Characterization of a Tumor-Microenvironment-Relevant Gene Set Based on Tumor Severity in Colon Cancer and Evaluation of Its Potential for Dihydroartemisinin Targeting.

Colon cancer (COAD) is a leading cause of cancer mortality in the world. Most patients with COAD die as a result of cancer cell metastasis. However, the mechanisms underlying the metastatic phenotype of COAD remain unclear. Instead, particular features of the tumor microenvironment (TME) could predict adverse outcomes including metastasis in patients with COAD, and the role of TME in governing COAD progression is undeniable. Therefore, exploring the role of TME in COAD may help us better understand the molecular mechanisms behind COAD progression which may improve clinical outcomes and quality of patients. Here, we identified a Specific TME Regulatory Network including AEBP1, BGN, POST, and FAP (STMERN) that is highly involved in clinical outcomes of patients with COAD. Comprehensive in silico analysis of our study revealed that the STMERN is highly correlated with the severity of COAD. Meanwhile, our results reveal that the STMERN might be associated with immune infiltration in COAD. Importantly, we show that dihydroartemisinin (DHA) potentially interacts with the STMERN. We suggest that DHA might contribute to immune infiltration through regulating the STMERN in COAD. Taken together, our data provide a set of biomarkers of progression and poor prognosis in COAD. These findings could have potential prognostic and therapeutic implications in the progression of COAD.

Apoptotic activities of brusatol in human non-small cell lung cancer cells: Involvement of ROS-mediated mitochondrial-dependent pathway and inhibition of Nrf2-mediated antioxidant response.

Brusatol occurs as a characteristic bioactive principle of Brucea javanica (L.) Merr., a traditional medicinal herb frequently employed to tackle cancer in China. This work endeavored to unravel the potential anti-cancer activity and action mechanism of brusatol against non-small cell lung cancer (NSCLC) cell lines. The findings indicated that brusatol remarkably inhibited the growth of wild-type NSCLC cell lines (A549 and H1650) and epidermal growth factor receptor-mutant cell lines (PC9 and HCC827) in a dose- and time-related fashion, and profoundly inhibited the clonogenic capability and migratory capacity of PC9 cells. Treatment with brusatol resulted in significant apoptosis in PC9 cells, as evidenced by Hoechst 33342 staining and flow cytometric analysis. The apoptotic effect was closely related to induction of G0-G1 cell cycle arrest, stimulation of reactive oxygen species (ROS) and malondialdehyde, decrease of glutathione levels and disruption of mitochondrial membrane potential. Furthermore, pretreatment with N-acetylcysteine, a typical ROS scavenger, markedly ameliorated the brusatol-induced inhibition of PC9 cells. Western blotting assay indicated that brusatol pronouncedly suppressed the expression levels of mitochondrial apoptotic pathway-associated proteins Bcl-2 and Bcl-xl, accentuated the expression of Bax and Bak, and upregulated the protein expression of XIAP, cleaved caspase-3/pro caspase-3, cleaved caspase-8/pro caspase-8, and cleaved PARP/total PARP. In addition, brusatol significantly suppressed the expression of Nrf2 and HO-1, and abrogated tBHQ-induced Nrf2 activation. Combinational administration of brusatol with four chemotherapeutic agents exhibited marked synergetic effect on PC9 cells. Together, the inhibition of PC9 cells proliferation by brusatol might be intimately associated with the modulation of ROS-mediated mitochondrial-dependent pathway and inhibition of Nrf2-mediated antioxidant response. This novel insight might provide further evidence to buttress the antineoplastic efficacy of B. javanica, and support a role for brusatol as a promising anti-cancer candidate or adjuvant to current chemotherapeutic medication in the therapy of EGFR-mutant NSCLC.

Patchouli Oil Attenuates High Fat Diet-induced Non-alcoholic Hepatic Steatosis.

Non-alcoholic fatty liver disease (NAFLD) is one of the most common chronic liver diseases worldwide. Nevertheless, no first-line therapy exists. Hepatic steatosis is the earliest stage of NAFLD, which is characterized by an accumulation of hepatic lipids. Patchouli oil (PO), which is isolated from the well-known Chinese herb named Pogostemon cablin (Blanco) Benth. (Lamiaceae), inhibits hepatic lipid accumulation effectively. However, its potential ability for the treatment of NAFLD had not been reported before. Thus, the objective of this study was to investigate the effectiveness of PO against hepatic steatosis and its underlying mechanisms. We used a high fat diet (HFD)-induced hepatic steatosis model of rats to estimate the effect of PO against NAFLD. Hematoxylin-eosin and oil red O staining were used to analyze the hepatic histopathological changes. ELISA, RT-qPCR, and Western blotting analysis were applied to evaluate the parameters for hepatic steatosis. Our results showed that PO significantly attenuated the lipid profiles and the serum enzymes, evidenced by quantitative and histopathological analyses. It also markedly down-regulated the expression of sterol regulatory element-binding protein 1 (SREPB-1c) with its downstream factors in de novo lipogenesis. And, likewise, in lipid export by very low-density lipoproteins (VLDL), related molecules were dramatically improved. Furthermore, PO observably normalized the aberrant peroxisome proliferator-activated receptor α (PPAR-α) signal in fatty acids oxidation. In conclusion, PO exerted a preventing effect against HFD-induced steatosis and might be due to decrease de novo lipogenesis, promote export of lipids, as well as owing to improve fatty acids oxidation.

Huang-Lian-Jie-Du extract ameliorates atopic dermatitis-like skin lesions induced by 2,4-dinitrobenzene in mice via suppression of MAPKs and NF-κB pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Huang-Lian-Jie-Du Decoction (HLJDD), is a well-known traditional Chinese herbal formula first written in the Tang dynasty. In Chinese medicine practice, HLJDD is commonly prescribed to treat various inflammatory skin diseases, such as atopic dermatitis (AD) and psoriasis. AIM OF THE STUDY: The present study aimed at investigating the therapeutic effect of HLJDD extract (HLJDE) and to elucidate the underlying molecular mechanisms of action in the 1-chloro-2,4-dinitrobenzene (DNCB)-induced AD-like mice. MATERIALS AND METHODS: Female Balb/c mice were sensitized with DNCB for three days. After sensitization, mice were challenged with DNCB every three days and orally administrated with HLJDE (150, 300 and 600 mg/kg) daily from day 14 to day 29 for consecutive 16 days. At the end of experiment, the clinical AD scores of the mice were calculated to evaluate the therapeutic effect of HLJDE, and serum, ears and dorsal skin of the mice were collected for unravelling molecular mechanisms. RESULTS: HLJDE significantly reduced the clinical symptoms in the AD-like mice by inhibiting eosinophil and mast cell infiltration, suppressing the production of Th2-associated cytokine (IL-4) and pro-inflammatory cytokines (TNF-α). In addition, HLJDE significantly suppressed the NF-κB and MAPKs pathways. Moreover, HLJDE was able to accentuate filaggrin expression in the skin lesion when compared to the sensitized mouse without treatment. CONCLUSION: HLJDE significantly improved the AD-like symptoms on the DNCB-sensitized mice through mitigating the production of inflammatory mediators via suppressing MAPKs and NF-κB pathways. Additionally, the elevated expression of filaggrin in the skin lesion by HLJDE contributes to the recovery of dysfunctional skin barrier on the DNCB-sensitized mice.

Exploring brusatol as a new anti-pancreatic cancer adjuvant: biological evaluation and mechanistic studies.

Pancreatic cancer is highly resistant to chemotherapeutic agents and is known to have a poor prognosis. The development of new therapeutic entities is badly needed for this deadly malignancy. In this study, we demonstrated for the first time that brusatol, a natural quassinoid isolated from a Chinese herbal medicine named Bruceae Fructus, possessed potent cytotoxic effect against different pancreatic adenocarcinoma cell lines. Its anti-pancreatic cancer effect was comparable to that of the first-line chemotherapeutic agents such as gemcitabine and 5-fluorouracil, with a more favorable safety profile. In addition, brusatol showed a synergistic anti-proliferative effect toward PANC-1 and Capan-2 cell lines when combined with gemcitabine or 5-fluorouracil. The results of flow cytometry suggested that brusatol combination treatment with gemcitabine or 5-fluorouracil was able to cause cell cycle arrest at G2/M phase, and accentuate apoptosis in PANC-1 cells. Moreover, brusatol deactivated gemcitabine/5-fluorouracil-induced NF-κB activation. Western blot analysis and qRT-PCR results showed that brusatol significantly down-regulated the expression of vimentin and Twist, and markedly stimulated the expression of E-cadherin, the key regulatory factors of the epithelial-mesenchymal transition process. Furthermore, treatment with combination of brusatol and gemcitabine or 5-fluorouracil significantly reduced in vivo tumor growth when compared with treatment of either brusatol or gemcitabine/5-fluorouracil alone. Taken together, these results have amply demonstrated that brusatol is a potent anti-pancreatic cancer natural compound, and the synergistic anti-pancreatic cancer effects of brusatol and gemcitabine/5-fluorouracil observed both in vitro and in vivo are associated with the suppression of epithelial-mesenchymal transition process, indicating that brusatol is a promising adjunct to the current chemotherapeutic regimen.

Characterization of brusatol self-microemulsifying drug delivery system and its therapeutic effect against dextran sodium sulfate-induced ulcerative colitis in mice.

Brusatol (BR) is one of the main bioactive components derived from Brucea javanica, a medicinal herb historically used in the treatment of dysenteric disorders (also known as ulcerative colitis(UC)). Due to its poor aqueous solubility, a novel brusatol self-microemulsifying drug delivery system (BR-SMEDDS) nanoformulation with smaller size, higher negative zeta potential and drug content, and excellent stability was developed. The appearance of BR-SMEDDS remained clear and transparent, and transmission electron microscopy showed microemulsion droplets to be spherical with homogeneous distribution. Pharmacokinetic parameters indicated that oral bioavailability was greatly improved by BR-SMEDDS as compared with aqueous suspension. Meanwhile, the anti-colitis activity of BR-SMEDDS was evaluated on dextran sodium sulfate (DSS)-induced colitis mice model. The result illustrated that the nano-formation significantly reduced the body weight loss, recovered colon length, decreased disease activity index and microscopic score, regulated immune-inflammatory cytokines, diminished oxidative stress and repressed the colonic expression of myeloid differentiation factor 88 (MyD88), toll-like receptor 4 (TLR4) and nuclear factor kappa B p65 (NF-κB p65) proteins. Our findings demonstrated for the first time that BR could effectively attenuate colonic inflammation in mice, at least partially, via favorable regulation of anti-oxidative and anti-inflammatory status and inhibition of the TLR4-linked NF-κB signaling pathway. The BR nano-formulation was superior to BR suspension and sulphasalazine, in treating experimental UC, and exhibited similar effect with azathioprine, with much smaller dosage. The enhanced anti-UC effect of BR might be intimately associated with the improved pharmacokinetic property by SMEDDS. The developed nano-delivery system might thus be a promising candidate for colitis treatment.

Anti-inflammatory and anti-allergic effects and underlying mechanisms of Huang-Lian-Jie-Du extract: Implication for atopic dermatitis treatment.

ETHNOPHARMACOLOGICAL RELEVANCE: Huang-Lian-Jie-Du Decoction (HLJDD), a well-known Chinese herbal formula recorded in the Tang dynasty, is composed of Coptidis rhizoma (Huang-Lian), Scutellariae radix (Huang-Qin), Phellodendri Chinensis cortex (Huang-Bai) and Gardenia fructus (Zhi-Zi). It has clinical efficacy of purging fire for removing toxin and is commonly used for the treatment of disease including Alzheimer's disease, stroke and gastrointestinal disorders. HLJDD is also frequently applied for the treatment of various skin diseases, such as atopic dermatitis (AD) and various types of eczema. The aim of this study is to investigate the anti-inflammatory and anti-allergic actions of Huang-Lian-Jie-Du ethanolic extract (HLJDE) and to elucidate underlying molecular mechanisms of action using relevant in vitro experimental models. MATERIALS AND METHODS: The anti-inflammatory effects of HLJDE were investigated through evaluating the change of nitric oxide (NO) and the production of several cytokines and chemokines in lipopolysaccharide (LPS)-stimulated RAW264.7 cell line. Expression of mitogen-activated protein kinases (MAPKs), NF-κB p65 phosphorylation, inhibitor-κBα (IκBα) degradation were further investigated to elucidate its anti-inflammatory molecular mechanisms. Meanwhile, the anti-allergic activities of HLJDE was also evaluated using antigen-induced RBL-2H3 cell line. ß-hexosaminidase and histamine release and selected cytokines and chemokines were measured to evaluate the anti-allergic activities of HLJDE. In addition, intracellular Ca(2+)level, MAPKs and Lyn phosphorylation were further investigated to reveal its anti-allergic molecular mechanisms. RESULTS: HLJDE could significantly suppress the secretion of NO, IL-1ß, IL-4, MCP-1 and GM-CSF in RAW264.7 cells in a dose-dependent manner. In addition, HLJDE also markedly reduced the phosphorylation of MAPKs, and inhibited the transcriptional activity of NF-κB and IκBα degradation. Furthermore, HLJDE exerted marked anti-allergic activity through inhibiting the release of ß-hexosaminidase and histamine. The release of cytokines and chemokines (IL-4, TNF-α, MCP-1) from activated RBL-2H3 cells were also attenuated by pretreatment with HLJDE. The inhibitory effects on intracellular Ca(2+)level, and reduced phosphorylation of MAPKs and Lyn are believed to be the anti-allergic mechanisms. CONCLUSIONS: HLJDE exerted significant anti-inflammatory and anti-allergic effects through suppressing the production of allergic and inflammatory mediators via the NF-κB and MAPKs inactivation and IκBα degradation in the LPS-stimulated RAW24.7 cells, inactivation of MAPKs and Lyn pathway in antigen-induced RBL-2H3 cells. The present study provides in vitro experimental evidence to support the use of HLJDE for the clinical treatment of AD.

Protective effects of pogostone from Pogostemonis Herba against ethanol-induced gastric ulcer in rats.

We examined the protective effect of pogostone (PO), a chemical constituent isolated from Pogostemonis Herba, on the ethanol-induced gastric ulcer in rats. Administration of PO at doses of 10, 20 and 40 mg/kg body weight prior to ethanol ingestion effectively protected the stomach from ulceration. The gastric lesions were significantly ameliorated by all doses of PO as compared to the vehicle group. Pre-treatment with PO prevented the oxidative damage and the decrease of prostaglandin E2 (PGE2) content. In addition, PO pretreatment markedly increased the mucosa levels of glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT), and decreased gastric malonaldehyde (MDA), relative to the vehicle group. In the mechanistic study, significant elevation of non-protein-sulfhydryl (NP-SH) was observed in the gastric mucosa pretreated by PO. Analysis of serum cytokines indicated that PO pretreatment obviously elevated the decrease of interleukin-10 (IL-10) level, while markedly mitigated the increment of interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) secretions in ethanol-induced rats. Taken together, these results strongly indicate that PO could exert a gastro-protective effect against gastric ulceration, and the underlying mechanism might be associated with the stimulation of PGE2, improvement of antioxidant and anti-inflammatory status, as well as preservation of NP-SH.

Fingerprinting and simultaneous determination of alkaloids in Picrasma quassioides from different locations by high performance liquid chromatography with photodiode array detection.

A simple and sensitive method was developed and validated for profiling and simultaneous quantitation of seven alkaloids (6-hydroxy-ß-carboline-1-carboxylic acid, ß-carboline-1-carboxylic acid, ß-carboline-1-propanoic acid, 3-methylcanthin-5,6-dione, 5-hydroxy-4-methoxycanthin-6-one, 1-methoxycarbony-ß-carboline, and 4,5-dimethoxycanthin-6-one) in Picrasma quassioide grown in different locations by high-performance liquid chromatography with photodiode array detection. The analysis was conducted on a Phenomenex Gemini C(18) column at 35°C with mobile phase of 25 mM aqueous ammonium acetate (pH 4.0, adjusted by glacial acetate acid) and acetonitrile. A common fingerprint chromatograph under 254 nm consisting of 27 peaks was constructed for the evaluation of the similarities among 31 P. quassioide samples. Samples from Guangdong and Guangxi Provinces were found to be within group linkage and showed significant difference from that of Jiangxi Province origin by using principal component analysis and hierarchical clustering analysis. In addition, the seven alkaloids were identified by electrospray ionization mass spectrometry and comparing with reference standards and literature data. All of them were determined simultaneously using the established HPLC method. This rapid and effective analytical method could be employed for quality assessment of P. quassioide, as well as pharmaceutical products containing this herbal material.

[Fingerprint research and multi-component quantitative analysis of Kumu injection by HPLC].

OBJECTIVE: To establish the HPLC chromatographic fingerprint of Kumu injection and to simultaneously determine the contents of three beta-carboline alkaloids, comprehensively evaluating the immanent quality of Kumu injection. METHOD: The chromatographic analysis was performed on a Phenomenex Gemini C18 ( 4.6 mm x 250 mm, 5 microm) column with the gradient elution solvent system composed of methanol and 30 mmol x L(-1) aqueous ammonium acetate (adjusted with glacial acetic acid to pH 4.5). Similarity evaluation system for chromatographic fingerprint of traditional Chinese medicine (2004 A) was used in data analysis. RESULT: Sixteen co-possessing peaks were selected as the fingerprints of Kumu injection, and 7 peaks were identified by chemical reference substances. There were good similarities between the standard fingerprint chromatogram and each fingerprint chromatogram from the eleven samples for their similarity coefficients were not less than 0.9. Three kinds of beta-carboline alkaloids were separated well. The correlation coefficients were 0.999 9. The linear ranges of three components were 0.020 0-0.300 0, 0.102 0-1.530 0, 0.015 2-0. 228 0 microg, respectively, and the average recoveries ranged were from 99.5% to 102%. CONCLUSION: The method of fingerprint combined with quantitative analysis is sensitive, selective, and provide scientific basis for quality control of Kumu Injection.