| Tripterygium wilfordii Hook.f (TWHF), a traditional Chinese herbal medicine, shows great therapeutic effect on several autoimmune disease and inflammatory disease such as rheumatoid arthritis and asthma. However, TWHF and its preparation could trigger severe side effect, especially hepatic and kidney injuries, which infer the big risk in clinic use. Besides, triptolide was regarded as main bioactive substance for its therapeutic and toxic effect in TWHF. Recent study indicated that TP play inhibitory effect on inflammation through two pathways, activating apoptosis of T cells and downregulating multiple inflammatory factor associated with IL. Current opinion suggested that "oxidative stress" and "cell apoptosis" were two main theories of TWHF toxicity. However, it was still unclear how TWHF induced therapeutic and toxic effect.Our previous study demonstrated that TP could regulate sphingolipid metabolism to inhibit delayed type hypersensitivity. So it was estimated that sphingolipids metabolism may play a regulatory role in the TP-induced therapeutic and toxic mechanism in liver and kidney. To illustrate the mechanism by TWHF therapeutic and toxic effect, we utilized the strategy of targeted sphingolipidomics and transcriptomics to understand the regulatory effect of TP and TWHF preparations on the sphingolipid metabolism in targeted organs. With this strategy we tried to uncover the molecular mechanism for therapeutic and toxic effect of TP and TWHF preparations from the aspects of sphingolipid metabolism, discover potential biomarkers which could effectively indicate the therapeutic effect and/or warn the toxic effect, to help reasonable use of TWHF preparation in the clinic.First, we established a mice model of DTH, and TP at different dose and TWHF preparations from different manufactures were exposed to mice with DTH response. The inhibitory effect of these samples above on DTH response were measured using the traditional ear-swelling index. Besides, biochemical parameters (ALT, AST, BUN), oxidative stress related parameters (SOD, MDA, GSH) and pathological exam were utilized to evaluate the degree of their toxic effect on liver and kidney. Results showed that TP at low dose and TGT at both low and high dose all showed a great inhibitory effect on DTH reaction. However, TP at high dose did not have obvious suppression on for DTH reaction, And it even led to obvious pathological abnormality in liver. In contrast, it was observed that high-dose TGT induced great pathological change in both liver and kidney. Moreover, high-dose TP and TGT could resulted in abnormal change in biochemical parameters and oxidative stress status.Then, we made use of the developed HPLC-MS/MS platform for sphingolipid analysis to determine the sphingolipids levels in liver, kidney, spleen, thymus and plasma in DTH model exposure to TP at different dose and TGT preparations at different manufacturers. Furthermore, we discovered abnormal sphingolipid metabolism and potential biomarkers related to therapeutic and toxic effects of TP and TGT using advanced statistical and bioinformation method. Results indicated that the therapeutic and toxic effect of TP were closely associated with sphingolipids metabolism in a dose-dependent manner. Obviously TP induced the pertubance of sphingolipids subspecies. Furthermore, TP and TGT could influence not only the levels of sphingolipids subspecies, but also the fractions of sphingolipids with different length acyl chain and degree of unsaturation significantly in the body. TP at low dose mainly induced the downregulation of dhCer, Cer and Cer-1-P, but high-dose TP and TGT could resulted in the increase of Cer, SM and Sph. Besides, TP and TGT both induced the increase of fractions of sphingolipids with long chain acyl chain and the change of sphingolipids with different degree of saturation. TP at low and middle dose could upregulate the fractions of saturated HexCer, while the increase compositions of saturated Cer was observed when exposure to high-dose TP. Meanwhile, low-dose TGT could result in the downregulation of fractions of saturated dhCer and SM, and high-dose TGT led to the decrease of compositions of saturated Cer and HexCer. Spearman and OPLS-DA analysis were utilized to find potential sphingolipid biomarkers which are highly associated with TP toxic effect for tissue toxicity and/or therapeutic effect.3 dhCers,4 Cers,3 HexCers,2 Sphingosine bases,1 Cer-1-P was selected as potential biomarkers indicative of therapeutic effect of TP, and 5 dhCers,6 Cers,7 HexCers,4SMs,1 Sphingosine bases were regarded as potential biomarkers for TP toxicity; 5 dhCers,5 Cers,3 HexCers,5 SMs,2 Sphingosine bases,1 Cer-1-P was selected as potential biomarkers indicative of therapeutic effect of TGT, and 6 dhCers,7 Cers,9 HexCers,8 SMs,3 Sphingosine bases and 1 Cer-1-P were regarded as potential biomarkers for TP toxicity.Based on the sphingolipidomics results above, transcriptional level of 29 related enzymes were analyzed using fluorescent quantitative real time PCR method. The real PCR study showed that many enzymes with significant change at mRNA expression level were observed, including CerS1, Smpd1,2, Asah1,2 in liver, and Degs1, Smpd1, 2, Asah1,2 in kidney and Smpd1,2 in spleen. Especially, the change of 6 Ceramide Synthase (CerSs) maybe caused the change of fractions of sphingolipids with long acyl chain (C16-C20) and ones with very long chain (C22-C24). Biomarkers discovered using bioinformatics analysis and key enzymes were closely related to therapeutic and toxic effects of TP and TGT. In all, TP and TGT could both change the level and compositions of sphingolipids, which may be important molecular mechanism for its therapeutic and toxic effect. Moreover, potential biomarkers discovered in current study could be beneficial to the evaluation of therapeutic and toxic effect of TWHF, and key enzymes may be potential target of therapeutic and toxic effect of TWHF.Secondly, the metabolite profiling of TP and TGT in liver, kidney and plasma of mice with DTH response were analyzed to understand the in vivo substance basis of their regulatory effects on sphingolipids synthesis and metabolism. The first step was to analyze the compositions of TGT preparations systematically to obtain the information of its gradients and its contents comprehensively. Complexity of TGT components and similarity among many components were both great challenges for the chromatography separation. So, integrated analytical strategy combined advanced comprehensive two-dimensional chromatography with quadrupole time-of-flight mass spectrometry was developed to analyze the components in TGT samples. Methanol was used for extraction of TGT components from the preparations, and the extracted sample was injected into LC×LC chromatographic system. In first dimension (’D), C8 column was selected as separation column. Water and methanol was applied to mobile phase A and B of gradient elution respectively. In second dimension (2D), C18 column was utilized to separate the elute from 1D chromatography. Water containing 0.1% formic acid and acetonitrile including 0.08% formic acid were used as mobile phase A and B of 2D separation respectively. Integrated gradient mode with both shift gradient and full gradient program were used as separation program in 2D. ESI ion source was used for detection in positive mode with mass scan range of 100-1000 m/z to collect MS and MS/MS data. According to high accurate mass data above and related documentaries information,92 and 132 constituents were detected in TGT preparations from Hubei and Hunan manufacturers respectively, most of which belonged to diterpenoids, triterpenoids and alkaloids. There are 42 common constituents detected in two kinds of TGT preparations, and compositions of two kinds of TGT preparations showed great discrepancy.42 common constituents covered total contents of 48.0% and 45.8% in two preparations respectively. The contents of common constituents varied in two kinds of TGT samples. Compared to conventional one-dimensional chromatography, comprehensive two-dimensional chromatography owned special advantage of higher peak capacity, resolving power, and it could eliminate the interference between different constituents to the largest extent. There was obvious discrepancies in both chemical components and contents of two TGT preparations. Besides, previous toxic test demonstrated that both TGT preparations showed severe toxic effect on the liver and kidney, and the effect of Hunan Xieli TGT was more severe than that of Hubei Huangshi preparation. Results inferred that more attentions should be paid to discrepancies of compositions and contents of TGT preparations. Comprehensive two-dimensional chromatography with quadrupole time-of-flight mass spectrometry herein provides a rapid, effective, sensitive method for analysis and identification of components in TGT samples, and the data from this method established important foundation for the study of quality evaluation and toxicological research of TWHF and its preparations. This study uncovered the obvious discrepancies in the types and contents of components in TGT samples from different manufacturers, which help understand the discrepancies in therapeutic and toxic effect of TGT from different manufacturers from the point of substances basis. Finally, we studied the metabolites of TP and TGT in liver and kidney using HPLC analysis coupled with QTOF mass, based on previous study of TGT compositions. Biological samples were all pretreated using solid-phase extraction cartridges. A Restek ultra AQ C18 column was used for the chromatographic separation. The mobile phase system consisted of aqueous solvent A with 2mM ammonium acetate and 0.05% formic acid and organic solvent B with 0.05% formic acid in acetontrile. Gradient elution mode was used at the flow rate of 0.4 mL/min. Mass analysis instrument was equipped with electrospray ionization source. Mass data was collected using positive mode with mass scan range of:m/z 100-1000. In order to study the GSH-conjungated metabolites of TP, we utilized the in vitro liver microsome incubation to obtain the sample with high abundance GSH-conjungated metabolites of TP in the first step, and the information of 6 GSH-conjungated metabolites were obtained including the related chromatographic and mass patterns. On the basis of previous above, we systematically analyzed the metabolite profiling of TP and their time course in body from low dose to high dose, from normal status to DTH reaction. Results indicated that the major metabolic pathway of TP included Phase I metabolism, such as hydroxylation, desaturation, hydrolysis,’"hydroxylation+hydrolysis" and Phase Ⅱ metabolism containing conjungation of Cys, GSH, Glu. Results demonstrated that liver was main organ for metabolism of TP, and 45 major metabolites were detected in liver, especially including 14 GSH or Cys conjugated metabolites. Besides,1 and 3 GSH or Cys conjugated metabolites were detected in kidney and plasma, respectively. In all, GSH or Cys conjungation were crucial metabolic pathway, and corresponding metabolites maybe caused the therapeutic and toxic effect of TP in vivo. Besides, TP underwent rapid metabolism. The biggest number of metabolites were detected at 10 min and 30 min after exposure to TP, and the number was decreased after 30 min. We further studied the metabolites of TP and TGT in liver and kidney, based on previous study of TGT compositions. We also analyzed the constituents in the organ and related bioactive metabolites after oral administration of TGT using advanced HPLC-QTOF platform. Results indicated that 42 and 30 constituents from TGT were detected in live and kidney, respectively. Moreover 58 and 45 kinds of related metabolites were discovered in liver and kidney respectively. All these constituents and their metabolites detected in organ, which established the basis for the further study of the effective and toxic compounds of TWHF. Besides, all the data is beneficial to the clinic safety and the quality control of TGT preparations.
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