High-throughput screening of ancient forest plant extracts shows cytotoxicity towards triple-negative breast cancer.

According to the World Health Organization, women's breast cancer is among the most common cancers with 7.8 million diagnosed cases during 2016-2020 and encompasses 15 % of all female cancer-related mortalities. These mortality events from triple-negative breast cancer are a significant health issue worldwide calling for a continuous search of bioactive compounds for better cancer treatments. Historically, plants are important sources for identifying such new bioactive chemicals for treatments. Here we use high-throughput screening and mass spectrometry analyses of extracts from 100 plant species collected in Chinese ancient forests to detect novel bioactive breast cancer phytochemicals. First, to study the effects on viability of the plant extracts, we used a MTT and CCK-8 cytotoxicity assay employing triple-negative breast cancer (TNBC) MDA-MB-231 and normal epithelial MCF-10A cell lines and cell cycle arrest to estimate apoptosis using flow cytometry for the most potent three speices. Based on these analyses, the final most potent extracts were from the Amur honeysuckle (Lonicera maackii) wood/root bark and Nigaki (Picrasma quassioides) wood/root bark. Then, 5 × 106 MDA-MB-231 cells were injected subcutaneously into the right hind leg of nude mice and a tumour was allowed to grow before treatment for seven days. Subsequently, the four exposed groups received gavage extracts from Amur honeysuckle and Nigaki (Amur honeysuckle wood distilled water, Amur honeysuckle root bark ethanol, Nigaki wood ethanol or Nigaki root bark distilled water/ethanol (1:1) extracts) in phosphate-buffered saline (PBS), while the control group received only PBS. The tumour weight of treated nude mice was reduced significantly by 60.5 % within 2 weeks, while on average killing 70 % of the MDA-MB-231 breast cancer cells after 48 h treatment (MTT test). In addition, screening of target genes using the Swiss Target Prediction, STITCH, STRING and NCBI-gene database showed that the four plant extracts possess desirable activity towards several known breast cancer genes. This reflects that the extracts may kill MBD-MB-231 breast cancer cells. This is the first screening of plant extracts with high efficiency in 2 decades, showing promising results for future development of novel cancer treatments.

Host ALDH2 deficiency aggravates nonalcoholic steatohepatitis through gut-liver axis.

Nonalcoholic steatohepatitis (NASH) is the major cause of liver dysfunction. Animal and population studies have shown that mitochondrial aldehyde dehydrogenase (ALDH2) is implicated in fatty liver disease. However, the role of ALDH2 in NASH and the underlying mechanisms remains unclear. To address this issue, ALDH2 knockout (ALDH2-/-) mice and wild-type littermate mice were fed a methionine-and choline-deficient (MCD) diet to induce a NASH model. Fecal, serum, and liver samples were collected and analyzed to investigate the impact of the gut microbiota and bile acids on this process. We found that MCD-fed ALDH2-/- mice exhibited increased serum pro-inflammation cytokines, hepatic inflammation and fat accumulation than their wild-type littermates. MCD-fed ALDH2-/- mice exhibited worsened MCD-induced intestinal inflammation and barrier damage, and gut microbiota disorder. Furthermore, mice receiving microbiota from MCD-fed ALDH2-/- mice had increased severity of NASH compared to those receiving microbiota from MCD-fed wild-type mice. Notably, the intestinal Lactobacillus was significantly reduced in MCD-fed ALDH2-/- mice, and gavage with Lactobacillus cocktail significantly improved MCD-induced NASH. Finally, we found that ALDH2-/- mice had reduced levels of bile salt hydrolase and specific bile acids, especially lithocholic acid (LCA), accompanied by downregulated expression of the intestinal FXR-FGF15 pathway. Supplementation of LCA in ALDH2-/- mice upregulated intestinal FXR-FGF15 pathway and alleviated NASH. In summary, ALDH2 plays a critical role in the development of NASH through modulation of gut microbiota and bile acid. The findings suggest that supplementing with Lactobacillus or LCA could be a promising therapeutic approach for treating NASH exacerbated by ALDH2 deficiency.

Systems pharmacology-based mechanism exploration of Acanthopanax senticosusin for Alzheimer's disease using UPLC-Q-TOF-MS, network analysis, and experimental validation.

BACKGROUND: Alzheimer's disease (AD) is a neurodegenerative disease, characterized by progressive cognitive dysfunction and memory loss. However, the disease-modifying treatments for AD are still lacking. Traditional Chinese herbs, have shown their potentials as novel treatments for complex diseases, such as AD. PURPOSE: This study was aimed at investigating the mechanism of action (MOA) of Acanthopanax senticosusin (AS) for treatment of AD. METHODS: In this study, we firstly identified the chemical constituents in Acanthopanax senticosusin (AS) utilizing ultra-high performance liquid chromatography coupled with Q-TOF-mass spectrometry (UPLC-Q-TOF-MS), and next built the drug-target network of these compounds. We also performed the systems pharmacology-based analysis to preliminary explore the MOA of AS against AD. Moreover, we applied the network proximity approach to identify the potential anti-AD components in AS. Finally, experimental validations, including animal behavior test, ELISA and TUNEL staining, were conducted to verify our systems pharmacology-based analysis. RESULTS: 60 chemical constituents in AS were identified via the UPLC-Q-TOF-MS approach. The systems pharmacology-based analysis indicated that AS might exert its therapeutic effects on AD via acetylcholinesterase and apoptosis signaling pathway. To explore the material basis of AS against AD, we further identified 15 potential anti-AD components in AS. Consistently, in vivo experiments demonstrated that AS could protect cholinergic nervous system damage and decrease neuronal apoptosis caused by scopolamine. CONCLUSION: Overall, this study applied systems pharmacology approach, UPLC-Q-TOF-MS, network analysis, and experimental validation to decipher the potential molecular mechanism of AS against AD.

Covalent Modification of Proteins by Osthole Reactive Metabolites using Proteomic Approaches.

BACKGROUND: Osthole (OST) is a bioactive natural coumarin derived from the plant Cnidium monnieri (L.) Cusson fruit (She Chuang Zi), which has various pharmacological and biological activities. OST contains an α,ß- unsaturated lactone, which is an electrophilic group that tends to be metabolized into reactive metabolites (RMs). Then, RMs are able to covalently modify nucleophilic amino acid (AA) residues of target proteins. However, few researchers considered the contribution of the covalent modification induced by OST or its metabolites. OBJECTIVE: This study aims to investigate the metabolic profile and the metabolites-protein modification of OST. METHODS: The metabolites of OST were qualitatively identified using UHPLC-Q-TOF-MS. The RMs modification patterns and potentially modified AA residues were confirmed by UHPLC-Q-TOF-MS using rat liver microsomes (RLMs) and model AAs. Finally, the modified peptides derived from high-abundance microsomal peptides were separated via nano-LC-Orbitrap-MS, and then RM-modified proteins were identified using a proteome discoverer. RESULTS: In the presence of RLMs, OST could rapidly be metabolized within 1 h and hardly identified at 4 h. We detected 10 OST metabolites, 13 OST metabolites-NAC (N-acetyl cysteine) adducts, 3 NAL (N-acetyl lysine) adducts, and 11 GSH (glutathione) adducts. Furthermore, 16 RM-modified protein targets were identified, many of which are included in the essential biological processes of OST's anti-Alzheimer's disease (AD) and anti-tumor. CONCLUSION: This study provides a novel perspective on the molecular mechanism of OST's pharmacological activities, as well as identifies potential targets for further development and application of OST and other Natural products (NPs).

Ferulic acid prevents Diosbulbin B-induced liver injury by inhibiting covalent modifications on proteins.

Diosbulbin B (DIOB) has been reported to cause serious liver injury. However, in traditional medicine, DIOB-containing herbs are highly safe in combination with ferulic acid (FA)-containing herbs, suggesting potential neutralizing effect of FA on the toxicity of DIOB. DIOB can be metabolized to generate reactive metabolites (RMs), which can covalently bind to proteins and lead to hepatoxicity. In the present study, the quantitative method was firstly established for investigating the correlation between DIOB RM-protein adducts (DRPAs) and hepatotoxicity. Then, we estimated the detoxication effect of FA in combination with DIOB and revealed the underlying mechanism. Our data indicated that the content of DRPAs positively correlate with the severity of hepatotoxicity. Meanwhile, FA is able to reduce the metabolic rate of DIOB in vitro. Moreover, FA suppressed the production of DRPAs and decreased the serum alanine/aspartate aminotransferase (ALT/AST) levels elevated by DIOB in vivo. Thus, FA can ameliorate DIOB-induced liver injury through reducing the production of DRPAs.

Combination of chemical profiling and network pharmacology analysis to investigate the potential mechanism of Li-Zhong-Xiao-Pi granules in the treatment of gastric precancerous lesions.

Li-Zhong-Xiao-Pi granules (LZXP) are effective for treating gastric precancerous lesions (GPL) in traditional Chinese medicine. However, the active compounds of LZXP and their potential therapeutic mechanism in GPL remained unclarified. The purpose of this study is to investigate the chemical composition and potential targets of LZXP. Based on the accurate masses, ion fragments, and literature data, a total of 128 compounds were identified in the LZXP sample using ultra-performance liquid chromatography and quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) in both positive and negative ion modes, and 28 of these compounds were exactly determined by comparison with authentic reference standards. Meanwhile, 11 typical components were quantified via UPLC during a 24 min period. The linearity, accuracy, stability and recovery of the method were all proven. Through the network pharmacological analysis, six chemicals (quercetin, 4'-hydroxywogonin, sinensetin, 5, 7, 8, 3', 4'-pentamethoxyflavanone, 8-gingerdione and quercetin) were identified as the active ingredients, and five LZXP targets (AKT1, CYP1B1, PTGS2, MMP9 and EGFR) were found to be the crucial molecules in the treatment of GPL. This study provides a systematic and applicable method for the rapid screening and identification of the chemical constituents from LZXP, and an effective understanding the mechanism of LZXP in the treatment of GPL.

Ample dietary fat reduced the risk of primary vesical calculi by inducing macrophages to engulf budding crystals in mice.

Although primary vesical calculi is an ancient disease, the mechanism of calculi formation remains unclear. In this study, we established a novel primary vesical calculi model with d,l-choline tartrate in mice. Compared with commonly used melamine and ethylene glycol models, our model was the only approach that induced vesical calculi without causing kidney injury. Previous studies suggest that proteins in the daily diet are the main contributors to the prevention of vesical calculi, yet the effect of fat is overlooked. To assay the relationship of dietary fat with the formation of primary vesical calculi, d,l-choline tartrate-treated mice were fed a high-fat, low-fat, or normal-fat diet. Genetic changes in the mouse bladder were detected with transcriptome analysis. A high-fat diet remarkably reduced the morbidity of primary vesical calculi. Higher fatty acid levels in serum and urine were observed in the high-fat diet group, and more intact epithelia in bladder were observed in the same group compared with the normal- and low-fat diet groups, suggesting the protective effect of fatty acids on bladder epithelia to maintain its normal histological structure. Transcriptome analysis revealed that the macrophage differentiation-related gene C-X-C motif chemokine ligand 14 (Cxcl14) was upregulated in the bladders of high-fat diet-fed mice compared with those of normal- or low-fat diet-fed mice, which was consistent with histological observations. The expression of CXCL14 significantly increased in the bladder in the high-fat diet group. CXCL14 enhanced the recruitment of macrophages to the crystal nucleus and induced the transformation of M2 macrophages, which led to phagocytosis of budding crystals and prevented accumulation of calculi. In human bladder epithelia (HCV-29) cells, high fatty acid supplementation significantly increased the expression of CXCL14. Dietary fat is essential for the maintenance of physiological functions of the bladder and for the prevention of primary vesical calculi, which provides new ideas for the reduction of morbidity of primary vesical calculi.

Radix Tetrastigma Extracts Enhance the Chemosensitivity in Triple-Negative Breast Cancer Via Inhibiting PI3K/Akt/mTOR-Mediated Autophagy.

OBJECTIVE: Drug resistance in tumors is one of the major factors that leads to chemotherapy failure. This study aims to investigate the effect of Radix Tetrastigma extracts (RTEs) on Taxol-induced autophagy and the chemosensitivity against drug resistance in triple-negative breast cancer (TNBC). METHODS: Taxol-resistant MDA-MB-468 (MDA-MB-468/Taxol) cells were induced and treated with RTEs and/or Taxol. Mice were subcutaneously inoculated with MDA-MB- 468/Taxol cells to establish xenograft models. The associated protein levels were measured by western blotting. Flow cytometry, CCK-8 and EdU assay were performed to detect cell apoptosis, viability, and proliferation, respectively. RESULTS: In MDA-MB-468/Taxol cells, RTEs & Taxol treatment increased cell apoptosis, reduced cell viability and proliferation, up-regulated anti-autophagy marker LC3I/LC3II ratio, and enhanced mTOR level. With RTEs & Taxol treatment, mTOR silencing downregulated LC3I/LC3II ratio, increased cell viability and proliferation, and reduced cell apoptosis, while mTOR overexpression showed the opposite results. PI3K inhibitor reduced AKT and mTOR levels, and the effects on cell activities were similar to the results of mTOR silencing. After RTEs & Taxol injection, xenograft tumor was smaller, and AKT, mTOR, LC3I/LC3II ratio and apoptotic marker cleaved caspase-3 were increased. CONCLUSION: RTEs enhanced the chemosensitivity of resistant TNBC cells to Taxol through inhibiting PI3K/Akt/mTOR-mediated autophagy. MICRO: RTEs exerted anti-tumor effects in various cancers, and this study determined its role in TNBC. Taxol-resistant MDA-MB-468 cells were induced and xenograft models were established. We found that RTEs inhibited autophagy of MDA-MB-468/Taxol cells and reduced tumor growth. Inhibition of PI3K/Akt/mTOR pathway promoted autophagy of MDA-MB-468/Taxol cells. We may provide a new potential strategy for TNBC treatment.

A triterpenoid-enriched extract of bitter melon leaves alleviates hepatic fibrosis by inhibiting inflammatory responses in carbon tetrachloride-treated mice.

Liver fibrosis is a progression of chronic liver disease characterized by excess deposition of fibrillary collagen. The aim of this study was to investigate the protective effect of a triterpenoid-enriched extract (TEE) from bitter melon leaves against carbon tetrachloride (CCl4)-induced hepatic fibrosis in mice. Male ICR mice received TEE (100 or 150 mg kg-1) by daily oral gavage for one week before starting CCl4 administration and throughout the entire experimental period. After intraperitoneal injection of CCl4 for nine weeks, serum and liver tissues of the mice were collected for biochemical, histopathological and molecular analyses. Our results showed that TEE supplementation reduced CCl4-induced serum aspartate aminotransferase and alanine aminotransferase activities. Histopathological examinations revealed that CCl4 administration results in hepatic fibrosis, while TEE supplementation significantly suppressed hepatic necroinflammation and collagen deposition. In addition, TEE supplementation decreased α-smooth muscle actin (α-SMA)-positive staining and protein levels of α-SMA and transforming growth factor-ß1. TEE-supplemented mice had lower mRNA expression levels of interleukin-6, tumor necrosis factor-α, and toll-like receptor 4. Moreover, TEE (150 mg kg-1) supplementation significantly reduced intrahepatic inflammatory Ly6C+ monocyte infiltration. We demonstrated that TEE could ameliorate hepatic fibrosis by regulating inflammatory cytokine secretion and α-SMA expression in the liver to reduce collagen accumulation.

Pectin supplement significantly enhanced the anti-PD-1 efficacy in tumor-bearing mice humanized with gut microbiota from patients with colorectal cancer.

Background: Anti-PD-1-based immunotherapy has emerged as a promising therapy for several cancers. However, it only benefits a small subset of colorectal cancer (CRC) patients. Mounting data supports the pivotal role of gut microbiota in shaping immune system. Pectin, a widely consumed soluble fiber, has been reported to ameliorate the imbalance of gut microbiota. Therefore, we aimed to explore the effect and the underlying mechanisms of pectin in improving anti-PD-1 mAb efficacy. Methods: The C57BL/6 mice were treated with a broad-spectrum antibiotic (ATB) cocktail to depleted endogenous gut microbiota and subsequently humanized with feces from healthy controls or newly diagnosed CRC patients. The antitumor efficacies of anti-PD-1 mAb combined with or without pectin were assessed using these mice. Flow cytometry and immunohistochemistry (IHC) were conducted to investigate the tumor immune microenvironment after treatment. The gut microbiota profiles and short-chain fatty acids (SCFAs) levels were determined by 16S ribosomal RNA (16S rRNA) gene sequencing and gas chromatography-mass spectrometry (GC-MS), respectively. The effect of gut microbiota on anti-PD-1 mAb efficacy after pectin supplement was further tested by fecal microbiota transplantation (FMT). Results: The anti-PD-1 mAb efficacy was largely impaired in the mice humanized with feces from newly diagnosed CRC patients compared to those from healthy controls. However, pectin significantly enhanced the anti-PD-1 mAb efficacy in the tumor-bearing mice humanized with CRC patient gut microbiota. Flow cytometry and IHC analysis revealed increased T cell infiltration and activation in the tumor microenvironment of mice treated with anti-PD-1 mAb plus pectin. In vivo depletion of CD8+ T cells diminished the anti-tumor effect of anti-PD-1 mAb combined with pectin. 16S rRNA gene sequencing showed that pectin significantly increased gut microbial diversity and beneficially regulated microbial composition. In addition, we identified unique bacterial modules that were significantly enriched in the anti-PD-1 mAb + pectin group, which composed of butyrate-producing bacteria indicative of good response to immunotherapy. Meanwhile, GC-MS showed that pectin altered the level of SCFA butyrate. Furthermore, butyrate, a main product of dietary fiber in gut microbial fermentation, was found to be sufficient to promote T cells infiltration and thus enhance the efficacy of anti-PD-1 mAb. In addition, FMT demonstrated the effects of pectin were dependent on gut microbiota. Importantly, the beneficial effects of pectin were confirmed in the mice humanized with gut microbiota from patient with resistance to anti-PD-1 mAb. Conclusion: Pectin facilitated the anti-PD-1 mAb efficacy in CRC via regulating the T cell infiltration in the tumor microenvironment, which was potentially mediated by the metabolite butyrate.

The effects and safety of omega-3 fatty for acute lung injury: a systematic review and meta-analysis.

BACKGROUND: Several randomized controlled trials (RCTs) have compared the treatment of acute lung injury (ALI) with omega-3 fatty, yet the results remained inconsistent. Therefore, we attempted this meta-analysis to analyze the role of omega-3 fatty in the treatment of ALI patients. METHODS: We searched PubMed databases from inception date to October 31, 2019, for RCTs that compared the treatment of ALI with or without omega-3 fatty. Two authors independently screened the studies and extracted data from the published articles. Summary mean differences (MD) with 95% confidence intervals (CI) were calculated for each outcome by fixed- or random-effects model. RESULTS: Six RCTs with a total of 277 patients were identified, of whom 142 patients with omega-3 fatty acid treatment and 135 patients without omega-3 fatty treatment. Omega-3 fatty treatments significantly improve the PaO2 (MD = 13.82, 95% CI 8.55-19.09), PaO2/FiO2 (MD = 33.47, 95% CI 24.22-42.72), total protein (MD = 2.02, 95% CI 0.43-3.62) in ALI patients, and omega-3 fatty acid treatments reduced the duration of mechanical ventilation (MD = - 1.72, 95% CI - 2.84 to - 0.60) and intensive care unit stay (MD = - 1.29, 95% CI - 2.14 to - 0.43) in ALI patients. CONCLUSIONS: Omega-3 fatty can effectively improve the respiratory function and promote the recovery of ALI patients. Future studies focused on the long-term efficacy and safety of omega-3 fatty use for ALI are needed.

Comparative effectiveness of different forms of traditional Chinese medicine for treatment of post-stroke depression: Protocol for network meta-analysis of randomized controlled trials.

BACKGROUND: Traditional Chinese medicine (TCM) therapy is effective for post-stroke depression (PSD). TCM therapy encompasses various forms of practices. However, the comparative effectiveness of these therapies is still not clear. Here, we provide a network meta-analysis protocol to compare the effects of different types of TCM therapy on PSD, using both direct and indirect evidence. METHODS: Twelve databases investigation will be conducted through the keywords from their inception to June 1, 2019. At least 2 independent reviewers will identify eligible articles. EndNote X7 software is utilized to manage the literatures and RevMan V.5.3 (The Cochrane Collaboration) software is for data processing throughout the review. The package "netmeta" (version 0.5-0) in R (version 3.0.2, The R Foundation for Statistical Computing) will be used to perform network meta-analysis (NMA). In addition, the overall quality of evidence is evaluated by GRADEPro software, and Cochrane Collaboration Risk of Bias Tool is employed for the methodological quality. Generally speaking, this review protocol is reported according to the preferred reporting items for systematic review and meta-analysis protocols 2015 guidelines. RESULTS: According to this protocol, it will provide evidence in support of, or against, the hypothesis that TCM therapy for PSD is more effective than pharmacotherapy. The results of this study will also provide evidence on relative efficacy of different forms of TCM. Furthermore, this analysis will show which form(s) of TCM therapy is (are) the most effective. CONCLUSION: The results will help PSD doctors and patients choose the treatment regimen which is effective, time-saving and economical. PROSPERO REGISTRATION NUMBER: CRD42016041594.

Generation and characterization of a monoclonal antibody against human BCL6 for immunohistochemical diagnosis.

BACKGROUND: Human B-cell lymphoma 6 (BCL6) gene, usually coding protein of 706 amino acids, is closely associated with large B cell lymphoma. Researches showed that protein mutation or change of expression levels usually happened in the mounting non-hodgkin lymphoma (NHL). Thus BCL6 is considered to be involved in germinal center (GC)-derived lymphoma. RESULTS: The BCL61-350 gene codons were optimized for prokaryotic system. After expression of BCL61-350 in E. coli, the BCL61-350 protein was purified with Ni column. Then the BCL61-350 protein, mixing with QuickAntibody-Mouse5W adjuvant, was injected into Balb/c mice. After immunization and cell fusion, a stable cell line named 1E6A4, which can secrete anti-BCL6 antibody, was obtained. The isotype of 1E6A4 mAb was determined as IgG2a, and the affinity constant reached 5.12×1010 L/mol. Furthermore, the specificity of the mAb was determined with ELISA, western blot and immunohistochemistry. Results indicated that the 1E6A4 mAb was able to detect BCL6 specifically and sensitively. CONCLUSIONS: BCL61-350 antigen has been successfully generated with an effective and feasible method, and a highly specific antibody named 1E6A4 against BCL6 has been screened and characterized in this study, which was valuable in clinical diagnosis.

The Chinese Herbal Formula PAPZ Ameliorates Behavioral Abnormalities in Depressive Mice.

Major depressive disorder (MDD) is a chronic mental disorder characterized by mixed symptoms and complex pathogenesis. With long history of practical application, traditional Chinese medicine (TCM) offers many herbs for the treatment and rehabilitation of chronic disease. In this study, we developed a modified Chinese herbal formula using Panax ginseng, Angelica Sinensis, Polygala tenuifolia Willd, and Ziziphi spinosae Semen (PAPZ), based on an ancient TCM prescription. The antidepressant effects of PAPZ were investigated with a corticosterone (CORT) model of depression in mice. Our results showed that administration of PAPZ ameliorated depression-like phenotypes in the CORT model. An anatomic study showed that chronic PAPZ administration upregulated the protein expression of brain-derived neurotrophic factor (BDNF) in hippocampal tissue. The enzyme activity of superoxide dismutase was enhanced in hippocampal tissue, in line with a decreased malondialdehyde level. Taken together, these findings suggested that PAPZ has therapeutic effects in a mice depression model through increasing protein expression of BDNF and improving the anti-oxidation ability of the brain.

Naoxuekang, Xinnaoshutong and Xuesaitong capsules for treating stroke: a protocol for a randomised controlled trial.

INTRODUCTION: After stroke, hemiplegia, dysphasia and facial paralysis can manifest during the convalescent period. Currently, no Chinese patent medicine (CPM) is previously reported to cure each of these symptoms primarily, and thus, there are no relevant instructions for the use of CPM. This study presents a new approach based on comparative effectiveness research to distinguish the curative effects of three CPMs that are often used in stroke convalescence to determine the ideal medicine for the treatment of each symptom. METHODS AND ANALYSIS: In this multicentre and double-blind clinical trial, stratified randomisation is used to group the patients according to their primary symptoms (hemiplegia, dysphasia and facial paralysis). Three strata will be enrolled, with 80 eligible participants included in each stratum. Each stratum will be randomly and equally divided into four groups, and each group will receive one of the following treatments: Naoxuekang, Xinnaoshutong (XNST), Xuesaitong (XST) or placebo. This study will include two stages: the initial treatment period (30 days) and a follow-up period (180 days). Three replicates for each data point will be completed during this trial. The first visit will occur on day 0 after enrolment, the second visit on day 30±2 and the third visit on day 210±5. The Delphi technique is adopted to achieve index weighting, which ensures that the evaluation outcome is patient oriented. The weighted index value will be computed as the final measurement index of the outcome. ETHICS AND DISSEMINATION: This study has been approved by the Medical Ethics Committee of Tianjin University of Traditional Chinese Medicine (registration number TJUTCM-EC20160007). The results will be offered for publication in peer-reviewed journals. TRIAL REGISTRATION NUMBER: This trial was registered with the Chinese Clinical Trial Registry (ChiCTR-IOR-17010397). The date of registration was 11 January 2017.

Osthole improves an accelerated focal segmental glomerulosclerosis model in the early stage by activating the Nrf2 antioxidant pathway and subsequently inhibiting NF-κB-mediated COX-2 expression and apoptosis.

Inflammatory reactions and oxidative stress are implicated in the pathogenesis of focal segmental glomerulosclerosis (FSGS), a common chronic kidney disease with relatively poor prognosis and unsatisfactory treatment regimens. Previously, we showed that osthole, a coumarin compound isolated from the seeds of Cnidium monnieri, can inhibit reactive oxygen species generation, NF-κB activation, and cyclooxygenase-2 expression in lipopolysaccharide-activated macrophages. In this study, we further evaluated its renoprotective effect in a mouse model of accelerated FSGS (acFSGS), featuring early development of proteinuria, followed by impaired renal function, glomerular epithelial cell hyperplasia lesions (a sensitive sign that precedes the development of glomerular sclerosis), periglomerular inflammation, and glomerular hyalinosis/sclerosis. The results show that osthole significantly prevented the development of the acFSGS model in the treated group of mice. The mechanisms involved in the renoprotective effects of osthole on the acFSGS model were mainly a result of an activated Nrf2-mediated antioxidant pathway in the early stage (proteinuria and ischemic collapse of the glomeruli) of acFSGS, followed by a decrease in: (1) NF-κB activation and COX-2 expression as well as PGE2 production, (2) podocyte injury, and (3) apoptosis. Our data support that targeting the Nrf2 antioxidant pathway may justify osthole being established as a candidate renoprotective compound for FSGS.

Osthole mitigates progressive IgA nephropathy by inhibiting reactive oxygen species generation and NF-κB/NLRP3 pathway.

Renal reactive oxygen species (ROS) and mononuclear leukocyte infiltration are involved in the progressive stage (exacerbation) of IgA nephropathy (IgAN), which is characterized by glomerular proliferation and renal inflammation. The identification of the mechanism responsible for this critical stage of IgAN and the development of a therapeutic strategy remain a challenge. Osthole is a pure compound isolated from Cnidiummonnieri (L.) Cusson seeds, which are used as a traditional Chinese medicine, and is anti-inflammatory, anti-apoptotic, and anti-fibrotic both in vitro and in vivo. Recently, we showed that osthole acts as an anti-inflammatory agent by reducing nuclear factor-kappa B (NF-κB) activation in and ROS release by activated macrophages. In this study, we examined whether osthole could prevent the progression of IgAN using a progressive IgAN (Prg-IgAN) model in mice. Our results showed that osthole administration resulted in prevention of albuminuria, improved renal function, and blocking of renal progressive lesions, including glomerular proliferation, glomerular sclerosis, and periglomerular mononuclear leukocyte infiltration. These findings were associated with (1) reduced renal superoxide anion levels and increased Nrf2 nuclear translocation, (2) inhibited renal activation of NF-κB and the NLRP3 inflammasome, (3) decreased renal MCP-1 expression and mononuclear leukocyte infiltration, (4) inhibited ROS production and NLRP3 inflammasome activation in cultured, activated macrophages, and (5) inhibited ROS production and MCP-1 protein levels in cultured, activated mesangial cells. The results suggest that osthole exerts its reno-protective effects on the progression of IgAN by inhibiting ROS production and activation of NF-κB and the NLRP3 inflammasome in the kidney. Our data also confirm that ROS generation and activation of NF-κB and the NLRP3 inflammasome are crucial mechanistic events involved in the progression of the renal disorder.

[Simultaneous determination of paeoniflorin, ferulic acid, prim-O-glucosylcimifugin and 4'-O-beta-glucopyranosyl-5-O-methylvisamminol in zhengtian pills by HPLC].

To simultaneously determine paeoniflorin, ferulic acid, prim-O-glucosylcimifugin and 4'-O-beta-glucopyranosyl-5-O-methylvisamminol in Zhengtian pills. In the test, Insertil ODS-C18 column (4.6 mm x 250 mm, 5 microm) was adopted, with acetonitrile-0.05% phosphoric acid solution as the mobile phase for gradient elution. The flow rate was 1.0 mL x min(-1), the column temperature was 30 degrees C and the detection wavelength was 230 nm. According to the results of the test, paeoniflorin, ferulic acid, prim-O-glucosylcimifugin and 4'-O-beta-glucopyranosyl-5-O-methylvisamminol showed good linear relations between peak areas and sample sizes in 11.37-170.5, 2.188-32.82, 2.896-43.44, and 3.000-45.00 mg x L(-1) (r = 0.999 9, 0.999 9, 1.000 0, 1.000 0), respectively. The average recoveries (n = 6) were 102.3% (RSD 1.2%), 99.71% (RSD 1.9%), 101.2% (RSD 1.2%), and 99.40% (RSD 2.4%), respectively. The above four components were determined in five batches of samples by using the established method, with satisfactory results. The method was so simple, accurate and highly reproducible that it could be used for quality control of the four components in Zhengtian pills.

[Inhibition of Jumi extraction on growth of human cervical cancer cell line HeLa].

OBJECTIVE: To explore the inhibition of Jumi (traditional Chinese medicine) extraction on the growth of human cervical cancer cell line HeLa. METHODS: Nude mouse model of human cervical cancer HeLa cell transplantation was established. The nude mice bearing cancer were randomly divided into control group and Jumi treated groups with different concentration (0.001, 0.002, 0.005, 0.01 mg/ml). The growth of cervical cancer cell in experimental mice were measured. Cultured HeLa cells were incubated in culture media with or without Jumi extract for 48 hours. Cell proliferation rate, cell apoptosis, caspase-3/7 and caspase-6 activity were determined by MTT colorimetric assay, flow cytometry analysis and spectrophotometric detection, respectively. RESULTS: With the increase of the concentration of Jumi extract, tumor-bearing mice tumor inhibition rate gradually increased. The proliferation of cultured HeLa cells were significantly inhibited by Jumi extract in a dose-dependent manner. IC50 was 0.004 mg/ml. Apoptosis rates in the cells treated with Jumi extract were higher than those of the control group. Compared with the control group, except for lower Jumi treated group (0.001 mg/ml), caspase-3/7 and caspase-6 activity were significantly increased in the all Jumi treated groups. CONCLUSION: Jumi extract can inhibit the proliferation of human cervical cancer cell line HeLa in vitro in a dose-dependent manner and promote cell apoptosis through caspase-3, caspase-7 and caspase-6 pathway.

Sorafenib and its derivative SC-49 sensitize hepatocellular carcinoma cells to CS-1008, a humanized anti-TNFRSF10B (DR5) antibody.

BACKGROUND AND PURPOSE: Previously, we have shown that sorafenib sensitizes hepatocellular carcinoma (HCC) to apoptosis induced by TNF-related apoptosis-inducing ligand (TNFSF10; TRAIL). Here, we report that sorafenib and SC-49 sensitize HCC cells to CS-1008, a novel anti-human death receptor 5 (TNFRSF10B) antibody. EXPERIMENTAL APPROACH: HCC cell lines (PLC5, Huh-7, and Hep3B) were treated with CS-1008 and/or sorafenib and analysed in terms of apoptosis and signal transductions. KEY RESULTS: SC-49 is a sorafenib derivative, which is devoid of kinase inhibitory activity. Both sorafenib and SC-49 down-regulated the phosphorylation of STAT3 at Tyr(705) and subsequently reduced the levels of STAT3-regulated proteins, Mcl-1, survivin and cylcin D1, in CS-1008-treated HCC cells. Knockdown of STAT3 by RNA interference overcame apoptotic resistance to CS-1008 in HCC cells, and ectopic expression of STAT3 in HCC cells abolished the sensitizing effects of sorafenib and SC-49 on CS-1008-induced apoptosis, indicating that inhibition of STAT3 mediates the enhancing effects of these compounds when combined with CS-1008. Importantly, inhibition of SHP-1 by adding a specific SHP-1 inhibitor reduced the effects of SC-49 and CS-1008 on p-STAT3 and apoptosis, whereas co-treatment of CS-1008 with SC-49 increased the activity of SHP-1. These data indicate that the combined effects of CS-1008 and SC-49 on HCC are mediated by SHP-1. Moreover, the combination of CS-1008 and SC-49 inhibited HCC xenograft tumour growth in vivo. CONCLUSIONS AND IMPLICATIONS: Sorafenib and its derivative SC-49 sensitize HCC cells to the antitumour effects of CS-1008 through SHP-1-dependent inactivation of STAT3.