| [Backgound]Triptolide is a diterpenoid isolated from the plant Tripterygium wilfordii Hook. f (TWHf), a member of the Celastraceae family and it is one of the main active components in TWHf. Many studies have shown that triptolide has various functions including immunosuppressive, anti-cancer, and contraceptive activities. The ethanol extract, ethyl acetate extract, and other extracts of TWHf containing triptolide have been used for the treatment of rheumatoid arthritis and autoimmune diseases clinically and triptolide was deemed to account for the immunosuppressive activity of the extracts. However, triptolide has severe toxicities on digestive, urogenital, blood circulatory, reproductive system and bone marrow, which have largely limited its clinical application. Furthermore, among acute poisoning of triptolide cases which result in fatal consequences, acute renal dysfunction/failure is the most important cause of death, and a subchronic toxicological study on mice also suggested that kidney is one of the most common target organs of triptolide-induced toxicity. However, the mechanism of renal injury caused by triptolide remains unclear. Therefore, elucidation of the possible mechanisms of nephrotoxicity is essential from a safety point of view.[Aims]We want to set up a model of triptolide-induced acute renal injury in SD rats, and study the possible mechanisms of triptolide-induced acute nephrotoxicity, as well as the preventive effect of antioxidant vitamin C on triptolide-induced acute nephrotoxicity in rats. It consists of the following:1. Set up the model of triptolide-induced acute renal injury in SD rats.2. Study on the role of oxidative stress in triptolide-induced acute nephrotoxicity in rats.3. Study the role of apoptosis of renal tubular cells in triptolide-induced acute nephrotoxicity and possible mechanisms in rats.4. Study the preventive function of vitamin C on triptolide-induced acute nephrotoxicity and possible mechanisms in rats.[Methods]1.30 male SD rats were randomly divided into a control group and two testing groups (triptolide a and triptolide b). The rats of two testing groups received peritoneal injection of triptolide solution at doses of lmg/kg of body weight and 2mg/kg of body weight respectively, and the rats of the control received peritoneal injection of 0.9% physiological saline instead. Testing rats were killed 48h after injection; blood samples were collected and both kidneys were removed. The PUN and Pcr concentrations were measured, and renal histology was examined by HE and Masson’s trichrome staining. The activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), as well as renal content of reactive oxygen species (ROS) and malondialdehyde (MDA) were measured.2. The experimental design and the methods for the detection of PUN and Pcr concentrations did not differ compared with the first part. TUNEL staining was performed to evaluate apoptosis of renal tissue. Renal expression of apoptosis related proteins Bcl-2, Bax, Bid, Bad, Fas and FasL, as well as corresponding regulating genes were assessed by immunohistochemical staining, Western Blotting and real-time PCR.3.40 male SD rats were randomly divided into four groups:the first group (control group) received a single peritoneal injection of 0.9% physiological saline on day 8; the second group (vitamin C group) first received an oral administration of vitamin C (dissolved in distilled water) at a dose of 250 mg/kg of body weight per day for 7 days continually, and then received a single peritoneal injection of 0.9% physiological saline on day 8 as the first group; the third group (triptolide group) received a single peritoneal injection of triptolide solution at a dose of 1 mg/kg of body weight on day 8; the forth group (Vitamin C+triptolide group) first received an oral administration of vitamin C (dissolved in distilled water) at a dose of 250 mg/kg of body weight per day for 7 days continually, and then received a single peritoneal injection of triptolide solution at a dose of 1 mg/kg of body weight on day 8 as the third group. All the rats were killed by cervical dislocation on day 10. The renal superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities, as well as renal ROS and malondialdehyde (MDA) content were measured to evaluate oxidative damage level. Blood samples were collected for biochemical analysis, and both kidneys were removed. Plasma creatinine (Pcr) concentrations and plasma urea nitrogen (PUN) concentrations were measured to assess renal function. Histological examination of renal tissue was done by H&E staining and viewed under common light microscope. TUNEL staining was performed to assess apoptosis of renal tubular cells. Renal expression of apoptosis related proteins Bcl-2, Bax, Bid, Bad, Fas and FasL, as well as corresponding regulating genes were assessed by Western Blotting and real-time PCR.[Results]1. A single large dose injection of triptolide resulted in both morphological and functional injury dose dependently in a short time; furthermore, triptolide shock could lead to depletion of renal SOD and GSH-Px activities and increases of renal ROS and MDA content in a dose dependent manner, which correlated well with the degree of renal injury. 2. After injection of triptolide, rats presented increased number of apoptotic tubular cells corresponding to the increases of PUN and Pcr concentrations. Furthermore, increased expression of Bax, Bid, Bad, Fas and FasL was detected at both protein and mRNA levels, but the expression of Bcl-2 did not differ around the three groups; nevertheless, the ratio of Bax to Bcl-2 increased at both protein and mRNA levels.3. Pretreatment with the antioxidant vitamin C significantly ameliorated triptolide-induced depletion in renal SOD and GSH-Px activities, caused marked normalization of renal ROS and MDA content and also inhibited the triptolide-induced apoptosis of renal tubular cells and renal injury. Compared with the triptolide group, the expression patterns of Bax, Bid and Bad, as well as the ratio of Bax to Bcl-2 at both protein and mRNA levels were decreased; however, there were no statistical differences of the expression patterns of Fas and FasL between the two groups.[Conclusion]1. Kidney is an important target organ of triptolide-induced toxicities, and oxidative stress contributes to the mechanism of triptolide-induced acute nephrotoxicity.2. Apoptosis of tubular cells plays a key role in the pathogenesis of triptolide-induced acute nephrotoxicity, and Bcl-2 family, as well as Fas and FasL, are involved in this process.3. Pretreatment with the antioxidant, oral administration of vitamin C (250 mg/kg of body weight, dissolved in distilled water) per day for 7 days continually, significantly reduced the generation of oxidative stress and effectively inhibited the triptolide-induced apoptosis of renal tubular cells and renal injury by regulating the expression patterns of Bcl-2 family members.4. The protective effect of vitamin C could provide a new insight into the potential therapeutic solution to triptolide-induced renal injury. |
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