Targets of Tripterygium glycosides in systemic lupus erythematosus treatment: A network-pharmacology study.

OBJECTIVE: We aimed to explore the underlying mechanism of Tripterygium glycosides (TGs) in treating systemic lupus erythematosus (SLE) through network-pharmacology approach. METHODS: The protein targets of TGs' three active ingredients (triptolide, tripterine, and wilforlide) and SLE were identified by database search. Then, the intersection of the two groups was studied. The drug-target network between the active ingredients of TGs and the overlapping genes was constructed, visualized, and analyzed with Cytoscape software. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment were performed to analyze these genes. Finally, we validated our predictions of the potential targets through docking study. RESULTS: A total of 55 overlapping genes were discovered. Results suggested that the TGs' mechanism in SLE treatment was associated with heat shock protein family A member 5, heat shock protein family A member 8, eukaryotic translation elongation factor 1 alpha 1, and so forth with their related 4042 gene network, which regulated ribosome, spliceosome, viral carcinogenesis, Epstein-Barr virus infection signaling, and so forth. Molecular-docking analysis proved that hydrogen bonding was the main form of interaction. CONCLUSIONS: Our research provided the protein targets affected by TGs in SLE treatment. The key targets (CASP3, MAPK1, HIF1A, and so forth) involving 4042 proteins became the multitarget mechanism of TGs in SLE treatment.

Detection and quantification of the anti-obesity drug celastrol in murine liver and brain.

Celastrol is a natural pentacyclic triterpene extracted from the roots of Tripterygium wilfordi (thunder god vine). Celastrol was reported as a powerful anti-obesity drug with leptin sensitizing properties that decreases food consumption and mediates body weight loss when administered to diet-induced obese mice at 100 µg/kg body weight. The weight lowering properties of celastrol are likely mediated by the CNS, in particular, by the hypothalamus, but the final proof for the accumulation of celastrol in the brain and hypothalamus remains to be established. Here, we aimed to demonstrate that intraperitoneal celastrol administration at 100 µg/kg can rapidly reach the brain and, in particular, the hypothalamus of mice. We developed and validated a sensitive liquid chromatography mass spectrometry method for the quantitative determination of celastrol in murine tissues, namely liver, brain and hypothalamus. Chow-fed lean mice were randomly assigned to the vehicle vs. celastrol groups, injected with saline or 100 µg/kg body weight of celastrol, and sacrificed 30 min or 120 min post injection. Celastrol was extracted from homogenized tissue using ethyl acetate as organic solvent, and quantified using a matrix-matched calibration curve with glycyrrhetinic acid as internal standard. Liver celastrol concentrations were 32.60 ± 8.21 pg/mg and 40.52 ± 15.6 pg/mg, 30 and 120 min after injection, respectively. We found 4.70 ± 0.31 pg/mg celastrol after 30 min, and 16.22 ± 3.33 pg/mg after 120 min in whole brain lysates, and detectable amounts in the hypothalamus. These results corroborate the validity of our methodology, demonstrate the accumulation of celastrol in the brain of mice injected intraperitoneally with a dose of 100 µg/kg, and confirm the CNS as possible site of action for the weight lowering properties of celastrol.

Friedelane-type triterpene cyclase in celastrol biosynthesis from Tripterygium wilfordii and its application for triterpenes biosynthesis in yeast.

Celastrol is a promising bioactive compound isolated from Tripterygium wilfordii and has been shown to possess many encouraging preclinical applications. However, the celastrol biosynthetic pathway is poorly understood, especially the key oxidosqualene cyclase (OSC) enzyme responsible for cyclisation of the main scaffold. Here, we report on the isolation and characterisation of three OSCs from T. wilfordii: TwOSC1, TwOSC2 and TwOSC3. Both TwOSC1 and TwOSC3 were multiproduct friedelin synthases, while TwOSC2 was a ß-amyrin synthase. We further found that TwOSC1 and TwOSC3 were involved in the biosynthesis of celastrol and that their common product, friedelin, was a precursor of celastrol. We then reconstituted the biosynthetic pathway of friedelin in engineered yeast constructed by the CRISPR/Cas9 system, with protein modification and medium optimisation, leading to heterologous production of friedelin at 37.07 mg l-1 in a shake flask culture. Our study was the first to identify the genes responsible for biosynthesis of the main scaffold of celastrol and other triterpenes in T. wilfordii. As friedelin has been found in many plants, the results and approaches described here have laid a solid foundation for further explaining the biosynthesis of celastrol and related triterpenoids. Moreover, our results provide insights for metabolic engineering of friedelane-type triterpenes.

A PRISMA-compliant systematic review and network meta-analysis on the efficacy between different regimens based on Tripterygium wilfordii Hook F in patients with primary nephrotic syndrome.

BACKGROUND: The present study aims to comprehensively determine the efficacy of different therapy regimens based on Tripterygium wilfordii Hook F (TwHF) for patients with primary nephrotic syndrome (PNS) using network meta-analysis method. METHODS: Seven electronic databases were searched to identify randomized controlled trials (RCTs) that compared the differences between different therapy regimens based on TwHF for patients with PNS. The risk of bias in included RCTs was evaluated according to the Cochrane Handbook version 5.2.0. Network meta-analysis was performed to compare different regimens. Primary outcomes were complete remission rate and total remission rate. The secondary outcomes were hr urinary protein excretion, serum albumin, serum creatinine, and urea nitrogen. Data analysis was performed using R software. RESULTS: A total of 40 studies involving 2846 patients with PNS were included. Compared with prednisone, the improvement in total remission rate and complete remission rate was associated with TwHF alone (odds ratio [OR] = 4.80, 95% credible intervals [CrI]: 2.20-10.00; OR = 6.30, 95% CrI: 2.90-13.00, respectively), TwHF+prednisone (OR = 2.10, 95% CrI: 1.30-3.50; OR = 2.40, 95% CrI: 1.50-3.80, respectively), TwHF+CPA (OR = 12.00, 95% CrI: 1.10-150.00; OR = 16.00, 95% CrI: 1.60-170.00, respectively), and TwHF+Cyclosporine A (OR = 28.00, 95% CrI: 3.20-250.00; OR = 35.00, 95% CrI: 4.50-270.00, respectively). Compared with TwHF alone, TwHF+prednisone showed less benefit in improving total remission rate and complete remission rate (OR = 0.44, 95% CrI: 0.21-0.91; OR = 0.38, 95% CrI: 0.19-0.77, respectively). TwHF alone, TwHF+prednisone could significantly reduce hr urinary protein excretion (MD = -0.69, 95% CrI: -1.30 to -0.14; MD = -1.00, 95% CrI: -1.90 to -0.14, respectively) and increase serum albumin (MD = 5.90, 95% CrI: 2.50-9.30; MD = 3.40, 95% CrI: 1.30-5.50, respectively) when compared to prednisone alone. TwHF alone showed significant reduction in serum creatinine when compared to CPA (MD = -19.00, 95% CrI: -37.00 to -0.56). CONCLUSIONS: TwHF alone, the addition TwHF to prednisone showed more benefit in improving total and complete remission rate, hr urinary protein excretion, serum albumin, and serum creatinine.

A MDR transporter contributes to the different extracellular production of sesquiterpene pyridine alkaloids between adventitious root and hairy root liquid cultures of Tripterygium wilfordii Hook.f.

KEY MESSAGE: TwMDR1 transports sesquiterpene pyridine alkaloids, wilforine and wilforgine, into the hairy roots of T. wilfordii Hook.f. resulting in low secretion ratio of alkaloids. Hairy roots (HRs) exhibit high growth rate and biochemical and genetic stability. However, varying secondary metabolites in HR liquid cultures mainly remain in root tissues, and this condition may affect cell growth and cause inconvenience in downstream extraction. Studies pay less attention to adventitious root (AR) liquid cultures though release ratio of some metabolites in AR liquid cultures is significantly higher than that of HR. In Tripterygium wilfordii Hook.f., release ratio of wilforine in AR liquid cultures reached 92.75 and 13.32% in HR on day 15 of culture. To explore potential roles of transporters in this phenomenon, we cloned and functionally identified a multidrug resistance (MDR) transporter, TwMDR1, which shows high expression levels in HRs and is correlated to transmembrane transportation of alkaloids. Nicotiana tabacum cells with overexpressed TwMDR1 efficiently transported wilforine and wilforgine in an inward direction. To further prove the feasibility of genetically engineered TwMDR1 and improve alkaloid production, we performed a transient RNAi experiment on TwMDR1 in T. wilfordii Hook.f. suspension cells. Results indicated that release ratios of wilforine and wilforgine increased by 1.94- and 1.64-folds compared with that of the control group, respectively. This study provides bases for future studies that aim at increasing secretion ratios of alkaloids in root liquid cultures in vitro.

[Effects of elicitors on growth of adventitious roots and contents of secondary metabolites in Tripterygium wilfordii Hook. f].

To study the effects of the extract of fungal elicitor, AgNO3, MeJA and yeast on the growth and content of secondary metabolites of adventitious roots in Tripterygium wilfordii. The above elicitors were supplemented to the medium, the growth and the content of secondary metabolites were measured. When the medium was supplemented with the elicitor Glomerella cingulata or Collectotrichum gloeosporioides, the content of triptolide was increased by 2.24 and 1.93-fold, the alkaloids content was increased by 2.02 and 2.07-fold, respectively. The optimal concentration of G. cingulata was 50 µg/mL for accumulation of triptolide, alkaloids and for the growth of adventitious roots. AgNO3 inhibited the growth of adventitious roots and the accumulation of the alkaloids, whereas it (at 25 µmol/L) increased the accumulation of triptolide by 1.71-fold compared to the control. The growth of adventitious roots, the contents of triptolide and alkaloids were increased 1.04, 1.64 and 2.12-folds, respectively when MeJA was at 50 µmol/L. When the concentration of yeast reached 2 g/L, the content of triptolide increased 1.48-folds. This research demonstrated that supplementation of AgNO3 and yeast enhanced the biosynthesis of triptolide in adventitious roots and the synergism of G. cingulata and MeJA could promote the biosynthesis of both triptolide and alkaloids.

Cloning and characterisation of the gene encoding 3-hydroxy-3-methylglutaryl-CoA synthase in Tripterygium wilfordii.

Tripterygium wilfordii is a traditional Chinese medical plant used to treat rheumatoid arthritis and cancer. The main bioactive compounds of the plant are diterpenoids and triterpenoids. 3-Hydroxy-3-methylglutaryl-CoA synthase (HMGS) catalyses the reaction of acetoacetyl-CoA to 3-hydroxy-3-methylglutaryl-CoA, which is the first committed enzyme in the mevalonate (MVA) pathway. The sequence information of HMGS in Tripterygium wilfordii is a basic resource necessary for studying the terpenoids in the plant. In this paper, full-length cDNA encoding HMGS was isolated from Tripterygium wilfordii (abbreviated TwHMGS, GenBank accession number: KM978213). The full length of TwHMGS is 1814 bp, and the gene encodes a protein with 465 amino acids. Sequence comparison revealed that TwHMGS exhibits high similarity to HMGSs of other plants. The tissue expression patterns revealed that the expression level of TwHMGS is highest in the stems and lowest in the roots. Induced expression of TwHMGS can be induced by MeJA, and the expression level is highest 4 h after induction. The functional complement assays in the YML126C knockout yeast demonstrated that TwHMGS participates in yeast terpenoid biosynthesis.

[Effects of exogenous abscisic acid (ABA) on the photosynthesis and chlorophyll fluorescence parameters of Tripterygium wilfordii seedlings exposed to low temperature].

Taking one year-old Tripterygium wilfordii cutting seedlings as test materials, this paper studied the effects of foliar spraying 0, 5, 10, 15, 20, and 25 mg x L(-1) of abscisic acid (ABA) on the leaf photosynthesis and chlorophyll fluorescence characteristics of the seedlings under low temperature stress. Spraying 20 mg x L(-1) of ABA increased the cold- resistance of the seedlings significantly, manifesting in the slowing down of the decrease amplitudes of leaf net photosynthetic rate (P(n)), transpiration rate (T(r)), stomatal conductance (g(s)), and intercellular CO2 concentration (C(i)) and the increase of photosynthetic capacity. After 6 days exposure to low temperature, the initial fluorescence (F(o)) decreased with increasing concentration of applied ABA, the maximum fluorescence (F(m)) and maximal photochemical yield (F(v)/F(m)) increased, the actual photochemical efficiency of system II (phi(PSII)) and photochemical quenching coefficient (q(P)) increased after an initial decrease, and the non-photochemical quenching coefficient (q(N)) showed a 'decreasing-increasing-decreasing' trend. The P(n), g(s), q(P), F(m), and F(v)/F(m) reached their peak values at 20 mg x L(-1) of ABA. In all treatments, with the increase of photosynthetically active radiation (PAR), the relative electron transport rate (rETR) increased first and decreased then, reached the peak when the PAR was 395 micromol x m(-2) x s(-1), and the peak value of the rETR in treatments 25 and 20 mg x L(-1) of ABA was 17.1% and 5.2% higher than that of the control, respectively. The light response curves of the psi(PSII) decreased with increasing PAR, whereas those of q(N) performed in adverse.