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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.

Glioblastoma is one of the most common and lethal intracranial malignant, and is still lack of ideal treatments. Kaempferol is a major nutrient found in various edible plants, which has exhibited the potential for the treatment of glioblastoma. However, the specific anti-glioma mechanism of kaempferol is yet to be studied. Herein, we aim to explore the mechanisms underlying the anti-glioma activity of kaempferol. Our results demonstrated that kaempferol suppresses glioma cell proliferation in vitro and inhibits tumor growth in vivo. Moreover, kaempferol raises ROS and decreases mitochondrial membrane potential in glioma cells. The high levels of ROS induce autophagy then ultimately trigger the pyroptosis of glioma cells. Interestingly, when we used 3-MA to inhibit autophagy, we found that the cleaved form of GSDME was also decreased, suggesting that kaempferol induces pyroptosis through regulating autophagy in glioma cells. In conclusion, this study revealed kaempferol possesses good anti-glioma activity by inducing ROS, and subsequently leads to autophagy and pyroptosis, highlighting its clinical potentials as a natural nutrient against glioblastoma.

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.

// Zheng Lu 1, 2, 4 , Zheng-Quan Lai 1 , Albert W.N. Leung 1 , Po Sing Leung 3 , Zhao-Shen Li 2 and Zhi-Xiu Lin 1 1 School of Chinese Medicine, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, China 2 Department of Gastroenterology, Changhai Hospital, The Second Military Medical University, Shanghai, China 3 School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, China 4 Liver Cirrhosis Diagnosis and Treatment Center, Beijing 302 Hospital, Beijing, China Correspondence to: Zhi-Xiu Lin, email: linzx@cuhk.edu.hk Keywords: pancreatic cancer, brusatol, gemcitabine, 5-fluorouracil, combination therapy Received: September 24, 2016 Accepted: April 17, 2017 Published: May 10, 2017 ABSTRACT 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.

(-)-Patchouli alcohol (PA), the major active principle of Pogostemonis Herba, has been reported to have anti-Helicobacter pylori and gastroprotective effects. In the present work, we aimed to investigate the possible protective effect of PA on H. pylori urease (HPU)-injured human gastric epithelial cells (GES-1) and to elucidate the underlying mechanisms of action. Results showed that pre-treatment with PA (5.0, 10.0, 20.0μM) was able to remarkably ameliorate the cytotoxicity induced by 17.0U/mg HPU in GES-1 cells. Flow cytometric analysis on cellular apoptosis showed that pre-treatment with PA effectively attenuated GES-1 cells from the HPU-induced apoptosis. Moreover, the cytoprotective effect of PA was found to be associated with amelioration of the HPU-induced disruption of MMP, attenuating oxidative stress by decreasing contents of intracellular ROS and MDA, and increasing superoxide dismutase (SOD) and catalase (CAT) enzymatic activities. In addition, pre-treatment with PA markedly attenuated the secretion of nitric oxide (NO) and pro-inflammatory cytokines such as interleukin-2 (IL-2), interleukin-4 (IL-4) and tumor necrosis factor-α (TNF-α), whereas elevated the anti-inflammatory cytokine interleukin-13 (IL-13) in the HPU-stimulated GES-1 cells. Molecular docking assay suggested that PA engaged in the active site of urease bearing nickel ions and interacted with important residues via covalent binding, thereby restricting the active urease catalysis conformation. Our experimental findings suggest that PA could inhibit the cellular processes critically involved in the pathogenesis of H. pylori infection, and its protective effects against the HPU-induced cytotoxicity in GES-1 cells are believed to be associated with its anti-apoptotic, antioxidative, anti-inflammatory and HPU inhibitory actions.

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.

Colorectal cancer (CRC) is a common and life-threatening type of malignant cancer, which is associated with a high mortality rate. Cisplatin (CDDP) is a commonly used chemotherapy drug with significant side effects. Brusatol (BR) is one of the principal chemical compounds isolated from the Chinese herb Bruceae Fructus, which has been reported to markedly inhibit the proliferation of numerous cancer cell lines. The present study aimed to investigate the possible synergistic anticancer effects of CDDP combined with BR on CT-26 cells, and to evaluate the underlying mechanisms of action. The growth inhibitory effects of BR, CDDP, and BR and CDDP cotreatment on CT-26 cells were assessed by MTT assay. Cell apoptosis were determined by flow cytometry and western blot analysis. The results indicated that compared with single-agent treatment, cotreatment of CT-26 cells with CDDP and BR synergistically inhibited cell proliferation and increased cellular apoptosis. Furthermore, treatment of CT-26 cells with CDDP and BR resulted in a marked increase in the release of cytosolic cytochrome c, decreased expression of procaspase-3 and procaspase-9, and upregulation of the B-cell lymphoma 2 (Bcl-2)-associated X protein/Bcl-2 ratio compared with treatment with BR or CDDP alone. These results strongly suggested that the combination of CDDP and BR was able to produce a synergistic antitumor effect in CRC cells, thus providing a solid foundation for further development of this combination regimen into an effective therapeutic method for CRC.

β-Patchoulene (β-PAE) is a tricyclic sesquiterpene isolated from patchouli oil. According to our previous study, β-PAE has anti-inflammatory activity in vivo; however, its anti-inflammatory response still remains unconfirmed in vitro. Therefore, this study is committed to demonstrate the anti-inflammatory effect of β-PAE on lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. According to our results, pre-treatment with β-PAE significantly decreased the protein and messenger RNA (mRNA) levels of pro-inflammatory cytokines including tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and IL-1β while increased the expressions of anti-inflammatory cytokines like IL-10 in a dose-dependent manner. In addition, real-time polymerase chain reaction (PCR) also revealed that β-PAE could interrupt the mRNA expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) and thus decreased the levels of nitric oxide (NO) and prostaglandin E2 (PGE2) in LPS-stimulated RAW264.7 macrophages. In conclusion, these results indicated that β-PAE exerted potent anti-inflammatory activity by maintaining the balance between pro- and anti-inflammatory cytokines as well as suppressing iNOS and COX-2 signaling pathways.

A new alkaloid, 6-hydroxy-β-carboline-1-carboxylic acid (1), together with six known alkaloids, including β-carboline-1-carboxylic acid (2), β-carboline-1-propanoic acid (3), 1-methoxycarbonyl-β-carboline (4), 3-methylcanthin-5,6-dione (5), 5-hydroxy-4-methoxycanthin-6-one (6), and 4,5-dimethoxycanthin-6-one (7), were isolated from the stem of Picrasma quassioides. Their structures were elucidated on the basis of spectroscopic data. Compounds 1–7 were screened for their cytotoxic activities against five cancer cell lines such as CT26.WT, K-562, SGC-7901, Hep G2, and A-549. Among the seven alkaloids tested, only compound 2 exhibited moderate inhibitory activities against K-562 and SGC-7901 cell lines.

A simple and sensitive method was developed and validated for profiling and simultaneous quantitation of seven alkaloids (6-hydroxy-β-carboline-1-carboxylic acid, β-carboline-1-carboxylic acid, β-carboline-1-propanoic acid, 3-methylcanthin-5,6-dione, 5-hydroxy-4-methoxycanthin-6-one, 1-methoxycarbony-β-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.

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. β-hexosaminidase and histamine release and selected cytokines and chemokines were measured to evaluate the anti-allergic activities of HLJDE. In addition, intracellular Ca2+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-1β, 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 β-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 Ca2+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.

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.

The transcription factor Nrf2 regulates the basal and inducible expression of a battery of cytoprotective genes. Whereas numerous Nrf2-inducing small molecules have been reported, very few chemical inhibitors of Nrf2 have been identified to date. The quassinoid brusatol has recently been shown to inhibit Nrf2 and ameliorate chemoresistance in vitro and in vivo. Here, we show that brusatol provokes a rapid and transient depletion of Nrf2 protein, through a posttranscriptional mechanism, in mouse Hepa-1c1c7 hepatoma cells. Importantly, brusatol also inhibits Nrf2 in freshly isolated primary human hepatocytes. In keeping with its ability to inhibit Nrf2 signaling, brusatol sensitizes Hepa-1c1c7 cells to chemical stress provoked by 2,4-dinitrochlorobenzene, iodoacetamide, and N-acetyl-p-benzoquinone imine, the hepatotoxic metabolite of acetaminophen. The inhibitory effect of brusatol toward Nrf2 is shown to be independent of its repressor Keap1, the proteasomal and autophagic protein degradation systems, and protein kinase signaling pathways that are known to modulate Nrf2 activity, implying the involvement of a novel means of Nrf2 regulation. These findings substantiate brusatol as a useful experimental tool for the inhibition of Nrf2 signaling and highlight the potential for therapeutic inhibition of Nrf2 to alter the risk of adverse events by reducing the capacity of nontarget cells to buffer against chemical and oxidative insults. These data will inform a rational assessment of the risk:benefit ratio of inhibiting Nrf2 in relevant therapeutic contexts, which is essential if compounds such as brusatol are to be developed into efficacious and safe drugs., Highlights • Nrf2 regulates the expression of numerous cytoprotective genes. • We show that the quassinoid brusatol rapidly and transiently inhibits Nrf2 signaling. • Brusatol sensitizes mammalian cells to chemical stress provoked by electrophiles. • Brusatol inhibits Nrf2 independent of its key regulatory mechanisms. • Therapeutic inhibition of Nrf2 could enhance drug-induced adverse effects.

A simple and sensitive method was developed and validated for profiling and simultaneous quantitation of seven alkaloids (6-hydroxy-β-carboline-1-carboxylic acid, β-carboline-1-carboxylic acid, β-carboline-1-propanoic acid, 3-methylcanthin-5,6-dione, 5-hydroxy-4-methoxycanthin-6-one, 1-methoxycarbony-β-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.

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.

Purpose: To develop and validate a simple and selective high performance liquid chromatography photo diode array mass spectrometry (HPLC-PDA-MS/MS) method for simultaneous determination and confirmation of seven major active alkaloids (6-Hydroxy-ß-Carboline-1-carboxylic acid, ß-Carboline-1- carboxylic acid, ß-Carboline-1-propanoic acid, 3-Methylcanthin-5,6-dione, 4-Methoxy-3-methylcanthine- 5,6-dione, 5-Hydroxy-4-methoxycanthin-6-one, 4,5-Dimethoxycanthin-6-one) in Kumu injections (KMIs)Methods: For the analysis of the preparation, the optimal chromatographic condition was achieved on a Phenomenex Gemini C18 column with gradient elution of 25 mM aqueous ammonium acetate (pH = 4.0 adjusted by glacial acetate acid) and acetonitrile with flow rate at 1.0 mL/min, column temperature at 35oC and detection wavelengths at 245, 260 and 271 nm.Results: Excellent linear behavior over the investigated concentration ranges was observed with regression coeffcient (R2) > 0.9997 for all analytes. Intra- and inter-day precisions for all studied constituents ranged from 0.20 to 1.80 %. Recoveries of the assayed constituents were in the range of 98.73 to 100.34 %. The results showed the contents of these seven marker compounds differed significantly among different batches of KMIs both from the same and different manufacturers.Conclusion: The validated method was reliable, accurate, repeatable and can be applied to routine quality assessment of these active components in KMIs.Keywords: Alkaloids, High performance liquid chromatography, Photo diode array, Mass spectrometry, Kumu injection, Quality control