The Anti-Tumor Effect and Mechanism of Triterpenoids in Rhus chinensis Mill. on Reversing Effector CD8+ T-cells Dysfunction by Targeting Glycolysis Pathways in Colorectal Cancer.

Rhus chinensis Mill. is a traditional Chinese medicine (TCM) which is commonly used for cancer treatments. Our previous work had proven that triterpenoids of Rhus chinensis (TER) could effectively regulate glycolysis involved in colorectal cancer (CRC) and play an important role in the prevention of T-cells dysfunction. This study aimed to systematically investigate the effects and mechanisms of TER on glucose metabolism in CRC, while the regulatory mechanisms of TER on restoring T-cells function and activity in CRC were explored as well. The extract of triterpenoids from Rhus chinensis was obtained, and production of lactic acid and glucose uptake were assayed. Also, the expression of CD8+ T-cells surface markers, cytokines secreted by CD8+ T cells, and the expression of key glycolytic enzymes and glucose deprivation induced by tumor cells were further examined. Notably, results showed that TER prevented the dysfunction in CD8+ T cells by enhancing mTOR activity and subsequent cellular metabolism. Furthermore, our findings also demonstrated that TER promoted glycolytic gene expression in CD8+ T cells in vivo, and significantly inhibited tumor growth. Altogether, our studies suggested that TER not only reversed effector CD8+ T-cells dysfunction and enhanced T-cells recognition, but also improved tumor microenvironment, thereby providing new insight into the prevention and treatment of CRC with TCM.

Effects and mechanisms of fatty acid metabolism­mediated glycolysis regulated by betulinic acid­loaded nanoliposomes in colorectal cancer.

Although previous studies have demonstrated that triterpenoids, such as betulinic acid (BA), can inhibit tumor cell growth, their potential targets in colorectal cancer (CRC) metabolism have not been systematically investigated. In the present study, BA­loaded nanoliposomes (BA­NLs) were prepared, and their effects on CRC cell lines were evaluated. The aim of the present study was to determine the anticancer mechanisms of action of BA­NLs in fatty acid metabolism­mediated glycolysis, and investigate the role of key targets, such as acyl­CoA synthetase (ACSL), carnitine palmitoyltransferase (CPT) and acetyl CoA, in promoting glycolysis, which is activated by inducing hexokinase (HK), phosphofructokinase­1 (PFK­1), phosphoenolpyruvate (PEP) and pyruvate kinase (PK) expression. The results demonstrated that BA­NLs significantly suppressed the proliferation and glucose uptake of CRC cells by regulating potential glycolysis and fatty acid metabolism targets and pathways, which forms the basis of the anti­CRC function of BA­NLs. Moreover, the effects of BA­NLs were further validated by demonstrating that the key targets of HK2, PFK­1, PEP and PK isoenzyme M2 (PKM2) in glycolysis, and of ACSL1, CPT1a and PEP in fatty acid metabolism, were blocked by BA­NLs, which play key roles in the inhibition of glycolysis and fatty acid­mediated production of pyruvate and lactate. The results of the present study may provide a deeper understanding supporting the hypothesis that liposomal BA may regulate alternative metabolic pathways implicated in CRC adjuvant therapy.

Triterpenoids Extracted from Rhus chinensis Mill Act Against Colorectal Cancer by Inhibiting Enzymes in Glycolysis and Glutaminolysis: Network Analysis and Experimental Validation.

Background: Rhus chinensis Mill is a traditional Chinese medicine (TCM) mostly used to treat several cancer types. Although previous studies have found that certain ingredients of R. chinensis such as flavonoids can inhibit tumor cell proliferation [e.g. colorectal cancer (CRC)], systematic research on the mechanism underlying anticancer effect of active compounds like triterpenoids (TER) is lacking.Study Design: Herein, the concept of "network pharmacology primarily based on active compounds" was applied to explore the anticancer mechanisms of TER extract from R. chinensis. In this regard, potential targets and pathways of glycolysis and glutaminolysis form the basis for the anti-CRC effect of triterpenoids. Network pharmacology was used to predict several key proteins in the metabolic pathways, which were further verified via western blot and metabolomics methods.Results: Our results showed that the total TER in R. chinensis remarkably inhibited the proliferation and apoptosis of SW620 cells. The top 4 compounds of TER (viz., betulinic acid-BTA, betulonic acid-BTOA, betulin-BT, and semialactic acid-SA) were confirmed through the detection of UPLC-MS and analysis of cell proliferation assays. Mechanistically, this study revealed that TER plays an anti-CRC role through key targets, such as ENO1, ALDOA, PFKFB3, PKM2, and LDHA, as well as key glycolytic and glutaminolytic pathways.Conclusion: Collectively, these results have provided new insights into the mechanism underlying anti-CRC effect of triterpenoids extract obtained from R. chinensis, mainly through combination of compositional quantitative analysis, network pharmacology, and experimental verification.

Inhibitory ASIC2-mediated calcineurin/NFAT against colorectal cancer by triterpenoids extracted from Rhus chinensis Mill.

ETHNOPHARMACOLOGICAL RELEVANCE: Studies have shown that the etiology and pathogenesis of colorectal cancer are closely related to the tumor microenvironment, and the cancer tissue is still in the state of "energy deficit" and has to promote energy generation through high glycolysis. Rhus chinensis Mill is a Chinese herbal medicine used to treat various types of solid tumors in China. Colorectal cancer (CRC) is a heterogeneous disease group caused by abnormal changes in glucose metabolism resulted in lactic acid production, which remodels acidosis. AIM OF THE STUDY: Although previous studies have shown that the active compounds of Rhus chinensis Mill. can inhibit the proliferation of tumor cells, whether its triterpenoids could effectively regulate glycolysis involved in CRC have not been systematically investigated. MATERIALS AND METHODS: In this study, the extraction of triterpenoids extract from Rhus chinensis Mill. was obtained, and cell viability assay, the percentage of apoptosis for CRC cells were counted, and matrigel invasion assay and production of lactic acid and glucose uptake assay was determined. we further examined the expression of the key glycolytic enzymes and acid-sending ion channel (ASIC) family members of SW620 cells, and some key proteins in the glycolytic pathway were further verified. RESULTS: Notably, triterpenoids (TER) of Rhus chinensis Mill. showed effective anti-proliferative activity and significantly altered protein levels associated with CRC cell survival and glycolysis metabolism. TER could down-regulate the expression of ASIC2, in CRC SW620 cell line. Most importantly, the levels of ASIC2 and calcineurin/nuclear factor of activated T cells (NFAT) were also down-regulated by TER. Furthermore, inhibition of activated the ASIC2-mediated calcineurin/NFAT1 pathway and target gene transcript expression of MMP-2 and MMP-9 in parallel to reduce, and resulted in the reduced invasion ability by TER treatment. CONCLUSION: The potential pathways and targets that involved in glycolysis to excert the anti-CRC effects of main compounds in triterpenoids of Rhus chinensis Mill. were predicted by network pharmacology methods. Our findings thus provided rational evidence that inhibition of the ASIC2-induced calcineurin/NFAT pathway by triterpenoids in Rhus chinensis Mill. profoundly suppressed cell growth and invasion in CRC, which target alternative glycolysis in colorectal tumor cells, may be a useful adjuvant therapy in the treatment of colorectal cancer.

Effects of rosemary (Rosmarinus officinalis L.) extracts and dry ice on the physicochemical stability of omega-3 fatty-acid-fortified surimi-like meat products.

BACKGROUND: Lipid peroxidation entails major quality degradation in omega-3 (ω-3) fatty-acid-fortified surimi-like meat products upon storage. Currently, the use of label-friendly alternatives to synthetic antioxidants is encouraged in the industry. Hence, we aimed to examine the applicability of the hurdle-technology concept, using an 80% (v/v) ethanol solution to obtain rosemary extracts (REs) containing substantial amounts of polyphenol, and dry ice (DI) which can create a cryogenic environment, on the physicochemical stabilities of ω-3 fatty-acid (FA)-fortified meat products after manufacturing and storage periods. The polyphenolic profiles of the REs were also investigated. RESULTS: Carnosol and rosmarinic acid are major phenolic components in REs. Furthermore, DI addition during the chopping procedure increased (P < 0.05) whiteness values and hardness of products, while total ω-3 and ω-6 FAs were relatively well preserved (P < 0.05) in products with flaxseed oil premixed with RE. During 14-day storage at 4 °C, combined treatment with RE and DI decreased (P < 0.05) thiobarbituric acid reactive substance (TBARS) levels and the centrifugation loss of products. Single or combined treatment with RE and/or DI decreased (P < 0.05) TBARS levels in products after 60 days of storage at -20 °C. CONCLUSION: Due to the antioxidant-polyphenol profile of REs and a possible oxygen exclusion of DI treatment under atmospheric pressure during food manufacturing, application of the hurdle-technology concept, using treatment with both RE and DI, can reduce lipid peroxidation and maintain a greater water-holding capacity of ω-3 FA-fortified meat products upon storage. © 2019 Society of Chemical Industry.

Incarvine C suppresses proliferation and vasculogenic mimicry of hepatocellular carcinoma cells via targeting ROCK inhibition.

BACKGROUND: Studies have described vasculogenic mimicry (VM) as an alternative circulatory system to blood vessels in multiple malignant tumor types, including hepatocellular carcinoma (HCC). In the current study, we aimed to seek novel and more efficient treatment strategies by targeting VM and explore the underlying mechanisms in HCC cells. METHODS: Cell counting kit-8 (CCK-8) assay and colony survival assay were performed to explore the inhibitory effect of incarvine C (IVC) on human cancer cell proliferation. Flow cytometry was performed to analyze the cell cycle distribution after DNA staining and cell apoptosis by the Annexin V-PE and 7-AAD assay. The effect of IVC on Rho-associated, coiled-coil-containing protein kinase (ROCK) was determined by western blotting and stress fiber formation assay. The inhibitory role of IVC on MHCC97H cell VM formation was determined by formation of tubular network structures on Matrigel in vitro, real time-qPCR, confocal microscopy and western blotting techniques. RESULTS: We explored an anti-metastatic HCC agent, IVC, derived from traditional Chinese medicinal herbs, and found that IVC dose-dependently inhibited the growth of MHCC97H cells. IVC induced MHCC97H cell cycle arrest at G1 transition, which was associated with cyclin-dependent kinase 2 (CDK-2)/cyclin-E1 degradation and p21/p53 up-regulation. In addition, IVC induced apoptotic death of MHCC97H cells. Furthermore, IVC strongly suppressed the phosphorylation of the ROCK substrate myosin phosphatase target subunit-1 (MYPT-1) and ROCK-mediated actin fiber formation. Finally, IVC inhibited cell-dominant tube formation in vitro, which was accompanied with the down-regulation of VM-key factors as detected by real time-qPCR and immunofluorescence. CONCLUSIONS: Taken together, the effective inhibitory effect of IVC on MHCC97H cell proliferation and neovascularization was associated with ROCK inhibition, suggesting that IVC may be a new potential drug candidate for the treatment of HCC.

Optimization of storage condition for maintaining long-term viability of nematophagous fungus Esteya vermicola as biocontrol agent against pinewood nematode.

The fungus, Esteya vermicola has been proposed as biocontrol agent against pine wilting disease caused by Bursaphelenchus xylophilus. In this study, we reported the effects of temperature and different additives on the viability and biocontrol efficacy of E. vermicola formulated by alginate-clay. The viability of the E. vermicola formulation was determined for six consecutive months at temperature ranged from -70 to 25 °C. The fresh conidia without any treatment were used as control. Under the optimal storage conditions with E. vermicola alginate-clay formulation, the results suggested that E. vermicola alginate-clay formulation with a long shelf life could be a non-vacuum-packed formulation that contains 2 % sodium alginate and 5 % clay at 4 °C. Three conidial formulations prepared with additives of 15 % glycerol, 0.5 % yeast extract and 0.5 % herbal extraction, respectively significantly improved the shelf life. In addition, these tested formulations retained the same biocontrol efficacy as the fresh conidial against pinewood nematode. This study provided a tractable and low-cost method to preserve the shelf life of E. vermicola.

Protection of Veratrum nigrum L. var. ussuriense Nakai alkaloids against ischemia-reperfusion injury of the rat liver.

AIM: To investigate the protective effects and possible mechanisms of Veratrum nigrum L.var. ussuriense Nakai alkaloids (VnA) on hepatic ischemia/reperfusion (I/R) injury in rats. METHODS: Forty male Wistar rats were randomly divided into four experimental groups (n = 10 in each): (A) Control group (the sham operation group); (B) I/R group (pretreated with normal saline); (C) Small-dose (10 microg/kg) VnA pretreatment group; (D) Large-dose (20 microg/kg) VnA pretreatment group. Hepatic ischemia/reperfusion (Hepatic I/R) was induced by occlusion of the portal vein and the hepatic artery for 90 min, followed by reperfusion for 240 min. The pretreatment groups were administered with VnA intraperitoneally, 30 min before surgery, while the control group and I/R group were given equal volumes of normal saline. Superoxide dismutase (SOD) activity, myeloperoxidase (MPO) activity and nitric oxide (NO) content in the liver tissue at the end of reperfusion were determined and liver function was measured. The expression of intercellular adhesion molecule-1 (ICAM-1) and E-selectin (ES) were detected by immunohistochemical examinations and Western blot analyses. RESULTS: The results showed that hepatic I/R elicited a significant increase in the plasma levels of alanine aminotransferase (ALT: 74.53 +/- 2.58 IU/L vs 1512.54 +/- 200.76 IU/L, P < 0.01) and lactic dehydrogenase (LDH: 473.48 +/- 52.17 IU/L vs 5821.53 +/- 163.69 IU/L, P < 0.01), as well as the levels of MPO (1.97 +/- 0.11 U/g vs 2.57 +/- 0.13 U/g, P < 0.01) and NO (69.37 +/- 1.52 micromol/g protein vs 78.39 +/- 2.28 micromol/g protein, P < 0.01) in the liver tissue, all of which were reduced by pretreatment with VnA, respectively (ALT: 1512.54 +/- 200.76 IU/L vs 977.93 +/- 89.62 IU/L, 909.81 +/- 132.76 IU/L, P < 0.01, P < 0.01; LDH: 5821.53 +/- 163.69 IU/L vs 3015.44 +/- 253.01 IU/L, 2448.75 +/- 169.4 IU/L, P < 0.01, P < 0.01; MPO: 2.57 +/- 0.13 U/g vs 2.13 +/- 0.13 U/g, 2.07 +/- 0.05 U/g, P < 0.01, P < 0.01; NO: 78.39 +/- 2.28 micromol/g protein vs 71.11 +/- 1.73 micromol/g protein, 68.58 +/- 1.95 micromol/g protein, P < 0.05, P < 0.01). The activity of SOD (361.75 +/- 16.22 U/mg protein vs 263.19 +/- 12.10 U/mg protein, P < 0.01) in the liver tissue was decreased after I/R, which was enhanced by VnA pretreatment (263.19 +/- 12.10 U/mg protein vs 299.40 +/- 10.80 U/mg protein, 302.09 +/- 14.80 U/mg protein, P < 0.05, P < 0.05). Simultaneously, the histological evidence of liver hemorrhage, polymorphonuclear neutrophil infiltration and the overexpression of ICAM-1 and E-selectin in the liver tissue were observed, all of which were attenuated in the VnA pretreated groups. CONCLUSION: The results demonstrate that VnA pretreatment exerts significant protection against hepatic I/R injury in rats. The protective effects are possibly associated with enhancement of antioxidant capacity, reduction of inflammatory responses and suppressed expression of ICAM-1 and E-selectin.