| Part 1 Role of oxidative stress in long-term high-fat diet-induced pancreatic injuries in ratsObjective To study the effect of high-fat diet on pancreatic tissue and discuss its significances.Methods Wistar rats were divided into two groups, group control and group HFD. Control rats were fed normal diet, while HFD rats were fed high-fat diet. All rats were fed for 20 weeks. On the end of week 2 and 20, serum were detected for blood lipid, lipase, amylase and glucose. In the end of week20, pancreatic microcirculation was detected by intravital microscope and ink perfusion morphometry. Pancreatic tissues were obtained for histological detections including H&E staining, TEM, IHC staining, and Sirius red staining, and for RT-PCR and western blotting analyses. MDA and SOD were also measured.Results All rats got body weight increase during experimental period. No rat died. HFD rats developed hyperlipidemia . Pancreatic blood flow decreased significantly in HFD rats (639.70±104.63μm/s) in comparison to control group (756.08±98.98μm/s) (P = 0.008) . Inceased microvessel diameter and decreased MVD were also observed in HFD rats according to ink perfusion morphometry. In HFD rat, under light microscope, acinar cells' atrophy and vacuolization were common, enlarged space between lobules, inflammatory cells, and sparse collagen information were also observed. Ultrastructural histological alterations were also found by TEM. Sirius red staining showed collagen information in HFD group. NF-κB/p65, ICAM-1 and α-SMA expressed significantly higher in HFD rats than in control rats. And, MDA contents (42.77±16.53nmol/mgprot) and SOD activity (36.91±8.49 U/mgprot) changed obvious in HFD group in comparison to control group (MDA: 9.89±6.15 nmol/mgprot, P=0.022; SOD: 36.91±8.49U/mgprot, P=0.039). Additionally, the levels of NF-κB/p65, ICAM-1, TNF-α and α-SMA mRNA levels were all elevated significantly in HFD group than in control group (all P<0.05). NF-κB/p65, ICAM-1 and α-SMA proteins of HFD rats were higher significantly than those of control rats according to western blotting detection.Conclusions High-fat diet can induce pancreatic injuries via decreased pancreatic blood flow and oxidative stress through up-regulation of NF-κB/p65 and ICAM-1 expressions.Part 2 Roles of ICAM-1 and its antisense oligonucleotide in oxidative stress-induced pancreatic fibrogenesisObjective To study the roles of ICAM-1 and its antisense oligonucleotide in oxidative stress-induced pancreatic fibrogenesis.Methods Wistar rats were divided into 4 groups. Group N: normal control group, rats treated with dilated water (i.p., twice a week); group ASON: rats treated with DDC (750mg/kg i.p., twice a week) and ICAM-1 antisense oligonucleotide (0.5mg/kg i.p., twice a week); group Mix: rats treated with DDC (750mg/kg i.p., twice a week) and mixed ICAM-1 antisense oligonucleotide (0.5mg/kg i.p., twice a week); group DDC: rats treated with DDC (750mg/kg i.p., twice a week) only. All rats were treated for 6 weeks. Pancreatic tissues were obtained for histological detections including H&E staining, Sirius red staining, IHC staining, and RT-PCR and western blotting analyses.Results In DDC and Mix groups, acinar cells' atrophy and vacuolization, enlarged space between lobules, inflammatory cells infiltration and sparse collagen information were observed in H&E and Sirius red staining sections. While in normal control group and ASON group, these alterations were slight. For IHC staining, ICAM-1 and a-SMA were strongly expressed in DDC and Mix rats than in control and ASON rats. These alterations were also comfirmed by Western blotting detection. Additionally, the levels of ICAM-1, α-SMA and Con I mRNA were all higher in DDC and Mix groups than in control and ASON groups (all P<0.05).Conclusions Oxidative stress can induce pancreatic fibrogenesis through up-regulation of ICAM-1 expression, and ICAM-1 antisense oligonucleotide can inhibit this pathophysiological course.Part 3 Protective and therapeutic effects ofNF-κB inhibitor sulfasalazine on oxidative stress-induced pancreatic fibrogenesisObjective To study the protective and therapeutic effects of NF-κB inhibitor sulfasalazine on oxidative stress-induced pancreatic fibrogenesis.Methods Wistar rats were divided into 5 groups. Group N: normal control group, rats treated with dilated water (i.p., twice a week) for 10 weeks; group DS1: rats treated with DDC (750mg/kg i.p., twice a week) for 10 weeks and sulfasalazine (10mg/kg i.p., twice a week) for 10 weeks; group DS2: rat treated with DDC (750mg/kg i.p., twice a week) for 10 weeks and sulfasalazine (100mg/kg i.p., twice a week) for 10 weeks; group DS3: rats treated with DDC (750mg/kg i.p., twice a week) first for 10 weeks, then treated with sulfasalazine (100mg/kg i.p., three times a week) for 2 weeks; group DDC: rats were treated with DDC only (750mg/kg i.p., twice a week) for 10 weeks. Pancreatic tissues were obtained for histological detections including H&E staining, IHC staining , Sirius red staining, and RT-PCR and western blotting analyses.Results In DDC group, pancreatic tissue demonstrated obvious acinar cells' atrophy, vacuolization, inflammatory cells, and collagen information in H&E and Sirius red staining sections. These alterations were also found in DS1 rats with a little improvement. But in DS2 and DS3 groups, these changes were not obvious. The differences between DDC and DS2 or DS3 group were significant (P<0.05, respectively). For IHC staining, NF-κB/p65, ICAM-1 and α-SMA were strongly expressed in DDC group, while in DS2 and DS3 group, their expression were significantly decreased, and these alterations were in accordance with the results of Western blotting detection. On the other hand, the levels of NF-κB/p65, ICAM-1 , TNF-α and Con I mRNA in DS2 and DS3 rats were decreased significantly in comparison to DDC group (all P<0.05).Conclusions Sulfasalazine can inhibit or ameliorate oxidative stress-induced pancreatic injuries in rats via inhibition of NF-κB activation and its downstream molecules' expression.
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