| Background:Diabetic neuropathy is one of the most common chronic complications of diabetes. The most common form of diabetic neuropathy is nerve damage in the periphery, such as hands, toes and ankles, gloves-like or socks-like on the body surface. Paitents with diabetic peripheral neuropathies(DPN) often experience aberrant pain sensation, including spontaneous pain, hyperalgesia(severe pain with mild painful stimuli) and allodynia(pain with innocuous stimuli, e.g. light touch), known as diabetic neuropathic pain. Autonomic neuropathy mainly effects the function of cardiovascular, gastrointestinal and urogenital system. When the upper digestive tracts are involved, patients often experience abdominal pain or discomfort, nausea and vomiting, acid regurgitation, postprandial fullness, etc., known as diabetic gastric hypersensitivity. At present, the pathogenesis of diabetic neuropathy has not been fully elucidated and treatment is much insufficiency. The quality of life of patients with diabetic neuropathies are affected significantly. Therefore, to study the pathogenesis of diabetic neuropathy and search for new therapeutic targets are very urgent and important.Part one1. Specific Aims: to elucidate the role and mechanism of nuclear factor kappa B(NF-κB) in diabetic gastric hypersensitivity, including:(1)To establish an animal model of diabetic gastric hypersensitivity and to study its electrophysiological mechanism.(2)To investigate the role and mechanism of CBS-H2 S system in diabetic gastric hypersensitivity.(3)To explore the regulatory role and mechanism of NF-κB on the CBS-H2 S system.2. Methods:(1) Diabetes were induced by intraperitoneal(i.p.) injection of streptozotocin(STZ, 65 mg/kg) in adult female rats.(2) Behavior responses to graded gastric balloon distention was employed on normal and diabetic rats.(3) Patch clamp recordings were performed in vitro on single gastric specific DRG neurons acutely isolated from these rats labeled with dye Di I.(4) Expression of NF-κB, H2 S producing enzyme cystathionine-beta-synthetase(CBS), DNA methyltransferase(Dnmt3a 、 Dnmt3b), methyl Cp G binding domain protein MBDs(MBD2、MBD4) and DNA repair enzyme thymine DNA glycosylase(TDG) were measured with western blotting and(or) real-time PCR analysis.(5) The status of DNA methylation were determined by methylation specific PCR and bisulfite sequencing.(6) Fluorescent-immunohistochemistry was employed to detect co-expression of CBS with NF-κB in DRG neurons.(7) The level of NF-κB binding with cbs gene promotor was analyzed with chromatin immunoprecipitation assays.3. Results:(1) Diabetic rats were more sensitive to graded gastric balloon distention 2 weeks after STZ injection, and lasted for 5 weeks. The excitability of gastric-specific DRG neurons from diabetic rats was increased 4 weeks after STZ injection.(2) CBS and CBS m RNA expression of gastric DRGs in diabetic rats were greatly enhanced(P<0.05). I.p. injection of CBS inhibitor Amino-oxyacetic acid(AOAA) could attenuate diabetic gastric hyperalgesia. One week after i.p. injection of AOAA, the neuronal excitability was decreased. In addition, Na HS(donor of H2S) increased the excitability of the gastric-specific DRG neurons in normal rats.(3) NF-κB expression of gastric DRGs in diabetic rats was greatly enhanced. CBS and NF-κB were co-localized in gastric specific DRG neurons of rats. Chromatin immunoprecipitation assay showed that NF-κB could bind specifically to the region of cbs gene promoter in diabetic rats. Injection with NF-κB inhibitor(Pyrrolidine dithiocarbamate, PDTC) for 1 week significantly reduced the up-regulated CBS expression. cbs gene promoter region of gastric DRGs contained recognition sequence region of NF-κB was significantly demethylated in diabetes. DNMT3 a and DNMT3 b m RNA expression of gastric-specific DRGs decreased in diabetic rats.Part two1. Specific Aims: to elucidate the role and mechanism of NF-κB in diabetic hindpaw pain hypersensitivity, including:(1)To investigate whether NF-κB is involved in diabetic hind-paw pain hypersensitivity.(2)To investigate the role and mechanism of P2X3 receptor in diabetic hindpaw pain hypersensitivity.(3)To explore the regulatory role and mechanism of NF-κB on P2X3 receptor.2. Methods:(1) Diabetes were induced by intraperitoneal(i.p.) injection of streptozotocin(STZ, 65 mg/kg) in adult female rats.(2) Behavior responses to stimulation of von frey filament and thermal radiation were employed on normal and diabetic rats.(3) Patch clamp recordings were performed in vitro on single hind-paw specific DRG neurons acutely isolated from these rats labeled with dye Di I.(4) Expression of NF-κB, P2X3 receptor, DNA methyltransferase(Dnmt3a, Dnmt3b), methyl Cp G binding domain protein MBDs(MBD2, MBD4), DNA repair enzyme thymine DNA glycosylase(TDG) and DNA damage-inducible protein alpha(Gadd45α) were measured with western blotting and(or) real-time PCR analysis.(5) The status of DNA methylation were determined by methylation specific PCR and bisulfite sequencing.(6) Fluorescent-immunohistochemistry was employed to detect co-expression of P2X3 receptor with NF-κB in DRG neurons.(7) The binding level of NF-κB to the p2x3 r gene promotor was analyzed with chromatin immunoprecipitation assays.3. Results:(1) Diabetic hind-paw pain hypersensitivity presented 2 weeks after STZ injection, and lasted at least for 9 weeks.The expression of NF-κB in diabetic hind-paw specific DRGs was greatly enhanced. Diabetic hind-paw pain hypersensitivity was attenuated by treatment with PDTC and NF-κB si RNA lentiviral vector.(2) Diabetic hind-paw pain hypersensitivity was attenuated by treatment with suramin or A-317491. The expression and function of P2X3 receptor from hind-paw specific DRGs in diabetic rats was greatly enhanced.(3) NF-κB and P2X3 receptor were co-localized in hind-paw specific DRG neurons of rats. Chromatin immunoprecipitation assay showed an enhanced binding ability of NF-κB with p2x3 r gene promoter region in rats with diabetes. Inhibition of p65 signaling using PDTC or NF-κB si RNA lentiviral vector remarkably suppressed P2X3 receptor activities. p2x3 r gene promoter region was significantly demethylated in diabetic rats. DNMT3 b m RNA expression of hind-paw specific DRGs in diabetic rats was greatly decreased.Conclusion:1. Intraperitoneal injection of STZ can successfully induce diabetic gastric hypersensitivity and hind-paw pain hypersensitivity.2. Diabetic pain hypersensitivity is associated with the enhanced excitability of gastric and hind-paw specific DRG neurons. NF-κB increases the expression of CBS and P2X3 receptor from gastric and hind-paw specific DRGs, respectively. The enhanced CBS-H2 S and P2X3 receptor increase the excitability of gastric and hind-paw specific DRG neurons, respectively.3. The DNA demethylation at cbs and p2x3 r gene promoters and enhanced interaction with NF-κB contribute to CBS and P2X3 receptor expression upregulation and diabetic pain hypersensitivity. |
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