L glucose,17mmol/L glucose and17mmol/L mannitol DMEM medium for96hours, there was no significant difference in secretion levels of nerve growth factor by Schwann cells (P>0.05)(ANOVA F=0.447P=0.648.S-N-K pairwise comparison analysis M±SEM5.6G group:177.33±52.40pg/ml17G group:134.56±9.65pg/ml17M group:158.30±15.53pg/ml N=6).2)1,25(OH)2D3(D310-7mol/l)and solute ethanol treated Schwann cells respectively in5.6mmol/L glucose DMEM medium for48hours.1,25(OH)2D3group of nerve growth factor secretion significantly increased.(P<0.05)(ANOVA F=7.317P=0.006S-N-K pairwise comparison analysis M±SEM5.6G group:177.3±52.40pg/ml D3+5.6G group:340.29±30.91pg/ml ethanol+5.6G:161.28±17.90pg/mlN=6).3) The Schwann cells were cultured in5.6mmol/L glucose (5.6G),17mmol/L glucose (17G) and17mmol/L mannitol (17M) DMEM culture medium for48hours,combination with1,25(OH)2D3for additional48huours.Levels of nerve growth factor in high glucose group (17G) was significantly lower than the other two groups.(P<0.05)(ANOVA F=15.979P=0.000SNK pairwise comparison analysis of M±SEM:D3+5.6G group340.29±30.91pg/ml D3+17G group:151.09±10.43pg/ml D3+17M group:319.05±30.87pg/ml N=6).Conclusions: 1) High glucose does not directly affect secretion of nerve growth factor by Schwann cells.2)1,25(OH)2D3increased secretion of nerve growth factor by Schwann cells.3) This is the first time study found that high glucose weakened ability of1,25(OH)2D3to stimulate Schwann cells secreting nerve growth factor. Capter2Expression of synthesis and metabolic enzyme of1,25(OH)2D3in schwann cells and effect of high glucose on these eznymeObjective: To study whether1,25(OH)2D3anabolic genes express in schwann cells, and whether high glucose affect the expression of these enzymes to look for molecular mechanisms that high glucose weakened ability of1,25(OH)2D3to stimulate nerve growth factor secretion by Schwann cells.Methods:1) Design1,25(OH)2D3anabolic gene primers comprising:synthase25(OH) D3-24-hydroxylase (CYP27Al)gene,25(OH) D3-la-hydroxylase (CYP27B1)gene; metabolic enzymes:25(OH) D3-24-hydroxylase (CYP24A1) genes.Total RNA was extracted using RNA kit Fermatas company and then reversed transcribed and PCR amplified CDNA using the company Thermo kit. CYP27A1upstream primer5’-AGCCCTCTACACAGATGCCTTAAC-3’,downstream primer5’-CCCAGTT-ATTCATGTATCGCTTCC-3’, Annealing temperature57.5℃, PCR product length314bp;CYP27B1upstream primer5’-TACGCTCTCCTGGGCACTCTATGA-3’ downstream primer5’-AAGCGTCTCCCTATGCAACTTCGTT-3’, annealing temperature57.5℃, PCR product length of408bp;CYP24A1upstream primer5’-GCAGTGGACGACCGCGAACA-3’, downstream primer’-CGATGCACATTCTC-TTCCCGATGCC-3’,annealing temperature of60℃, PCR product length of412bp; β-actin primer5’-GAACCCTAAGGCCAACCGTGAA-3’, reverse primer5’-CCGCTATTGCCGATAGTGATG-3’,annealing temperature of55℃,PCR product length of433bp. Semi-quantitative PCR method was detected whether Schwann cells expressed these enzymes. PCR conditions:95℃3minutes,95℃30s,55℃30s,72℃30s for30cycles,72℃10minutes. Running glue and analysing image.2) Different concentrations of1,25(OH)2D3cultured Schwann cells for20hours, and the aforementioned known gene expression were detected.Each gene is divided into three groups:①A groups:control group②Group B:1,25(OH)2D3(10"8mol/L)③C group:ethanol5μL④D group:1,25(OH)2D3(10-7mol/L)⑤E group: ethanol50μL. Semi-quantitative RT-PCR analysis was used. Experiments were performed three times.3)17mmol/L glucose affect Schwann cells at20hours and96hours respectively. A. The effects of high glucose on the CYP24A1gene:the expression of known genes were detected, and each gene was divided into six groups:①5.6G1group②17G1group③17M1group④5.6G4group⑤17G4group⑥17M4group.(①②③represented at20hours,④⑤⑥represented at96hours) Semi-quantitative PCR experiments carried out three times. B. Effects of high glucose on the expression of CYP24A1protein:after Semi-quantitative PCR initial had screened changes of gene expression,and further used western blotting method. Experiments were performed three times.4)0.8mmol/L hydrogen peroxide acts on Schwann cells for20hours. A. Effect of hydrogen peroxide on the CYP24A1gene:divided into two groups:①5.6G group②5.6G+H2O2group. Using Semi-quantitative RT-PCR analysis, the experiments were performed twice. B.Effects of Hydrogen Peroxide on CYP24A1protein:the change was validated further by western blotting method, and experiments carried out three times.Results:1)CYP27A1, CYP27B1, CYP24A1genes express in Schwann cells.2)1,25(OH)2D3enhanced expression of CYP24A1gene:using PCR electrop hero grams to study gene/(3-actin gray value, One way ANOVA analysis,enhanced CYP24A1gene expressed in schwann cells treated with1,25(OH) 2D3for20hours. F value:118.237P=0.000, there was a significant difference between group B (0.99±0.07N=3), D group (1.87±0.07N=3) and the other three groups (A group:0.52±0.05; C group:0.47±0.04; E group:0.46±0.05N=3)(S-N-K).3) High glucose increases CYP24A1gene and protein expression:A.17mmol/L high glucose increased CYP24A1gene expression. using PCR electropherograms to study gene/β-actin gray value. One way ANOVA analysis, high glucose increased CYP24A1gene expression after20hours and96hours, F value:15.261P=0.000(5.6G1group0.07±0.01;17G1group0.15±0.0117M1group0.06±0.02;5.6G4group0.06±0.01;17G4group0.14±0.01;17M4group0.06±0.02N=3).B. High glucose upregulated CYP24A1protein.17G group was significantly higher than the other two groups (P＜0.05). One way ANOVA F=6.706P=0.030(S-N-K pairwise comparison5.6G group:0.40±0.0517G group:0.82±0.1117M group:0.44±0.09N=3).4) Hydrogen peroxide affected gene and protein expression of CYP24A1in Schwann cells.A. Hydrogen peroxide reduced CYP24A1gene expression in Schwann cells:after0.8mmol/L H2O2had affected Schwann cells for20hours, the CYP24A1genee expression were detected in Schwann cells. Electropherograms of PCR was used to analyse CYP24Al/β-actin gray value. The results showed that:hydrogen peroxide reduced CYP24A1gene expression in Schwann cells.Two independent samples T-test (t=7.881, P=0.001)(5.6G group:0.29±0.01;5.6G+H2O2group:0.14±0.02N=3).B. Hydrogen peroxide does not affect CYP24A1protein expression in Schwann cells: after0.8mmol/L H2O2had affected Schwann cells for20hours, Schwann cells CYP24A1protein was detected by western blot. Gel electropherograms was used to analyse CYP24Al/β-actin gray value. The results showed that:hydrogen peroxide does not affect the expression of CYP24A1protein of Schwann cells. Two independent samples T-test (t=0.445, P=0.679)(5.6G group:0.40±0.05;5.6G+H2O2group:0.36±0.07N=3). Conclusions:1)This is first time study discovered that CYP27A1, CYP27B1, CYP24A1genes expressed in Schwann cells, indicating that Schwann cells is target cells of1,25(OH)2D3. Furthermore itself may have a synthesis capability of metabolizing active vitamin D3, possibly through autocrine and paracrine manner increasing local1,25(OH)2D3concentration,and thereby affecting themselves and the surrounding tissue.2)This is first time study found that high glucose upregulated genes and protein expression of CYP24A1, and upregulation of CYP24A1may interfere with catabolism function of1,25(OH)2D3in the normal of Schwann cells, leading to1,25(OH)2D3normal physiological function disrupted.3) Increasing expression of CYP24A1gene and protein in high glucose condition, may be unrelated to reactive oxygen species. Chapter3CYP24A1inhibitors affect secretion of nerve growth factor by Schwann cells cultured in high glucose medium with1,25(OH)2D3suplementationObjective:To study whether CYP24A1inhibitor affect ability of1,25(OH)2D3stimulating nerve growth factor secretion in high glucose medium in Schwann cells.Methods:1) Effect of CYP24A1inhibitor genistein on CYP24A1protein in Schwann cells cultured in high glucose medium:the Schwann cells cultured in medium containing17mmol/L glucose for48hours, then schwann cells were treated with genistein (Gen50μmol/L) or solute dimethyl sulfoxide (DMSO) for an additional48hours.The experiments were divided into two groups:①17G+Gen group②17G+DMSO group. Using western blotting to detect CYP24A1protein expression. Experiments were performed three times.2) Effect of genistein on secretion of nerve growth factor by Schwann cells treated with1,25(OH)2D3in high glucose condition:Schwann cells were cultured in the medium containing17mmol/L glucose for48hours,then schwann cells were treated with1,25(OH)2D3and genistein or DMSO for an additional48hours.The experiments were divided into2groups:①D3+17G+Gen group,②D3+17G+DMSO group. The expression level of NGF protein was detected by ELISA and then to detect absorbance OD values obtained from microplate reader.Each experiment was set three wells.The experiment was performed twiceResults:1) Genistein reduced CYP24A1protein expression in schwann cells:after schwann cells were culcured in17mmol/L Glucose culture media for48hours, then schwann cells were treated with genistein (Gen50μmol/1) or solute dimethyl sulfoxide (DMSO) for anadditional48hours, gel electrophoresis analysis with CYP24Al/β-actin gray value (t=-3.030, P=0.0.039)(Independent samples T-test Mean±SEM17G+Gen group:0.55±0.0417G+DMSO group:0.83±0.09N=6).2) Genistein group (D3+17G+Gen) nerve growth factor secretion induced by1,25(OH)2D3increased significantly. Schwann cells cultured in the medium containing17mmol/L glucose for48hours,then schwann cells were treated with1,25(OH)2D3, and genistein or DMSO for an additional48hours (T=-2.99, P=0.027). Independent samples T-test(Mean±SEM D3+17G+Gen group;446.53±68.61pg/ml D3+17G+DMSO group:242.38±38.25pg/ml N=6).Conclusions:1)This is first time study found that genistein inhibited the expression of upregulated CYP24A1protein by high glucose in Schwann cells.2)For the first time,the study found that genistein could recover NGF secretion by high glucose-cultured SCs stimulated with1,25(OH)2D3. Capter4To construct lentivirus-mediated shRNA silencing CYP24A1gene Schwann cell model Objective:To construct lentivirus-mediated shRNA silencing CYP24A1gene Schwann cell modelMethods:Using lentiviral packaging shRNA,silence CYP24A1gene of Schwann cells: Synthesis of four shRNA vectors by company (CYP24A1-1,CYP24A1-2, CYP24A1-3,CYP24A1-4) and negative (empty vector),blank (no treatment Schwann cells) were divided to6groups:①CYP24A1-1②CYP24A1-2③CYP24A1-3④CYP24A1-4⑤negative control (NC group)⑥blank control (BC group). Step:1)To construct CYP24shRNA carrier2)To amplify and extract plasmid3)To transfect plasmid and screen effective carrier (western blot, PCR)4)To package Viral5)To construct cell model6)To detect interference effects (western blot, PCR).Results:1)CYP24Al-4,a transfected lentivirus vector,was screened for effective carrier.2)293Ta cells packaged with lentiviral were successful.3)Lentivirus infected Schwann cells and silenced the CYP24A1gene and protein were successfulConclusion:Lentivirus-mediated shRNA silencing Schwann cells CYP24A1gene model had construced successful Capter5RNA interference mediated by lentivirus silencing CYP24A1gene affected secretion levels of NGF stimulated by1,25(OH)2D3in Schwann cell Objective:To confirm that effect of high glucose on1,25(OH)2D3stimulating NGF secretion in Schwann cells is related to CYP24A1gene upregulation through CYP24A1gene silencing Schwann cells.Methods:The experiments were divided into groups:1)To research wether silencing CYP24A1gene will affect NGF secretion by Schwann cells:ordinary RSC96Schwann cells (5.6G group) and CYP24A1gene silencing Schwann cells (si5.6G group) were cultured in5.6mmol/L glucose DMEM medium for96hours, and then the supernatant was detected for NGF protein levels. The experiments were divided into two groups:①5.6G group②si5.6G group (siRNA interference group).2)To study whether silencing CYP24A1gene will affect NGF secretion by Schwann cells treated with1,25(OH)2D3:1,25(OH)2D3(D310-7mol/L) and the ethanol treated Schwann cells and CYP24A1gene silencing Schwann cells cultured in5.6mmol/L glucose DMEM medium for48hours,and then the supernatants were detected for NGF protein levels. The experiments were divided into four groups:①5.6G+D3group②5.6G+ET group③si5.6G+D3group④si5.6G+ET groups. Enzyme-linked immunosorbent assay (elisa) detected supernatants for levels of nerve growth factor and then detected absorbance OD values obtained from microplate readerfor. Each experiment seted three wells.The experiment was performed twice.3) To study whether the silencing CYP24A1gene affected NGF secretion by Schwann cells cultured in high glucose with1,25(OH)2D3suplementation:Ordinary Schwann cells (5.6G group) and silencing CYP24A1gene of Schwann cells (si5.6G group) were cultured in17mmol/L glucose (17G) and17mmol/L mannitol (17M) DMEM medium for48hours and then added1,25(OH)2D3(D310-7mol/L) and the corresponding solute ethanol (ET) for an additional48hours, respectively. Experiments were divided into four groups:①17G+D3group②17M+D3group③si17G+D3group④si17+D3group.Enzyme-linked immunosorbent assay (elisa) detected supernatants for levels of nerve growth factor and then detected absorbance OD values obtained from microplate readerfor. Each experiment seted three wells.The experiment was performed twice.Results:1) Silencing CYP24A1gene Schwann cells did not affect the normal secretion of NGF by Schwann cells:using analysis of two independent samples Ttest, there was no significant difference between the two groups.T=0.931, P=0.374(Mean±SEM, group5.6G:162.08±4.13; si5.6G group:156.76±3.95N=6).2) CYP24A1gene silencing increased secretion levels of NGF mediated by1,25(OH)2D3in Schwann cells:using One-Way ANOVA test, si5.6G+D3group increased significantly compared with the other three groups.(P=0.000)(Mean±SEM,5.6G+D3group:238.17±7.26;5.6G+ET group:201.16±5.28; si5.6G+D3group:278.82±9.5; si5.6G+ET group:204.56±7.55, N=6).3) Silencing CYP24A1gene reduces the effect of high glucose on the NGF secretion mediated by1,25(OH)2D3in Schwann cells:using analysis of Independent samples T-test,NGF level in si17G schwann cells increase significantly compared with17G schwann cells, t=-8.807P=0.000(17G group:145.49±6.75; si17G group:238.28±7.57; N=6); comparing differences between17G and17M in the secretion level of NGF, the differences had a significant reduction in silencing gene of Schwann cells compared with in ordinary Schwann cells., t=4.371p=0.001(17M-17G group:92.79±5.49; si17M-17G group:33.50±12.4; N=6).Conclusions:1) Silencing CYP24A1gene does not affect secretion of NGF by Schwann cells.2) Silencing CYP24A1gene increases secretion of NGF mediated by1,25(OH)2D3in Schwann cells.3) Silencing CYP24A1gene reduces the effect of high glucose on the NGF secretion mediated by1,25(OH)2D3in the Schwann cells.