Comparison Of Anti-inflammatory Activities Between Triptolide And Effective Parts Of Tripterygium Wilfordii For Glomerulonephritis

Tripterygium wilfordii Hook.F.(TWHF)is a member of the Celastraceae family,whose roots,leaves,flowers,buds and fruits are toxic.In vitro and in vivo research and clinical trials have found that,the extracts from TWHF have powerful multiple efficiencies,including anti-inflammation and anti-infection,immunosuppression and immunoregulation,and anti-tumor,which have been widely applied in the treatment of inflammatory diseases,immune systemic diseases and tumors.The limitation of TWHF's clinical application is due to the following reasons.Firstly,the crucial compounds obtained from TWHF are responsible for both its biological properties and toxicities with close relationship.On the other hand,the differences among the preparation,formula and production process of the commercial TWHF are resulting in the clear discrepancies among the content of the contained effective componds,biological activities and toxicities.In order to optimize the medicinal parts and growth years of TWHF,reduce the toxicity and enhance the efficacy,we investigated its appropriate extract obtained from Taining region.By utilizing the concept of the effective parts of Traditional Chinese Medicine,analyzing the extraction procedure and quantitatively detecting the representive components,the effective part of TWHF aiming at chronic glomerulonephritis was screened in vitro.Furthermore,the acute toxicitity,anti-inflammatory activity and the possible molecular mechanism of triptolide and the chosen effective part of TWHF were studied in vitro and in vivo to evaluate the relationship among the bioligical components in the effective part of TWHF.The main contents of this paper are as follows:(1)In order to optimize the medicinal parts and growth years of TWHF,the biological activities of the ethanol extracts from the whole roots and the debarked roots of TWHF with different growth years on human normal hepatocytes HL-7702,murine macrophages RAW 264.7,and rat glomerular mesangial cells HBZY-1 were investigated.Based on the ratio of IC50/EC50,the ethanol extracts from the whole roots with moderate growth years could be considered more rational and economical.Furthermore,by utilizing the concept of the effective parts of Traditional Chinese Medicine,analyzing the extraction procedure and quantitatively detecting the representive components,the extracts from TWHF mainly containing diterpenoids and sesqueteerpenoid alkaloids were preliminarily chosen as its effective part aiming at chronic glomerulonephritis.(2)In order to examine the anti-inflammatory activity of triptolide on LPS-induced RAW 264.7 in vitro,the production of NO and TNF-a.the protein expression of CD 14,TLR4,iNOS,COX-2,p-TAK1,p-JNK,p-ERK and p-p38.and activation of NF-?B p65 and IKB-? were evaluated.Results showed that triptolide significantly suppressed TNF-a level and NO release in dose-dependent manners.Moreover,it significantly decreased the protein expression of iNOS,COX-2,TLR4,p-ERK and p-p38.Degradation of IKB-a and nuclear translocation of NF-?B p65 were effectively blocked by triptolide.These results cumulatively indicated that triptolide has anti-inflammatory activity by suppressing the level of pro-inflammatory factor TNF-a and inhibiting the expression of TLR4 through MAPK and NF-?B signaling pathway.(3)The effective part of TWHF mainly containing diterpenoids and sesqueteerpenoid alkaloids can suppress NO and TNF-a production in LPS-induced RAW 264.7 in dose-dependent manners,by inhibiting the expression of TLR4 and TAK1,and interfering with MAPK and NF-?B signaling pathway,to exert its anti-inflammatory activity.Compared with triptolide alone,the effective parts of TWHF mainly containing diterpenoids and sesqueteerpenoid alkaloids with the same content of triptolide had the higher biological activity and safty on RAW 264.7,suggested that the interaction among triptolide,celastrol.wilforine and other biological components contained in this effective part may be present.(4)The result of acute toxicity in mice after oral administration demonstrated that the LD50 for triptolide and the effective part of TWHF mainly containing diterpenoids and sesqueteerpenoid alkaloids were 1.077 mg/kg(95%confidence interval(CI)=0.886-1.297 mg/kg)and 1.211 mg/kg(95%CI= 1.099-1.323 mg/kg)in mice,respectively.Both of triptolide(200 ?g/kg)and the effective part of TWHF mainly containing diterpenoids and sesqueteerpenoid alkaloids(500 ?g/kg)can clearly alleviate the pathological changes of kidney in C-BSA mice after 4-week treatment.They can also reduce 24-hour urinary protein level,decrease the blood urea nitrogen and creatine level in the serum but not influence ALT and AST level,which indicates that them had anti-inflammatory activity without hepatic toxicity.In a word,the effective parts of TWHF mainly containing diterpenoids and sesqueteerpenoid alkaloids were preliminarily chosen aiming at chronic glomerulonephritis according to the ratio of IC50/EC50,concept of the effective parts of Traditional Chinese Medicine,extraction procedure and quantitative analysis of the representive components.Due to its bioligocal activities on RAW 264.7 in vitro and the invloved molecular mechanism,acute toxicity on mice and effect on C-BSA mice.anti-inflammatory activity of the effective parts of TWHF mainly containing diterpenoids and sesqueteerpenoid alkaloids with the same content of triptolide was higher than that of triptolide alone,which can provide the new thought to reduce the toxicity and enhance the efficacy of TWHF.