The Research Of Antioxidation And Anti-inflammation Of Rosa Laevigata Michx.on Experimental Diabetic Nephropathy Rats

BackgroundDiabetic nephropathy (DN) is one of the most common microvascular complications of diabetes mellitus (DM), and is the leading death cause of end-stage renal disease and DM patients. Recently, Numerous reports suggest that oxidative stress is defined as an excessive production of reactive oxygen species (ROS) surpassing existing antioxidative defense mechanisms, and plays a critical role in the pathogenesis of diabetic nephropathy, which is the hot spot of DN control research. However, the mechanism is unclear. Studies showed that oxidative stress can interact with inflammation in diabetes, which cooperately with promoting the progression of DN. Numerous reports suggest that Nrf2/ARE signal pathway was the most important endogenous defence against oxidative stress, and Nrf2was the central moderator. Nuclear factor-kappa B(NF-κB), which defined as an hinge involved in regulating the express of inflammatory genes. There is strong evidence showed that there is a "cross-talk" between Nrf2and NF-κB, and they can cooperate with regulating the OS and inflammation. However, the effects of the two pathway on oxidative stress in DM is unknown, especially in DN. Rosa Laevigata Michx.(RLM) are known as the commonly used traditional Chinese medicine (TCM) Jin Ying Zi, has long been used for ant-inflammatory treatments and anti-oxidative treatments. Our previous studies showed, RLM fruit could significantly dameliorate the disorder of plasma blood glucose and blood lipid in diabetic rats, and protected the renal in diabetic rats by decreasing the OS injure, inhibiting the expression of NF-κB, MCP-1and TNF-α. But the really molecular mechanism of RLM acting the above role is unknown. Thus, baseing on the importance of Nrf2/ARE and NF-κB signal pathway in anti-oxidative and anti-inflammation, we detected the antioxidative effect of extract from RLM in vitro, and further expored the molecular mechanism of RLM extract on anti-oxidative and anti-inflammation via activation of Nrf2/ARE and inhibition of NF-κB signal pathway in diabetic nephropathy rats.MethodsWe detected the antioxidant activities of extracts from RLM in vitro.40SD rats, which were previously fed a high-fat/high-sucrose/high-cholesterol diet for2months, were administered STZ in a single dose of65mg/kg intraperitoneally (i.p.). After3days, the animals were considered diabetic if FBG levels were>14mmol/l, and then the diabetic rats were randomly selected and evenly divided into two groups comprising of15animals in each group as follows:Diabetic model group (DM, n=15), Diabetic+Rosa Laevigata Michx. group (DM+RLM, n=15, fed with aqueous extracts of RLM,5g/kg/d). And another twenty normal SD rats were divided two groups comprising of10animals in each group as follows:normal control group (Control, n=10), and normal control+Rosa Laevigata Michx. group (Control+RLM, n=10, fed with aqueous extracts of RLM,5g/kg/d). The aqueous extracts of RLM were daily injected into the stomach. Urine was collected from the rats housed in metabolic cages for24h. All animals were sacrificed at the end of the24th week. Blood was collected from a puncture of the inferior vena cava at the time of sacrifice. Serum samples were collected for the measurement of total cholesterol (TC), triglyceride (TG), blood urea nitrogen (BUN), serum creatinine (Scr), GHb, insulin, reactive oxygen species(ROS), malondialdehyde(MDA), total anti-oxidant capacity (T-AOC), superoxide dismutase(SOD), and IL-6, IL-1β, MCP-1levels. Kidney samples were rapidly excised, decapsulated, weighed, counted the KW/BW and frozen in liquid nitrogen or fixed in10%buffered formaldehyde solution for HE and PAS staining or glutaraldehyde (3%in PBS) for electronical microscopic, kidney homogenates (10%, w/v) were prepared with0.1M PBS and centrifuged at12000×g for10min. The supernatant was used to determine T-AOC, SOD, MDA, IL-6, IL-1β, MCP-1levels. The expression of Nrf2and its downtream genes HO-1, γ-GCS, the NF-κB pathway and IL-6, IL-1B and MCP-1were detection by immunohistochemical, wester blot or qRT-PCR.Results1. The extracts from RLM could both scavenge hydroxyl free radical (-OH) produced by Fenton reaction and superoxide anion free radical (O2-) produced by oxidation of pyrogallol in dose-dependent manner. They also had inhibitory effects on the spontaneous lipid peroxidation in liver and kidney homogenate with a dose-dependent manner, and significantly inhibited the hemolysis of red blood cells induced by H2O2.2. Extract from RLM significantly ameliorated the disorder of plasma blood glucose, dyslipidemia and renal dysfunction in diabetic nephropathy rats. RLM extract also obviously decreased the concentration of IL-6, IL-1β and MCP-1, and the levels of ROS and MDA in the plasma and the kidney homogenates of diabetic nephropathy rats, but significantly improved the activity of SOD and T-AOC.3. Morphological observation showed that, the glomerular volume of diabetic nephropathy rats was significantly greater than that of the normal control rats, and the glomerular basement membrane (GBM) thickening and podocyte foot process effacement were striking in diabetic nephropathy rats, but these conditions began to revert back to normal following RLM treatment, yet not reached the normal. There were no significant differences in kidney morphology between the normal control and NC+RLM groups, which therefore suggested that RLM treatment didn't lead to renal injury in rats.4. Compared with normal rats, the expression of Nrf2protein in the kidney were significantly decreased, and accompanied with down-regulating the expression of HO-1and y-GCS in diabetic nephropathy rats, but when treated with RLM extract, the expression of Nrf2, HO-1and y-GCS protein were obviously upregulated in the kidney which detected by western blot. And the similar results with western blot assay about Nrf2protien expression in the rats were detected by Immunohistochemistry. The mRNA expression of HO-1and y-GCS in the kidney were significantly upregulated in the diabetic nephropathy rats treatment with RLM extract by qRT-PCR.5,Compared with normal rats, the expression of NF-κB p65protein in the kidney were significantly increased, and accompanied with upregulation expression of IKKβ,down-regulation expression of IκBα in diabetic nephropathy rats, but when treated with RLM extract, the expression of NF-κB p65and IKKβ were obviously down-regulated, the expression of IκBα was upregulated in the kidney which detected by western blot. And the similar results with western blot assay about NF-κB p65protien expression in the rats were detected by Immunohistochemistry. The mRNA expression of IL-6, IL-1β and MCP-1in the kidney were significantly down-regulated in the diabetic nephropathy rats treatment with RLM extract by qRT-PCR.Conclusions1. Oxidative stress and inflammation exist in the diabetic renal tissues, which promote the progression of diabetic nephropathy, and the Nrf2/ARE and NF-κB signal pathway maybe play an important role in the development of diabetic nephropathy. 2. RLM is able to ameliorate the disorder of plasma blood glucose, dyslipidemia and renal dysfunction, and perform antioxidation and anti-inflammation in diabetic nephropathy rats.3. RLM retards the progression of diabetic nephropathy by inhibiting the oxidative stress via activation of Nrf2/ARE signal pathway.4. RLM retards the progression of diabetic nephropathy by restraining the inflammation via inhibiting the activation of NF-κB signal pathway.