| Tripterygium wilfordii Hook,f.is the xylem of Celastraceae wilfordii root.Its Congeneric plant also include Tripterygium Hypoglaucum(Levl.)Hukeda.,known as the Tripterygium Forretii Dials,Tripterygium Regelli Sprague et Takeda,and so on.Tripterygium is one of the Chinese herbal which has a long history of applications.Medicine and modern clinical studies showed that tripterygium and its preparations have anti-inflammatory,immunosuppressive and anti-fertility activity,and it has a efficacy of clinical treatment for a variety of difficult diseases such as rheumatoid arthritis,autoimmune glomerulonephritis spondylitis,lupus erythematosus,and nephrotic syndrome.In recent years,basic research on Tripterygium was focused on the pharmacological effects and toxicity of the ingredients and the preparation such as glycosides and triptolide.But the tripterygium composition is complex,the toxicity of its substances is far more than these.More than 300 kinds of active substances has been isolated by the plant Tripterygium till now,but there are mixed views on the toxicity of various ingredients.As the main excretory organ of the body,kidney is vulnerable to the damage of toxic substances,studies have shown that:of the tripterygium poisoning death cases,the kidney damage occurrence rate is 54%,and up to 38%mortality rate is due to renal failure.But because of the complicate composition of Tripterygium and the complexity of the pathogenesis of renal injury,there have not been a clear and complete study of this medicine both in domestic and abroad.In this study,we first administrate experimental animals by the different extraction of the Tripterygium to identify the toxic extraction of renal injury,then further explore the mechanism of renal injury of these exraction.This will help to reduce the toxicity and raise the effect of Tripterygium and ensure the safety and effective application of tripterygium and its preparations in clinic.This study includes the following sections:1.Mice were divided into eleven groups as follows:Control group,which was given isovolumetric physiological saline;high and low dose group of water exract;high and low dose group of alcohol extract;high and low dose group of acetidin extact and high and low dose group of n-butyl alcohol exract.After extracting different parts of Tripterygium,administration of different extraction parts was conducted for one week,two weeks,three weeks and four weeks respectively.The high dose groups were given extrats at the dosage of 16.25g/kg,and the low dose groups were given at the dosage of 3.25g/kg.After the last adminstration,detect the levels of BUN and Scr and LDH,TP,and ALB in serum of mice,taking the kidney for histopathology;.The results show that,there is not yet the mouse kidney damage after administrated of different tripterygium extrat for one week.But two weeks after the administration,serum BUN,Scr,and LDH concentration was significantly increased,and the concentration of TP and ALB was significantly reduced,the alcohol extract parts,ethyl acetate and n-butanol part show the most obvious changes.The indicators of water extraction has no significant changes,but compared with the control group,its BUN,Scr,and LDH concentration increased,and the concentration of TP and ALB also show a decreasing trend.the histopathological examination showed that the Tripterygium extract could induce medullary tubular epithelial nuclear pyknosis,and edema of enal tubular epithelial cell.The results show that all of the four extract parts have a toxic effect on kidney,and the toxicity of alcohol extract part and n-butanol part is strongest,the weakest extract part of toxicity is the aqueous extract.2.Taking the kidney of the more poison extract,detect Ang?(angiotensin 2),caspase-3(Caspase 3),GSH(glutathione)and TLR-4(Toll-like receptor 4)of kidney by immunohistochemisty method,and detec KIM-1(kidney injury molecule-1)and ROS(reactive oxygen clusters)by Western Bolt method.The results of Western Blot show that after administrated different Tripterygium extracts for four weeks,the expression of KIM-1 and ROS increased,and these changes gradually increased with increasing dosage.Besides the expression of KIM-1 increased significantly in the first two weeks,in the next two weeks its expression was higher than the first two weeks,but has no difference compare with the control group.Immunohistochemic shows that,the expression of Ang? decreased in the first two weeks,and in the next two weeks some the expression group increased;Tripterygium extracts could also decrease the expression of casepa-3,and the effect of acetidin extract part was strongest;besides,Tripterygium could decrease the expression of GSH in kidney but has no evident effect to TLR-4.Hence we come to the conclusion that,the injury of Tripterygium was acute kindney injury,and this concerned with the increase of the expression of ROS,the change of Ang?,apoptosis and lipid peroxidation.3.Take logarithmic phase HK-2 cells,and fed them with different concentrations of Tripterygium extract and ingredients for respectively 12 hours,24 hours and 48 hours,MTT assay was used to observe the effect of different extract fractions and different ingredients of Tripterygium on HK-2 cell viability and proliferation,and the Griess reagent colorimetric assay was used to detect the release of NO in the cells.The experimental results show that fed HK-2 cells with different concentrations of Tripterygium for 24 hours and 48 hours,the NO release of the are increased significantly,and there were significant differences compared with the control group.compared to the three other extracts,the NO release of the water extract is relatively less,and among the other three exracts of the alcohol extract parts,ethyl acetate and n-butanol part,NO release of the ethyl acetate extract part is slightly less.The results show that all of the four extract parts have a toxic effect on HK-2 cells,and the toxicity of alcohol extract part and n-butanol part to HK-2 cells is strongest,followed by the toxicity of ethyl acetate extract,and the weakest extract part of toxicity is the aqueous extract.4.Rats were divided into for groups as follows:high dose group,medium dose group and low-dose group,which were administrated water extract of Tripterygium at the dosage of 11.25g/kg,5.6 g/kg and 2.25 g/kg respectively,and the control group was given isovolumetric physiological saline.The four groups were administrated for one week,two weeks,three weeks,four weeks,six weeks and eight weeks.After the last adminstration,Collecting the urine of 24h,observing the color and record urinary volume,taking the blood from carotid arteries for biochemical detection,taking the kidney to weigh its weight and for histopathology.The results show that the Tripterygium extract can reduce the urinary volume,increases the kidney index,and after administered two weeks the serum urea nitrogen(BUN),creatinine(Scr)and K+concentrations increased significantly,serum white protein(ALB)and total protein(TP)concentrations were significantly reduced;and the histopathological examination showed that the Tripterygium extract could induce medullary tubular epithelial nuclear pyknosis,the renal tubules of the renal papilla visible individual tube and a slight expansion of varying degrees of renal tubulartubular injury.These results suggest that,the Tripterygium administration two weeks in rats will lead to the apparent kidney damage,and these changes gradually increased with increasing dosage and time. |
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