| Objective: Heymann nephritis is a classical animal nephritis model of idiopathic membranous nephropathy in human, and it has the clinical and pathological characteristics of human idiopathic membranous nephropathy. In recent years, numerous studies found that the Heymann nephritis pathogenesis related to complement activation, oxidation reaction, cytokine activation, endoplasmic reticulum stress and autophagy in many aspects. At the occurrence of Heymann nephritis, complement activated through a variety of ways to generate the membrane attack complex C5b-9, and induce a large number of reactive oxygen species. A large number of animal experiments confirmed that the accumulation of reactive oxygen species activate Nrf2/HO-1 signaling pathway which play a role in anti-oxidative stress, there by preventing lipid oxidation reactions. In addition, in the late phase of Heymann nephritis, the accumulation of extracellular matrix proteins such as ? collagen, fibronectin,becoming the main pathological changes. Studies confirmed that transforming growth factor ?1 promotes the formation of ECM plays an important role in mechanisms of glomerular sclerosis and fibrosis. In this way, the coordination of the oxidative reaction and the inflammatory factors together led to the pathological changes of the kidney. Exploring new treatment methods, blocking or inhibiting oxidation and inflammatory reaction, delaying renal function damage, has been a hot spot in the field of kidney disease. In earlier years, hydrogen gas was always thought to be a physiological inert gas. With further research, more and more evidence that the hydrogen selective anti-oxidation, anti-inflammatory, anti-apoptotic effects, but associated with kidney disease studies have reported little. With the further research, more and more evidences show that hydrogen has the function of selective oxidation, anti inflammation and anti apoptosis.This paper intends to through the establishment of active Heymann nephritis model in rats, using immunohistochemical staining, real-time quantitative PCR method for the detection of renal tissue nrf2, HO-1 and TGF ? 1, collagen type ? expression, and treated with hydrogen rich liquid 15 mg / kg to intervene, observe AHN rat kidney tissues oxidative stress, inflammation, and fibrosis, explore the rich hydrogen fluid for its function and mechanism of intervention, provides new thinking for the prevention and treatment of human IMN.Methods: 36 SD rats were Selected(healthy and clean, male), weighing about 200 ± 20 g, were randomly divided into normal control(NC group, n = 12), Heymann nephritis model group(HN group, n = 12), hydrogen-rich liquid intervention group(HRS group, n = 12). Adaptive feed after 1 week, the group of HN, HRS were given by intraperitoneal injection immunogen 2 ml(including renal cortical 0.5 g), prepared Heymann nephritis rat model. NC group rats were given intraperitoneal injection 2 ml of complete freund's adjuvant as normal controls. The frequency of Abdominal cavity injection was once every two weeks, a total of 6 times. Since the first time from the injection of the immunogen, HRS rats given daily(concentration of 0.8 mmol / L) of hydrogen-rich solution 15 ml / kg orally, NC group, HN group were given the same amount of normal saline daily. During the experiment, all rats were raised in the temperature(20 C-26 C), humidity(45%-70%) relatively constant environment,free access to food and water. At 4th and 8th weekend, the rats were placed in metabolic cages, 24-hour urine specimens were collected for measuring 24-hour urinary protein. In the 4th weekend after the injection of the immune original, quantitative of 24 hours urinary protein is higher than that of normal rats is the sign of nephritis formation. In the 12 th and 14 th weekend after the first injection of the immunogen, 6 rats in each group were randomly selected, measuring 24-hour urinary protein excretion, serum creatinine, blood urea nitrogen. Rat kidney tissue samples were collected and prepared by histopathology, to prepare for hematoxylin- observe pathological changes in rat kidney tissue under eosin(HE) staining, glycogen(PAS) staining and immunohistochemical staining; Immunohistochemical and real-time polymerase chain reaction(PCR) were used to detect the quantitative of Nrf- 2, HO- 1 and TGF- ?1, the expression of collagen type ?.Experimental data obtained with the mean ± standard deviation((?)±S),using SPSS 20.0 statistical analysis software for data, satisfy the normality and homogeneity of variance test data using single factor variance analysis, does not satisfy the normality and homogeneity of variance test data using non parametric test number analysis, P < 0.05 were considered to be statistically significant.Results:1 General situationThe diet of rats in group NC was normal, the hair color was bright and soft, and the growth and development was good. In HN group and HRS group, there was a gradual decrease in diet, slow reaction, and no light in the rats after intraperitoneal injection. The HRS rats were slightly better than the HN group rats.2 Biochemical indicators2.1 24 hours urinary proteinCompared with the NC group at the same time, HN group, HRS group rats 24 hours urinary protein levels were increased(P < 0.05). Among them, compared with the HN group, 4 weeks, HRS rats 24 hours urinary protein levels were not significantly changed(P > 0.05); 8 weeks, 12 weeks, 14 weeks, HRS group of 24 hours urinary protein levels were significantly decreased(P < 0.05).2.2 Serum creatinine and blood urea nitrogenCompared with the NC group, the serum creatinine and blood urea nitrogen levels were increased in the HN group and the HRS group(P < 0.05) in the 12 week and 14 weeks. Compared with the HN group, 12 weeks, hrs serum creatinine level showed no significant change(P > 0.05), and blood urea nitrogen levels appear decreased(P < 0.05); 14 weeks,HRS group serum creatinine, blood urea nitrogen and water appeared on average decreased significantly(P < 0.05).3 Histochemical changesLight microscope show, NC renal tissue of rats was normal, consistent with the basement membrane uniform, no thickening, no mesangial cells, mesangial matrix increase and other abnormalities. Compared with the NC group, HN group and HRS rat glomerular basement membrane showed varying degrees of thickening, endothelial cell cytoplasm swelling; tubular structural disorder, focal interstitial infiltration of lymphocytes and plasma cells. And the HN group than in HRS group kidney tissue lesions more severe than the 12 and 14 weekend pathological changes more obvious.4 The immunohistochemicalCompared the same period with NC group, 12 weeks, 14 weeks, the expression of nrf2, HO-1 and TGF ?1, collagen ? in HRS group and HN group were increased(P < 0.05); compared the same period with HN group, HRS group TGF ?1, type ? collagen expression levels decreased(P < 0.05), nrf2 and HO-1 expression increased(P < 0.05).5 The Real-time PCRCompared with the NC group over the same period, 12 weeks, 14 weekend, HRS group, HN group Nrf2 mRNA, HO-1 mRNA, TGF-?1 mRNA, ? collagen mRNA expression were increased(P <0.05); with the same period in HN compared to the group, HRS group TGF-?1 mRNA, ? collagen mRNA expression level decreased to varying degrees(P <0.05), while Nrf2 mRNA, HO-1 mRNA expression has increased(P <0.05).Conclusion:1 In the kidneys of HN rats, the expression of the antioxidant stress protein in Nrf2/HO-1 pathway is ncreased, as same as the expression of TGF-?1, ? collagen, indicating the presence of oxidative stress and fibrosis.2 Hydrogen rich liquid can increase the levels of Nrf2 and HO-1, and reduce the expression of TGF-?1 and ? collagen, thereby reducing the oxidative stress injury of the kidney tissue and delaying the renal fibrosis. |
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