Biosensor-based active ingredient recognition system for screening TNF-α inhibitors from lotus leaves.

Erhuangquzhi granules (EQG) have been clinically proven to be effective in nonalcoholic steatohepatitis (NASH) treatment. However, the active components and molecular mechanisms remain unknown. This study aimed to screen active components targeting tumor necrosis factor α (TNF-α) in EQG for the treatment of NASH by a surface plasmon resonance (SPR) biosensor-based active ingredient recognition system (SPR-AIRS). The amine-coupling method was used to immobilize recombinant TNF-α protein on an SPR chip, the specificity of the TNF-α-immobilized chip was validated, and nine medicinal herbs in EQG were prescreened. Nuciferine (NF), lirinidine (ID), and O-nornuciferine (NNF) from lotus leaves were found and identified as TNF-α ligands by UPLC‒MS/MS, and the affinity constants of NF, ID, and NNF to TNF-α were determined by SPR experiments (Kd = 61.19, 31.02, and 20.71 µM, respectively). NF, ID, and NNF inhibited TNF-α-induced apoptosis in L929 cells, the levels of secreted IL-6 and IL-1ß were reduced, and the phosphorylation of IKKß and IκB was inhibited in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. In conclusion, a class of new active small-molecule TNF-α inhibitors was discovered, which also provides a valuable reference for the material basis and mechanism of EQG action in NASH treatment.

iTRAQ Quantitative Proteomic Analysis of Different Expressed Proteins and Signal Pathways in Bakuchiol-Induced Hepatotoxicity.

Bakuchiol (BAK) is an abundant natural compound. BAK has been reported to have several biological activities such as anticancer, antiaging, anti-inflammatory, and prevention of bone loss. However, it causes hepatotoxicity, the mechanism of which is not known. In this study, we explored the mechanism of BAK hepatotoxicity by treating rats with 52.5 mg/kg and 262.5 mg/kg of BAK, administered continuously for 6 weeks. We examined the liver pathology and biochemical composition of bile to determine toxicity. Mechanisms of BAK hepatotoxicity were analyzed based on relative and absolute quantification (iTRAQ) protein equivalent signatures and validated in vitro using LO2 cells. iTRAQ analysis revealed 281 differentially expressed proteins (DEPs) in liver tissue of the BAK-treated group, of which 215 were upregulated, and 66 were downregulated. GO and KEGG enrichment analysis revealed that bile secretion, lipid metabolism, and cytochrome P450 signaling pathways were enriched in DEPs. Among them, peroxisome proliferator-activated receptor α (PPARα), farnesoid X receptor (FXR), and cholesterol 7α-hydroxylase (CYP7a1) were closely associated with the development and progression of BAK-induced hepatic metabolic dysfunction and abnormal bile metabolism. This study shows that BAK can induce hepatotoxicity through multiple signaling pathways.

Ganoderic acid improves 5-fluorouracil-induced cognitive dysfunction in mice.

5-Fluorouracil (5-FU) is a chemotherapeutic drug with a good anti-cancer effect on various types of cancers, such as colorectal cancer and breast cancer. However, previous studies have found that 5-FU could induce cognitive deficit in clinics. As ganoderic acid, isolated from Ganoderma lucidum, has a protective effect on neurons, this study investigated the effects of ganoderic acid (GA) against 5-FU-induced cognitive dysfunction with a series of behavioral tests and related indicators. Experimental results showed that GA significantly prevented the reduction of spatial and non-spatial memory in 5-FU-treated mice. In addition, GA not only ameliorated the damage to hippocampal neurons and mitochondrial structure, but also significantly improved abnormal protein expression of mitochondrial biogenesis related marker PGC-1α, and mitochondrial dynamics related markers MFN2, DRP1 and FIS1 in the hippocampi of 5-FU-treated mice. Moreover, GA could up-regulate the expression of neuronal survival and growth-related proteins, such as BDNF, p-ERK, p-CREB, p-Akt, p-GSK3ß, Nrf2, p-mTOR, and p-S6, in the hippocampi of 5-FU-treated mice. These results suggest that GA could prevent cognitive dysfunction in mice treated with 5-FU via preventing mitochondrial impairment and enhancing neuronal survival and growth, which provide evidence for GA as a promising adjunctive therapy for chemotherapy related cognitive impairment in clinics.

Ganoderic acid alleviates chemotherapy-induced fatigue in mice bearing colon tumor.

Chemotherapy-related fatigue (CRF) is increasingly being recognized as one of the severe symptoms in patients undergoing chemotherapy, which not only largely reduces the quality of life in patients, but also diminishes their physical and social function. At present, there is no effective drug for preventing and treating CRF. Ganoderic acid (GA), isolated from traditional Chinese medicine Ganoderma lucidum, has shown a variety of pharmacological activities such as anti-tumor, anti-inflammation, immunoregulation, etc. In this study, we investigated whether GA possessed anti-fatigue activity against CRF. CT26 tumor-bearing mice were treated with 5-fluorouracil (5-FU, 30 mg/kg) and GA (50 mg/kg) alone or in combination for 18 days. Peripheral and central fatigue-related behaviors, energy metabolism and inflammatory factors were assessed. We demonstrated that co-administration of GA ameliorated 5-FU-induced peripheral muscle fatigue-like behavior via improving muscle quality and mitochondria function, increasing glycogen content and ATP production, reducing lactic acid content and LDH activity, and inhibiting p-AMPK, IL-6 and TNF-α expression in skeletal muscle. Co-administration of GA also retarded the 5-FU-induced central fatigue-like behavior accompanied by down-regulating the expression of IL-6, iNOS and COX2 in the hippocampus through inhibiting TLR4/Myd88/NF-κB pathway. These results suggest that GA could attenuate 5-FU-induced peripheral and central fatigue in tumor-bearing mice, which provides evidence for GA as a potential drug for treatment of CRF in clinic.

Ellagic Acid Inhibits Trichophyton rubrum Growth via Affecting Ergosterol Biosynthesis and Apoptotic Induction.

BACKGROUND: Trichophyton rubrum, among other dermatophytes, is a major causative agent for superficial dermatomycoses like onychomycosis and tinea pedis, especially among pediatric and geriatric populations. Ellagic acid (EA) and shikonin (SK) have been reported to have many bioactivities, including antifungal activity. However, the mechanism of EA and SK on Trichophyton rubrum has not yet been reported. OBJECTIVES: The purposes of this study were to evaluate the antifungal activities of EA and SK against Trichophyton rubrum and to illuminate the underlying action mechanisms. METHODS: The effect of EA (64, 128, and 256 µg/mL) and SK (8, 4, and 2 µg/mL) on Trichophyton rubrum was investigated with different doses via detecting cell viability, ultrastructure with using a scanning electron microscope (SEM), cell apoptosis and necrosis by using the flow cytometry instrument technique (FCIT), and the ergosterol biosynthesis pathway-related fungal cell membrane key gene expressions in vitro. RESULTS: SEM detection revealed that the T. rubrum cell surface was shrivelled, folded, and showed deformation and expansion, visible surface peeling, and broken hyphae, and cell contents overflowed after being treated with EA and SK; the cell apoptosis rate was significantly increased in dose-dependent manner after T. rubrum was treated with EA and SK; the qPCR results showed that mRNA expression of MEP4 and SUB1 was downregulated in EA- and SK-treated groups. CONCLUSIONS: Overall, our results revealed the underlying antifungal mechanism of EA and SK, which may be related to the destruction of the fungal cell membrane and inhibition of C14 demethylase and the catalytic rate of squalene cyclooxidase in the ergosterol biosynthesis pathway via downregulation of MEP4 and SUB1, suggesting that EA and SK have the potential to be developed further as a natural antifungal agent for clinical use.

Bakuchiol Contributes to the Hepatotoxicity of Psoralea corylifolia in Rats.

Psoralea corylifolia L. (Fructus Psoraleae) is widely used in Asia, but there are concerns about hepatotoxicity caused by constituents such as psoralens and bakukiol. Bakuchiol (BAK) has antiinflammatory, antipyretic, antibacterial antiviral, anticancer, and estrogenic activity but appears to be hepatotoxic in in vitro tests. This study investigated the hepatotoxicity in vivo in rats. Using intragastrically administered bakuchiol at doses of 52.5 and 262.5 mg/kg for 6 weeks. Bodyweight, relative liver weight, biochemical indicators, histopathology, mRNA expression of CYP7A1, HMG-CoA reductase, BSEP, PPARα, SREBP-2, and MRP3 were measured. Many abnormalities were observed in the bakuchiol-treated groups including suppression of weight gain and food intake, change of some parameters in serum biochemistry, and increased weight of liver. The mRNA expression of CYP7A1, HMG-CoA reductase, PPARα, and SREBP-2 decreased in bakuchiol-treated group, the expression of BSEP increased in bakuchiol-treated low dosage, and the expression of BSEP decreased in bakuchiol-treated high dosage. In conclusion, we provide evidence for the first time that bakuchiol can induce cholestatic hepatotoxicity, suggesting potential hepatotoxicity. The mechanism may be related to effects on liver lipid metabolism, but further investigation is necessary. Copyright © 2017 John Wiley & Sons, Ltd.