Tripterygium hypoglaucum (Levl.) Hutch, a traditional Chinese medicinal herb with a long history of use, is widely distributed in China. One of its main active components, celastrol, has great potential to be developed into anti-cancer and anti-obesity drugs. Although it exhibits strong pharmacological activities, there is a lack of sustainable sources of celastrol and its derivatives, making it crucial to develop novel sources of these drugs through synthetic biology. The key step in the biosynthesis of celastrol is considered to be the cyclization of 2,3-oxidosqualene into friedelin under the catalysis of 2,3-oxidosqualene cyclases. Friedelin was speculated to be oxidized into celastrol by cytochrome P450 oxidases (CYP450s). Here, we reported a cytochrome P450 ThCYP712K1 from Tripterygium hypoglaucum (Levl.) Hutch that catalyzed the oxidation of friedelin into polpuonic acid when heterologously expressed in yeast. Through substrate supplementation and in vitro enzyme analysis, ThCYP712K1 was further proven to catalyze the oxidation of friedelin at the C-29 position to produce polpunonic acid, which is considered a vital step in the biosynthesis of celastrol, and will lay a foundation for further analysis of its biosynthetic pathway.
Anti-Obesity Agents ; Cytochrome P-450 Enzyme System/metabolism* ; Pentacyclic Triterpenes ; Squalene/analogs & derivatives* ; Tripterygium/metabolism* ; Triterpenes/metabolism*

Metabolic associated fatty liver disease (MAFLD) is a liver disease with hepatocyte steatosis caused by metabolic disorders, which is closely related to obesity, diabetes, metabolic dysfunction, and other factors. Its pathological process changes from simple steatosis, liver inflammation to non-alcoholic steatohepatitis (NASH), and then leads to liver fibrosis, cirrhosis, and liver cancer. At present, no specific therapeutics are available for treatment of MAFLD targeting its etiology. Celastrol is the main active component of the traditional Chinese medicine Celastrus orbiculatus Thunb. In recent years, it has been found that celastrol shows important medicinal value in regulating lipid metabolism, reducing fat and weight, and protecting liver, and then ameliorates MAFLD. This article reviews the related research progress of celastrol in the prevention and treatment of MAFLD, so as to provide a reference for the comprehensive development and utilization of celastrol.
Humans ; Non-alcoholic Fatty Liver Disease/metabolism* ; Liver/pathology* ; Pentacyclic Triterpenes/metabolism* ; Obesity

Cadmium (Cd) is a common heavy metal in the environment. Cd²⁺ may penetrate the blood-brain barrier and produce neurotoxicity, thus inducing various neurodegenerative diseases. Celastrol is an effective component of Tripterygium wilfordii Hook. F., which has many pharmacological effects such as anti-cancer and anti-inflammatory. Here we explored the effect of celastrol on the corresponding neurotoxicity induced by Cd²⁺. Cell proliferation test, cell membrane integrity test, and cell morphology were observed to analyze the effect of Cd²⁺ on the viability of HMC3. The neurotoxicity of Cd²⁺ and the effect of celastrol on the corresponding neurotoxicity induced by Cd²⁺ were analyzed by nitric oxide (NO) test, lipid peroxidation (MDA) test, and Western blotting. When the concentration of Cd²⁺ reached 40 μmol/L, the inhibition rate of HMC3 cell proliferation was (57.17±8.23)% (P < 0.01, n=5), compared with the control group. The cell activity continued to reduce when the Cd²⁺ concentration further increased. When the concentration of Cd²⁺ was higher than 40 μmol/L, the cell membrane of HMC3 was significantly damaged, and the damage was dose-dependent. Upon increasing the Cd²⁺ concentration, the cell morphology began to change and the adhesion also became worse. Cd²⁺ significantly increased the amount of NO released by HMC3 cells, while celastrol effectively inhibited the NO release of HMC3 cells induced by Cd²⁺. Cd²⁺ greatly increased the release of MDA in HMC3 cells, and the level of MDA decreased rapidly upon the addition of 10^-7 mol/L celastrol. Cd²⁺ increased the expression of p-PI3K protein, and the levels of p-PI3K protein and p-AKT protein were inhibited by the addition of celastrol (10^‒7 mol/L, 10^‒6 mol/L), thus preventing cell apoptosis. In conclusion, celastrol inhibits Cd²⁺ induced microglial cytotoxicity and plays a neuroprotective role.
Anti-Inflammatory Agents/pharmacology* ; Apoptosis ; Cadmium/toxicity* ; Nitric Oxide/pharmacology* ; Pentacyclic Triterpenes ; Phosphatidylinositol 3-Kinases ; Proto-Oncogene Proteins c-akt/metabolism* ; Triterpenes/pharmacology*

OBJECTIVETo study effects of three pentacyclic triterpenoids, oleanolic acid (OA), ursolic acid (UA) and asiatic acid (AA) on Ca(2+)-induced liver mitochondrial permeability transition (MPT).

METHODEffects of three compounds on liver MPT induced by Ca2+ were assessed by measuring the change in mitochondrial swelling, mitochondrial membrane potential and release of matrix Ca2+ in vitro.

RESULTObvious mitochondrial swelling, loss of mitochondrial membrane potential and release of matrix Ca2+ occurred after the addition of 50 micromol x L(-1) Ca2+. However, preincubation with 50 mg x L(-1) OA, UA or AA significantly blocked the above changes. In addition, it was also found that there are differences in the inhibitions of three compounds on liver MPT induced by Ca2+.

CONCLUSIONThree pentacyclic triterpenoids, OA, UA and AA, have significant mitochondrial protection through blocking on liver MPT and the inhibition on liver MPT of AA is stronger than that of UA and OA.

Animals ; Calcium ; metabolism ; pharmacology ; Membrane Potential, Mitochondrial ; drug effects ; Mice ; Mice, Inbred ICR ; Mitochondria, Liver ; drug effects ; metabolism ; Mitochondrial Membrane Transport Proteins ; drug effects ; Mitochondrial Swelling ; drug effects ; Pentacyclic Triterpenes ; pharmacology

Epithelial-mesenchymal transformation(EMT) exists in embryonic development and is closely related to cell migration and invasion. The increased EMT level in tumors showed that E-cadherin was replaced by N-cadherin, and the expression of interstitial markers such as α-SMA and vimentin was up-regulated. It has been reported that lupeol can reduce the expression of matrix metalloproteinase-2(MMP-2), matrix metalloproteinase-9(MMP-9) and N-cadherin to inhibit the metastasis of osteoma cells. However lupeol has been less studied in liver cancer. Therefore, this paper investigated the effect of lupanol on invasion and metastasis of human hepatoma cell line HepG2 and SK-HEP-1 and its possible mechanism. MTT assay and Annexin V/PI double staining were used to investigate the effect of lupeol on activity and apoptosis of HepG2 cells and SK-HEP-1 cells. Moreover, the effect of lupeol on the invasion of HepG2 cells and SK-HEP-1 cells were evaluated by Transwell assay. The expressions of E-cadherin, N-cadherin, α-SMA, vimentin and MMP-9 were measured by Western blot. The model of subcutaneous transplantation of nude mice and the lung metastasis model of H22 hepatocellular carcinoma cells were established to evaluate the efficacy of lupeol in vivo on tumor growth and lung metastasis by HE staining combined with immunohistochemical assay. The results showed that lupeol inhibited the activity and invasion of HepG2 cells and SK-HEP-1 cells in a dose-dependent manner and induced apoptosis. Western blot showed that the expression of E-cadherin, a landmark protein for EMT, was induced by lupeol, and the expressions of N-cadherin, α-SMA, vimentin and MMP-9 were decreased. In vivo experiments showed that lupeol inhibited tumor growth in mice bearing xenograft. In addition, immunohistochemical experiments confirmed that lupeol could up-regulate the expression of E-cadherin in tumor tissues of nude mice, reduce the expression of N-cadherin, and inhibit the metastasis of liver cancer H22 cells in the lungs of mice. The above results indicated that the mechanism of lupeol inhibiting the invasion and metastasis of HCC cells may be related to the regulation of EMT process.
Animals ; Carcinoma, Hepatocellular ; Cell Line, Tumor ; Cell Movement ; Epithelial-Mesenchymal Transition ; Gene Expression Regulation, Neoplastic ; Hep G2 Cells ; Humans ; Liver Neoplasms ; Matrix Metalloproteinase 2/metabolism* ; Mice ; Mice, Nude ; Neoplasm Invasiveness ; Pentacyclic Triterpenes

AIMTo investigate antioxidant potential of lupeol/mango pulp extract (MPE) in testosterone induced oxidative stress in prostate of male Swiss albino mice.

METHODSOral treatment of lupeol (1 mg/animal) and MPE (1 mL [20% w/v]/animal) was given separately to animals along with subcutaneous injection of testosterone (5 mg/kg body weight) consecutively for 15 days. At the end of the study period, the prostate was dissected out for the determination of reactive oxygen species (ROS) levels, lipid peroxidation and antioxidant enzymes status (catalase, superoxide dismutase, glutathione reductase, glutathione-S-transferase).

RESULTSIn testosterone treated animals, increased ROS resulted in depletion of antioxidant enzymes and increase in lipid peroxidation in mouse prostate. However, lupeol/MPE treatment resulted in a decrease in ROS levels with restoration in the levels of lipid peroxidation and antioxidant enzymes.

CONCLUSIONThe results of the present study demonstrate that lupeol/MPE are effective in combating oxidative stress-induced cellular injury of mouse prostate. Mango and its constituents, therefore, deserve study as a potential chemopreventive agent against prostate cancer.

Animals ; Antioxidants ; pharmacology ; Catalase ; metabolism ; Glutathione Reductase ; metabolism ; Glutathione Transferase ; metabolism ; Lipid Peroxidation ; drug effects ; Male ; Mangifera ; Mice ; Oxidative Stress ; drug effects ; Pentacyclic Triterpenes ; Plant Extracts ; administration & dosage ; Prostate ; drug effects ; enzymology ; Reactive Oxygen Species ; metabolism ; Superoxide Dismutase ; metabolism ; Testosterone ; administration & dosage ; Triterpenes ; administration & dosage

OBJECTIVETo investigate the protective effects and mechanism of triterpenoids on primarily cultured rat hepatocytes injured by D-galactosamine (D-GalN) or carbon tetrachloride (CCl4).

METHODSRat hepatocytes were isolated by two-step collagenase perfusion and cultured in RPMI 1640 medium. Protective effects of asiatic acid (AA) and beta-glycyrrhetinic acid (GA) were evaluated on hepatocytes injured by D-GalN (2 mmol/L) or CCl4 (10 mmol/L). Cell morphology was observed by light microscope, cell viability was measured by MTT assay, AST and LDH were determined by an automatic analyzer. Fluorescence assay was applied to test reactive oxygen species (ROS), nitric oxide end products (NOx) and reduced glutathione (GSH), and JC-1 staining was used to determine mitochondria membrane potential (DeltaPsim).

RESULTSAST and LDH in medium were decreased when treated with AA and GA after D-GalN injury (P<0.05), furthermore AA enhanced the hepatocyte viability (P<0.05). Moreover, AA and GA significantly reduced ROS and NOx generation, and ameliorated DeltaPsim lost induced by D-GalN. AA also inhibited GSH decrease due to D-GalN and CCl4 treatment.

CONCLUSIONBoth AA and GA could protect hepatocytes from D-GalN and CCl4 injuries, which is associated with reducing intracellular ROS and NOx, reversing GSH depression and ameliorating DeltaPsim lost.

Animals ; Carbon Tetrachloride ; toxicity ; Cell Survival ; drug effects ; Cells, Cultured ; Galactosamine ; toxicity ; Glycyrrhetinic Acid ; pharmacology ; Hepatocytes ; cytology ; drug effects ; metabolism ; Male ; Membrane Potential, Mitochondrial ; drug effects ; Nitric Oxide ; metabolism ; Pentacyclic Triterpenes ; Protective Agents ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Reactive Oxygen Species ; metabolism ; Triterpenes ; pharmacology

To study the protective effect and preliminary mechanisms of asiatic acid against oxygen-glucose deprivation/reoxygenation (OGD/R) injury of PC12 cells, Na2S2O4 combined with low glucose induced damage of PC12 cells was served as OGD/R injury model in vitro. MTT method was used to evaluate cell survival. Ultraviolet spectrophotometry was performed to determine lactate dehydrogenase (LDH) leakage, lactic acid (LD) content, intracellular superoxide dismutase (SOD), malonyldialdehyde (MDA), and cellular Caspase-3 activity. Flow cytometry was applied to assay cell apoptosis. Na2S2O4 combined with low glucose induced significant cell survival rate decreasing compared with normal cells. Cell survival rate increasing, LDH leakage alleviating, LD producing inhibiting, SOD activity promotion, MDA content reducing, cell apoptotic rate decreasing and Caspase-3 activity inhibiting were observed when cells were preincubated with different concentration of asiatic acid (10, 1 and 0.1 micromol x L(-1)). Evident protective effect of asiatic acid against OGD/R injured PC12 cells was verified in our experiment, and the possible mechanisms were related to eliminating free radicals and inhibiting cell apoptosis.
Animals ; Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Cell Survival ; drug effects ; Centella ; chemistry ; Dose-Response Relationship, Drug ; Glucose ; metabolism ; L-Lactate Dehydrogenase ; metabolism ; Lactic Acid ; metabolism ; Malondialdehyde ; metabolism ; Neuroprotective Agents ; administration & dosage ; pharmacology ; Oxygen ; metabolism ; PC12 Cells ; Pentacyclic Triterpenes ; administration & dosage ; isolation & purification ; pharmacology ; Plants, Medicinal ; chemistry ; Rats ; Reperfusion Injury ; metabolism ; Superoxide Dismutase ; metabolism

The Goto-Kakizaki (GK) rat is a spontaneous type 2 diabetic animal model, which is characterized by a progressive loss of beta islet cells with fibrosis. In the present study, the hypoglycemic effect of asiatic acid (AA) in GK rats was examined. GK rats receiving AA at a daily dose of 25 mg·kg(-1) for four weeks showed a significant reduction in blood glucose levels. Age-matched normal Wistar rats were given 0.5% sodium carboxymethyl cellulose (CMC-Na) solution for the same periods and used as control. Compared to the normal Wistar rats, GK rats treated with AA showed improvement in insulin resistance partially through decreasing glucose level (P < 0.01) and insulin level (P < 0.05). Furthermore, the results of immunohistochemistry indicate that AA treatment reduced islet fibrosis in GK rats. Fibronectin, a key protein related to islet fibrosis, was over-expressed in GK rats, which was reversed significantly by AA treatment (P < 0.05). These findings suggest that AA has a beneficial effect on lowering blood glucose levels in GK rats and improves fibrosis of islets in diabetes, which may play a role in the prevention of islets dysfunction.
Animals ; Blood Glucose ; metabolism ; Centella ; chemistry ; Diabetes Mellitus, Type 2 ; drug therapy ; pathology ; Disease Models, Animal ; Fibronectins ; metabolism ; Fibrosis ; Glucose Tolerance Test ; Hyperglycemia ; drug therapy ; pathology ; Insulin ; blood ; Insulin Resistance ; Islets of Langerhans ; drug effects ; pathology ; Male ; Pancreatic Diseases ; metabolism ; pathology ; prevention & control ; Pentacyclic Triterpenes ; pharmacology ; therapeutic use ; Phytotherapy ; Plant Extracts ; pharmacology ; therapeutic use ; Rats, Inbred Strains