Region-dependent effects of diabetes and insulin-replacement on neuronal nitric oxide synthase-and heme oxygenase-immunoreactive submucous neurons

AIM To investigate the intestinal segment-specific effects of diabetes and insulin replacement on the density of different subpopulations of submucous neurons. METHODS Ten weeks after the onset of type 1 diabetes samples were taken from the duodenum, ileum and colon of streptozotocin-induce diabetic, insulin-treated diabetic and sex-and age-matched control rats. Whole-mount preparations of submucous plexus were prepared from the different gut segments for quantitative fluorescent immunohistochemistry. The following double-immunostainings were performed: neuronal nitric oxide synthase(n NOS) and Hu C/D, heme oxygenase(HO) 1 and peripherin, as well as HO2 and peripherin. The density of n NOS-, HO1-and HO2-immunoreactive(IR) neurons was determined as a percentage of the total number of submucous neurons. RESULTS The total number of submucous neurons and the proportion of n NOS-, HO1-and HO2-IR subpopulations were not affected in the duodenal ganglia of control, diabetic and insulin-treated rats. While the total neuronal number did not change in either the ileum or the colon, the density of nitrergic neurons exhibited a 2-and 3-fold increase in the diabetic ileum and colon, respectively, which was further enhanced after insulin replacement. The presence of HO1-and HO2-IR submucous neurons was robust in the colon of controls(38.4%-50.8%), whereas it was significantly lower in the small intestinal segments(0.0%-4.2%, P < 0.0001). Under pathophysiological conditions the only alteration detected was an increase in the ileum and a decrease in the colon of the proportion of HO-IR neurons in insulin-treated diabetic animals. CONCLUSION Diabetes and immediate insulin replacement induce the most pronounced region-specific alterations of n NOS-, HO1-and HO2-IR submucous neuronal density in the distal parts of the gut.

Huangqin Decoction Delays Progress of Colitis-Associated Carcinogenesis by Regulating Nrf2/HO-1 Antioxidant Signal Pathwayin Mice

Objective:To investigate the effect of Huangqin Decoction(HQD)on nuclear factor erythroid 2 related-factor 2(Nrf2)/heme oxygenase(HO-1)signaling pathway by inducing the colitis-associated carcinogenesis(CAC)model mice with azoxymethane(AOM)/dextran sodium sulfate(DSS).Methods:The chemical components of HQD were analyzed by liquid chromatography-quadrupole-time-of-flight mass spectrometry(LC-Q-TOF-MS/MS)to determine the molecular constituents of HQD.Totally 48 C57BL/6J mice were randomly divided into 6 groups by a random number table,including control,model(AOM/DSS),mesalazine(MS),low-,medium-,and high-dose HQD(HQD-L,HQD-M,and HQD-H)groups,8 mice in each group.Except for the control group,the mice in the other groups were intraperitoneally injected with AOM(10 mg/kg)and administrated with 2.5%DSS orally for 1 week every two weeks(totally 3 rounds of DSS)to construct a colitis-associated carcinogenesis mouse model.The mice in the HQD-L,HQD-M and HQD-H groups were given HQD by gavage at doses of 2.925,5.85,and 11.7 g/kg,respectively;the mice in the MS group was given a suspension of MS at a dose of 0.043 g/kg(totally 11 weeks).The serum levels of malondialdehyde(MDA)and superoxide dismutase(SOD)were measured by enzyme-linked immunosorbent assay.The mRNA and protein expression levels of Nrf2,HO-1,and inhibitory KELCH like ECH-related protein 1(Keap1)in colon tissue were detected by quantitative real-time PCR,immunohistochemistry,and Westem blot,respectively.Results:LC-Q-TOF-MS/MS analysis revealed that the chemical constituents of HQD include baicalin,paeoniflorin,and glycyrrhizic acid.Compared to the control group,significantly higher MDA levels and lower SOD levels were observed in the model group(P<0.05),whereas the expressions of Nrf2 and HO-1 were significantly decreased,and the expression of Keap1 increased(P<0.01).Compared with the model group,serum MDA level was decreased and SOD level was increased in the HQD-M,HQD-H and MS groups(P<0.05).Higher expressions of Nrf2 and HO-1 were observed in the HQD groups.Conclusion:HQD may regulate the expression of Nrf2 and HO-1 in colon tissue,reduce the expression of MDA and increase the expression of SOD in serum,thus delaying the progress of CAC in AOM/DSS mice.

Protective Effects of Anthocyanins Extracted from Vaccinium Uliginosum on 661W Cells Against Microwave-Induced Retinal Damage

Objective: To study the protective effect of anthocyanins extracted from Vaccinium Uliginosum(VU)on retinal 661W cells against microwave radiation induced retinal injury. Methods: 661W cells were divided into 6 groups, including control, model [661W cells radiated by microwave(30 mW/cm2, 1 h)] and VU groups [661W cells pretreated with anthocyanins extracted from VU(25, 50, 100 and 200 μg/mL, respectively) for 48 h, and radiated by microwave 30 mW/cm2, 1 h]. After treatment with different interventions, the cell apoptosis index(AI)was determined using Heochst staining;contents of malonaldehyde(MDA), glutataione(GSH), and activity of superoxide dismutase(SOD) were measured. mRNA expressions of nuclear factor erythroid 2-related factor 2(Nrf2) and heme oxygenase 1(HO-1) were detected by real time quantitative polymerase chain reaction, and the expression of HO-1 protein was examined by Western blot analysis. Nucleus and cytoplasm were separated and Nrf2 protein expression was further verified by Western blot analysis. Results: There was significant difference in AI among the groups(F=322.83, P<0.05). Compared with the control group, AI was significantly higher in the model group and was lower in 4 VU-pretreated groups(P<0.05). Linear regression analysis showed the decline of AI was in a dose-dependent manner with VU treatment(r=0.8419, P<0.05). The MDA and GSH contents of 661W cells in VU-treated groups were significantly lower than the model group(P<0.05). Compared with the model group, the SOD activity in the VU-treated groups(50, 100 and 200 μg/mL) was significantly higher(all P<0.05). The Nrf2 and HO-1 mRNA expressions were slightly increased after irradiation, and obviously increased in 100 μg/mL VU-treated group. After irradiation, the relative expressions of HO-1 and Nrf2 proteins in nucleus were slightly increased(P<0.05), and the changes in cytoplasm were not obvious,whereas it was significantly increased in both nucleus and cytoplasm in the VU treatment groups. Conclusions:Anthocyanins extracted from VU could reduce apoptosis, stabilize cell membrane, and alleviate oxidant injury of mouse retinal photoreceptor 661W cells. The mechanism might be through activating Nrf2/HO-1 signal pathway and inducing HO-1 transcription and translation.