Inflammatory Milieu Related to Dysbiotic Gut Microbiota Promotes Tumorigenesis of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is an invasive primary liver cancer caused by multiple pathogenic factors and is a significant global health concern. With few effective therapeutic options, HCC is a heterogeneous carcinoma that typically arises in an inflammatory environment. Recent studies have suggested that dysbiotic gut microbiota is involved in hepatocarcinogenesis via multiple mechanisms. In this review, we discuss the effects of gut microbiota, microbial components, and microbiota-derived metabolites on the promotion and progression of HCC by feeding a persistent inflammatory milieu. In addition, we discuss the potential therapeutic modalities for HCC targeting the inflammatory status induced by gut microbiota. A better understanding of the correlation between the inflammatory milieu and gut microbiota in HCC may be beneficial for developing new therapeutic strategies and managing the disease.

Antihepatoma peptide, scolopentide, derived from the centipede scolopendra subspinipes mutilans.

BACKGROUND: Centipedes have been used to treat tumors for hundreds of years in China. However, current studies focus on antimicrobial and anticoagulation agents rather than tumors. The molecular identities of antihepatoma bioactive components in centipedes have not yet been extensively investigated. It is a challenge to isolate and characterize the effective components of centipedes due to limited peptide purification technologies for animal-derived medicines. AIM: To purify, characterize, and synthesize the bioactive components with the strongest antihepatoma activity from centipedes and determine the antihepatoma mechanism. METHODS: An antihepatoma peptide (scolopentide) was isolated and identified from the centipede scolopendra subspinipes mutilans using a combination of enzymatic hydrolysis, a Sephadex G-25 column, and two steps of high-performance liquid chromatography (HPLC). Additionally, the CCK8 assay was used to select the extracted fraction with the strongest antihepatoma activity. The molecular weight of the extracted scolopentide was characterized by quadrupole time of flight mass spectrometry (QTOF MS), and the sequence was matched by using the Mascot search engine. Based on the sequence and molecular weight, scolopentide was synthesized using solid-phase peptide synthesis methods. The synthetic scolopentide was confirmed by MS and HPLC. The antineoplastic effect of extracted scolopentide was confirmed by CCK8 assay and morphological changes again in vitro. The antihepatoma effect of synthetic scolopentide was assessed by the CCK8 assay and Hoechst staining in vitro and tumor volume and tumor weight in vivo. In the tumor xenograft experiments, qualified model mice (male 5-week-old BALB/c nude mice) were randomly divided into 2 groups (n = 6): The scolopentide group (0.15 mL/d, via intraperitoneal injection of synthetic scolopentide, 500 mg/kg/d) and the vehicle group (0.15 mL/d, via intraperitoneal injection of normal saline). The mice were euthanized by cervical dislocation after 14 d of continuous treatment. Mechanistically, flow cytometry was conducted to evaluate the apoptosis rate of HepG2 cells after treatment with extracted scolopentide in vitro. A Hoechst staining assay was also used to observe apoptosis in HepG2 cells after treatment with synthetic scolopentide in vitro. CCK8 assays and morphological changes were used to compare the cytotoxicity of synthetic scolopentide to liver cancer cells and normal liver cells in vitro. Molecular docking was performed to clarify whether scolopentide tightly bound to death receptor 4 (DR4) and DR5. qRT-PCR was used to measure the mRNA expression of DR4, DR5, fas-associated death domain protein (FADD), Caspase-8, Caspase-3, cytochrome c (Cyto-C), B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), x-chromosome linked inhibitor-of-apoptosis protein and Cellular fas-associated death domain-like interleukin-1ß converting enzyme inhibitory protein in hepatocarcinoma subcutaneous xenograft tumors from mice. Western blot assays were used to measure the protein expression of DR4, DR5, FADD, Caspase-8, Caspase-3, and Cyto-C in the tumor tissues. The reactive oxygen species (ROS) of tumor tissues were tested. RESULTS: In the process of purification, characterization and synthesis of scolopentide, the optimal enzymatic hydrolysis conditions (extract ratio: 5.86%, IC50: 0.310 mg/mL) were as follows: Trypsin at 0.1 g (300 U/g, centipede-trypsin ratio of 20:1), enzymolysis temperature of 46 °C, and enzymolysis time of 4 h, which was superior to freeze-thawing with liquid nitrogen (IC50: 3.07 mg/mL). A peptide with the strongest antihepatoma activity (scolopentide) was further purified through a Sephadex G-25 column (obtained A2) and two steps of HPLC (obtained B5 and C3). The molecular weight of the extracted scolopentide was 1018.997 Da, and the peptide sequence was RAQNHYCK, as characterized by QTOF MS and Mascot. Scolopentide was synthesized in vitro with a qualified molecular weight (1018.8 Da) and purity (98.014%), which was characterized by MS and HPLC. Extracted scolopentide still had an antineoplastic effect in vitro, which inhibited the proliferation of Eca-109 (IC50: 76.27 µg/mL), HepG2 (IC50: 22.06 µg/mL), and A549 (IC50: 35.13 µg/mL) cells, especially HepG2 cells. Synthetic scolopentide inhibited the proliferation of HepG2 cells (treated 6, 12, and 24 h) in a concentration-dependent manner in vitro, and the inhibitory effects were the strongest at 12 h (IC50: 208.11 µg/mL). Synthetic scolopentide also inhibited the tumor volume (Vehicle vs Scolopentide, P = 0.0003) and weight (Vehicle vs Scolopentide, P = 0.0022) in the tumor xenograft experiment. Mechanistically, flow cytometry suggested that the apoptosis ratios of HepG2 cells after treatment with extracted scolopentide were 5.01% (0 µg/mL), 12.13% (10 µg/mL), 16.52% (20 µg/mL), and 23.20% (40 µg/mL). Hoechst staining revealed apoptosis in HepG2 cells after treatment with synthetic scolopentide in vitro. The CCK8 assay and morphological changes indicated that synthetic scolopentide was cytotoxic and was significantly stronger in HepG2 cells than in L02 cells. Molecular docking suggested that scolopentide tightly bound to DR4 and DR5, and the binding free energies were-10.4 kcal/mol and-7.1 kcal/mol, respectively. In subcutaneous xenograft tumors from mice, quantitative real-time polymerase chain reaction and western blotting suggested that scolopentide activated DR4 and DR5 and induced apoptosis in SMMC-7721 Liver cancer cells by promoting the expression of FADD, caspase-8 and caspase-3 through a mitochondria-independent pathway. CONCLUSION: Scolopentide, an antihepatoma peptide purified from centipedes, may inspire new antihepatoma agents. Scolopentide activates DR4 and DR5 and induces apoptosis in liver cancer cells through a mitochondria-independent pathway.

Dysbiosis: The first hit for digestive system cancer.

Gastrointestinal cancer may be associated with dysbiosis, which is characterized by an alteration of the gut microbiota. Understanding the role of gut microbiota in the development of gastrointestinal cancer is useful for cancer prevention and gut microbiota-based therapy. However, the potential role of dysbiosis in the onset of tumorigenesis is not fully understood. While accumulating evidence has demonstrated the presence of dysbiosis in the intestinal microbiota of both healthy individuals and patients with various digestive system diseases, severe dysbiosis is often present in patients with digestive system cancer. Importantly, specific bacteria have been isolated from the fecal samples of these patients. Thus, the association between dysbiosis and the development of digestive system cancer cannot be ignored. A new model describing this relationship must be established. In this review, we postulate that dysbiosis serves as the first hit for the development of digestive system cancer. Dysbiosis-induced alterations, including inflammation, aberrant immune response, bacteria-produced genotoxins, and cellular stress response associated with genetic, epigenetic, and/or neoplastic changes, are second hits that speed carcinogenesis. This review explains the mechanisms for these four pathways and discusses gut microbiota-based therapies. The content included in this review will shed light on gut microbiota-based strategies for cancer prevention and therapy.

Pan-cancer analysis of oncogenic TNFAIP2 identifying its prognostic value and immunological function in acute myeloid leukemia.

BACKGROUND: Tumor necrosis factor alpha-induced protein 2 (TNFAIP2), a TNFα-inducible gene, appears to participate in inflammation, immune response, hematopoiesis, and carcinogenesis. However, the potential role of TNFAIP2 in the development of acute myeloid leukemia (AML) remains unknow yet. Therefore, we aimed to study the biological role of TNFAIP2 in leukemogenesis. METHODS: TNFAIP2 mRNA level, prognostic value, co-expressed genes, differentially expressed genes, DNA methylation, and functional enrichment analysis in AML patients were explored via multiple public databases, including UALCAN, GTEx portal, Timer 2.0, LinkedOmics, SMART, MethSurv, Metascape, GSEA and String databases. Data from The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO) and Beat AML database were used to determine the associations between TNFAIP2 expression and various clinical or genetic parameters of AML patients. Moreover, the biological functions of TNFAIP2 in AML were investigated through in vitro experiments. RESULTS: By large-scale data mining, our study indicated that TNFAIP2 was differentially expressed across different normal and tumor tissues. TNFAIP2 expression was significantly increased in AML, particularly in French-American-British (FAB) classification M4/M5 patients, compared with corresponding control tissues. Overexpression of TNFAIP2 was an independent poor prognostic factor of overall survival (OS) and was associated with unfavorable cytogenetic risk and gene mutations in AML patients. DNA hypermethylation of TNFAIP2 at gene body linked to upregulation of TNFAIP2 and inferior OS in AML. Functional enrichment analysis indicated immunomodulation function and inflammation response of TNFAIP2 in leukemogenesis. Finally, the suppression of TNFAIP resulted in inhibition of proliferation by altering cell-cycle progression and increase of cell death by promoting early and late apoptosis in THP-1 and U937AML cells. CONCLUSION: Collectively, the oncogenic TNFAIP2 can function as a novel biomarker and prognostic factor in AML patients. The immunoregulation function of TNFAIP2 warrants further validation in AML.

[Mechanism of combined treatment of rhein and emodin in Rhubarb for ulcerative colitis].

This study aimed to explore the efficacy and mechanism of combined rhein and emodin in the treatment of ulcerative colitis(UC) from the aspects of network pharmacology, animal inflammation improvement and molecular mechanism. Network pharmacology predicted that combined rhein and emodin acted on 52 potential targets, mainly participating in signaling pathways such as cancer, PI3 K/AKT, microRNAs in cancer and apoptosis. PI3 K/AKT signaling pathway has been reported to be closely related to UC, and the optimal candidate pathway for combined therapy. The UC mice model was established by dextran sodium sulfate, and then the modeled mice were randomly divided into control group, model group, rhein group, emodin group, rhein+emodin group and sulfasalazine group. After administration, compared with the conditions in model group, body weight, disease activity index(DAI) score, colon length, TNF-α, IL-6, IL-1ß and myeloperoxidase(MPO) of mice in rhein+emodin group were improved(P<0.01); colonic mucosal injury was significantly reduced; the expression of p-PI3 K/PI3 K and p-AKT/AKT proteins were down-regulated(P<0.01). All the above indices were better than those in the rhein/emodin group alone. The Jin's Q-values of the effect of combined rhein and emodin on colon length, TNF-α, IL-6, IL-1ß, MPO, p-PI3 K/PI3 K and p-AKT/AKT were all greater than 1.15, which indicated that there was obvious synergistic effect between rhein and emodin. In all, rhein and emodin have synergistic effect in the treatment of UC, and the mechanism may be related to the inhibition of PI3 K/AKT signaling pathway and the down-regulation of proinflammatory factors. They are the new components in the treatment of UC, which is worthy of attention.

Anti-melanogenesis effect from Wampee fruit pectin via α-MSH/TRY pathway in A375 cells.

BACKGROUND: Polysaccharides from wampee have been reported to process various biological activities, while the relationship between structure and bioactivities has been barely addressed. Pectin, an abundant water-soluble polysaccharide in wampee, showed significant antioxidant activity, which was associated with the anti-melanogenic activity. Therefore, this study investigated the physicochemical characteristics and the anti-melanogenesis effect of pectin extracted from wampee fruit in A375 cells. METHODS: The physicochemical characterization of pectin from wampee fruit was investigated by gel chromatography (GCP), FT-IR spectroscopy, and NMR spectroscopy methods. The anti-melanogenesis effects and mechanism were evaluated by mushroom tyrosine enzyme and human melanin cell model in vitro. RESULTS: The results showed that a molecular weight of 5.271 × 105 Da wampee fruit pectin (WFP) were mainly composed of mannose (Man), ribose (Rib), rhamnose (Rha), glucuronic acid (Glc A), glucose (Glc), galacturonic acid (Gal A), galactose (Gal), and arabinose (Ara), which linked with →4)-ß-D-Galp-(1 → units. The current study revealed that WFP could significantly suppress mushroom TRY activity in vitro. Furtherly, WFP significantly reduced intracellular and extracellular melanin formation in A375 melanoma cells depending on the presence of alpha-melanocyte stimulating hormone (α-MSH). TRY activity was only inhibited in α-MSH treated A375 cells. Western blot analysis demonstrated that WFP reverse α-MSH induced melanogenesis in A375 melanoma cells, including in down-regulated TRY, TYRP-1, TYRP-2, MITF and CREB expressions. CONCLUSION: These results indicated that WFP could inhibit α-MSH induced melanogenesis in A375 melanoma cells via α-MSH/TRY pathway. In conclusion, these data provided a new perspective to annotate WFP anti-melanogenesis activity mechanism.

Xihuang pills induce apoptosis in hepatocellular carcinoma by suppressing phosphoinositide 3-kinase/protein kinase-B/mechanistic target of rapamycin pathway.

BACKGROUND: The phosphoinositide 3-kinase/protein kinase-B/mechanistic target of rapamycin (PI3K/Akt/mTOR) signalling pathway is crucial for cell survival, differentiation, apoptosis and metabolism. Xihuang pills (XHP) are a traditional Chinese preparation with antitumour properties. They inhibit the growth of breast cancer, glioma, and other tumours by regulating the PI3K/Akt/mTOR signalling pathway. However, the effects and mechanisms of action of XHP in hepatocellular carcinoma (HCC) remain unclear. Regulation of the PI3K/Akt/mTOR signalling pathway effectively inhibits the progression of HCC. However, no study has focused on the XHP-associated PI3K/Akt/mTOR signalling pathway. Therefore, we hypothesized that XHP might play a role in inhibiting HCC through the PI3K/Akt/mTOR signalling pathway. AIM: To confirm the effect of XHP on HCC and the possible mechanisms involved. METHODS: The chemical constituents and active components of XHP were analysed using ultra-performance liquid chromatography-quadrupole time of flight mass spectrometry (UPLC-Q-TOF-MS). Cell-based experiments and in vivo xenograft tumour experiments were utilized to evaluate the effect of XHP on HCC tumorigenesis. First, SMMC-7721 cells were incubated with different concentrations of XHP (0, 0.3125, 0.625, 1.25, and 2.5 mg/mL) for 12 h, 24 h and 48 h. Cell viability was assessed using the CCK-8 assay, followed by an assessment of cell migration using a wound healing assay. Second, the effect of XHP on the apoptosis of SMMC-7721 cells was evaluated. SMMC-7721 cells were stained with fluorescein isothiocyanate and annexin V/propidium iodide. The number of apoptotic cells and cell cycle distribution were measured using flow cytometry. The cleaved protein and mRNA expression levels of caspase-3 and caspase-9 were detected using Western blotting and quantitative reverse-transcription polymerase chain reaction (RT-qPCR), respectively. Third, Western blotting and RT-qPCR were performed to confirm the effects of XHP on the protein and mRNA expression of components of the PI3K/Akt/mTOR signalling pathway. Finally, the effects of XHP on the tumorigenesis of subcutaneous hepatocellular tumours in nude mice were assessed. RESULTS: The following 12 compounds were identified in XHP using high-resolution mass spectrometry: Valine, 4-gingerol, myrrhone, ricinoleic acid, glycocholic acid, curzerenone, 11-keto-ß-boswellic acid, oleic acid, germacrone, 3-acetyl-9,11-dehydro-ß-boswellic acid, 5ß-androstane-3,17-dione, and 3-acetyl-11-keto-ß-boswellic acid. The cell viability assay results showed that treatment with 0.625 mg/mL XHP extract decreased HCC cell viability after 12 h, and the effects were dose- and time-dependent. The results of the cell scratch assay showed that the migration of HCC cells was significantly inhibited in a time-dependent manner by the administration of XHP extract (0.625 mg/mL). Moreover, XHP significantly inhibited cell migration and resulted in cell cycle arrest and apoptosis. Furthermore, XHP downregulated the PI3K/Akt/mTOR signalling pathway, which activated apoptosis executioner proteins (e.g., caspase-9 and caspase-3). The inhibitory effects of XHP on HCC cell growth were determined in vivo by analysing the tumour xenograft volumes and weights. CONCLUSION: XHP inhibited HCC cell growth and migration by stimulating apoptosis via the downregulation of the PI3K/Akt/mTOR signalling pathway, followed by the activation of caspase-9 and caspase-3. Our findings clarified that the antitumour effects of XHP on HCC cells are mediated by the PI3K/Akt/mTOR signalling pathway, revealing that XHP may be a potential complementary therapy for HCC.

[Effects of exercise intervention on mitochondrial-derived peptide MOTS-c in the germ cells of obese men].

Mitochondrial open reading frame of the 12S rRNA-c (MOTS-c) is a newly discovered mitochondrial-derived peptide with the main functions of promoting glucose metabolism and reducing adipose tissue. MOTS-c was found to be a substance that can mimic the motor effect and improve the motor ability. It is sex-related and the circulating MOTS-c level is decreased in obese males. Obesity can cause male reproductive dysfunction, while exercise can improve obesity-induced reduction of male reproductive function. This article discusses the effect of exercise intervention on the mitochondrial-derived peptide MOTS-c in the germ cells of obese men.

Shuyu pills inhibit immune escape and enhance chemosensitization in hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is characterized by dysregulation of the immune microenvironment and the development of chemoresistance. Specifically, expression of the programmed cell death protein 1 (PD-1)/programmed cell death 1 ligand 1 (PD-L1) axis, an immune checkpoint, may lead to tumour immune escape, resulting in disease progression. The latest research shows that tumour immune escape may be caused by the upregulation of PD-L1 mediated by hypoxia-inducible factor-1 alpha (HIF-1α), and simultaneous inhibition of HIF-1α and PD-L1 has the potential to enhance the host's antitumour immunity. Moreover, inhibition of the PD-1/PD-L1 axis may mitigate tumour chemoresistance. Shuyu pills (SYPs) contain immunity-enhancing and antitumour components, making them a potential HCC treatment. AIM: To investigate the efficacy of SYPs for HCC treatment via simultaneous HIF-1α and PD-L1 inhibition and the mechanism involved. METHODS: A subcutaneous xenograft tumour model was first established in BALB/c nude mice by the subcutaneous injection of 1 × 107 SMMC-7721 cells. Male mice (male, 5 weeks old; n = 24) were then randomly divided into the following four groups (n = 6): Control (0.9% normal saline), SYP (200 mg/kg), SYP + cisplatin (DDP) (200 mg/kg + 5 mg/kg DDP weekly via intraperitoneal injection), and DDP (5 mg/kg cisplatin weekly via intraperitoneal injection). The dose of saline or SYPs for the indicated mouse groups was 0.2 mL/d via intragastric administration. The tumour volumes and body weights of the mice were measured every 2 d. The mice were euthanized by cervical dislocation after 14 d of continuous treatment, and the xenograft tissues were excised and weighed. Western blot assays were used to measure the protein expression of HIF-1α, PD-1, PD-L1, CD4+ T cells, and CD8+ T cells in HCC tumours from mice. Quantitative reverse transcription polymerase chain reaction was used for real-time quantitative detection of PD-1, PD-L1, and HIF-1α mRNA expression. An immunofluorescence assay was conducted to examine the expression of CD4+ T cells and CD8+ T cells. RESULTS: Compared to mice in the control group, those in the SYP and SYP + DDP groups exhibited reduced tumour volumes and tumour weights. Moreover, the protein and mRNA expression levels of the oncogene HIF-1α and that of the negative immunomodulatory factors PD-1 and PD-L1 were decreased in both the SYP and SYP + DDP groups, with the decrease effects being more prominent in the SYP + DDP group than in the SYP group (HIF-1α protein: Control vs SYP, P = 0.0129; control vs SYP + DDP, P = 0.0004; control vs DDP, P = 0.0152, SYP + DDP vs DDP, P = 0.0448; HIF-1α mRNA: control vs SYP, P = 0.0009; control vs SYP + DDP, P < 0.0001; control vs DDP, P = 0.0003, SYP vs SYP + DDP, P = 0.0192. PD-1 protein: Control vs SYP, P = 0.0099; control vs SYP + DDP, P < 0.0001, SPY vs SYP + DDP, P = 0.0009; SYP + DDP vs DDP, P < 0.0001; PD-1 mRNA: control vs SYP, P = 0.0002; control vs SYP + DDP, P < 0.0001; control vs DDP, P = 0.0003, SPY vs SYP + DDP, P = 0.0003; SYP + DDP vs DDP, P = 0.0002. PD-L1 protein: control vs SYP, P < 0.0001; control vs SYP + DDP, P < 0.0001; control vs DDP, P < 0.0001, SPY vs SYP + DDP, P = 0.0040; SYP + DDP vs DDP, P = 0.0010; PD-L1 mRNA: Control vs SYP, P < 0.0001; control vs SYP + DDP, P < 0.0001; control vs DDP, P < 0.0001, SPY vs SYP + DDP, P < 0.0001; SYP + DDP vs DDP, P = 0.0014). Additionally, the quantitative and protein expression levels of CD4+ T cells and CD8+ T cells were simultaneously upregulated in the SYP + DDP group, whereas only the expression of CD4+ T cells was upregulated in the SYP group. (CD4+ T cell quantitative: Control vs SYP + DDP, P < 0.0001, SYP vs SYP + DDP, P = 0.0005; SYP + DDP vs DDP, P = 0.0002. CD4+ T cell protein: Control vs SYP, P = 0.0033; Control vs SYP + DDP, P < 0.0001; Control vs DDP, P = 0.0021, SYP vs SYP + DDP, P = 0.0004; SYP + DDP vs DDP, P = 0.0006. Quantitative CD8+ T cells: Control vs SYP + DDP, P = 0.0013; SYP vs SYP + DDP, P = 0.0347; SYP + DDP vs DDP, P = 0.0043. CD8+ T cell protein: Control vs SYP + DDP, P < 0.0001; SYP vs SYP + DDP, P < 0.0001; SYP + DDP vs DDP, P < 0.0001). Finally, expression of HIF-1α was positively correlated with that of PD-1/PD-L1 and negatively correlated with the expression of CD4+ T cells and CD8+ T cells. CONCLUSION: SYPs inhibit immune escape and enhance chemosensitization in HCC via simultaneous inhibition of HIF-1α and PD-L1, thus inhibiting the growth of subcutaneous xenograft HCC tumours.

Long-term outcomes of busulfan plus melphalan-based versus melphalan 200 mg/m2 conditioning regimens for autologous hematopoietic stem cell transplantation in patients with multiple myeloma: a systematic review and meta-analysis.

BACKGROUND: High-dose melphalan (HDMEL, 200 mg/m2) is considered as the standard conditioning regimen for autologous hematopoietic stem cell transplantation (auto-HSCT) in multiple myeloma (MM). However, whether the combination of melphalan with busulfan (BUMEL) conditioning outperforms HDMEL remains controversy. Accordingly, a systematic review and meta-analysis was carried out to compare the outcomes of HDMEL and BUMEL-based conditioning regimens in newly diagnosed MM patients having undergone auto-HSCT. METHODS: A systematic literature search was conducted in PubMed, Embase and Cochrane Library database until July 31, 2021, to identify all eligible studies comparing progression-free survival (PFS), overall survival (OS), optimal treatment response after auto-HSCT, duration of stem cell engraftment and incidence of toxic events between patients undergoing BUMEL-based and HDMEL conditioning regimens. Hazard ratio (HR), mean difference (MD) or odds ratio (OR) corresponding to 95% confidence interval (CI) were determined to estimate outcomes applying RevMan 5.4 software. Publication biases were assessed by performing Egger's test and Begg's test by Stata 15 software. RESULTS: Ten studies with a total of 2855 MM patients were covered in the current meta-analysis. The results of this study demonstrated that patients having received BUMEL-based regimen was correlated with longer PFS (HR 0.77; 95% CI 0.67~0.89, P = 0.0002) but similar OS (HR 1.08; 95% CI 0.92~1.26, P = 0.35) compared with those having received HDMEL. The differences of best treatment response after auto-HSCT and duration of neutrophil or platelet engraftment did not have statistical significance between the two groups of patients. With respect to adverse effects, the patients in BUMEL-based group were less frequently subject to gastrointestinal toxicity while the patients in HDMEL group less often experienced mucositis and infection. No significant difference was observed in hepatic toxicity between the two groups of patients. CONCLUSIONS: In the present study, BUMEL-based conditioning was identified as a favorable regimen for a better PFS and equivalent OS as compared with HDMEL, which should be balanced against higher incidences of mucositis and infection. BUMEL-based conditioning is likely to act as an alternative strategy to more effectively improve auto-HSCT outcomes in MM.

Exploring the Pharmacological Mechanisms of Xihuang Pills Against Prostate Cancer via Integrating Network Pharmacology and Experimental Validation In Vitro and In Vivo.

Background: Drug resistance is the major cause of increasing mortality in prostate cancer (PCa). Therefore, it an urgent to develop more effective therapeutic agents for PCa treatment. Xihuang pills (XHP) have been recorded as the efficient anti-tumor formula in ancient Chinese medical literature, which has been utilized in several types of cancers nowadays. However, the effect protective role of XHP on the PCa and its underlying mechanisms are still unclear. Methods: The active ingredients of XHP were obtained from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) and BATMAN-TCM. The potential targets of PCa were acquired from the Gene Cards and OMIM databases. R language and Perl language program were utilized to clarify the interaction between the PCa-related targets and the potential targets of XHP. The potential targets of XHP for prostate cancer were gathered from the Gene ontology and KEGG pathway. Furthermore, cell proliferation assays were verified by PC3 and LNCaP cells. The efficacy and potential mechanism tests were confirmed by the PCa PC3 cells and mice subcutaneous transplantation. The effects of PI3K/Akt/mTOR-related proteins on proliferation, apoptosis, and cell cycle of PCa cells were measured by the Cell Counting Kit-8(CCK8), TUNEL assay, real-time quantitative reverse transcription PCR (QRT-PCR), and Western Blotting, respectively. Results: The active components of four traditional Chinese medicines in XHP were searched on the TCMSP and Batman TCM database. The biological active components of XHP were obtained as OB ≥30% and DL ≥0.18. The analysis of gene ontology and KEGG pathway identified the PI3K/Akt/mTOR signaling pathway as the XHP-associated pathway. Collectively, the results of in vitro and in vivo experiments showed that XHP had the effect of inhibiting on the proliferation of PC3 and LNCaP cells. XHP promoted the apoptosis and restrained the cell cycle and invasion of the PC3 cells and subcutaneous transplantation. Meanwhile, the suppression of XHP on the level of expression of PI3K, Akt, and mTOR-pathway-related pathway proteins has been identified in a dose-dependent manner. Conclusion: PI3K/Akt/mTOR pathway-related pathway proteins were confirmed as the potential XHP-associated targets for PCa. XHP can suppress the proliferation of prostate cancer via inhibitions of the PI3K/Akt/mTOR pathway.

Diagnostic and Prognostic Potentials of Long Noncoding RNA ELF3-AS1 in Glioma Patients.

OBJECTIVE: Accumulating evidence implies that long noncoding RNAs (lncRNAs) play a crucial role in predicting survival for glioma patients. However, the potential function of lncRNA ELF3-antisense RNA 1 (ELF3-AS1) in tumors remained largely unclear. The aim of this study was to explore the expression of lncRNA ELF3-antisense RNA 1 (ELF3-AS1) and evaluate its functions in glioma patients. Patients and Methods. ELF3-AS1 expressions were examined by RT-PCR in 182 pairs of glioma specimens and adjacent normal tissues. The receiver operating characteristic (ROC) curve was performed to estimate the diagnostic value of ELF3-AS1. The chi-square tests were used to examine the associations between ELF3-AS1 expression and the clinicopathological characters. The overall survival (OS) and disease-free survival (DFS) were analyzed by log-rank test, and survival curves were plotted according to Kaplan-Meier. The prognostic value of the ELF3-AS1 expression in glioma patients was further analyzed using univariate and multivariate Cox regression analyses. Loss-of-function assays were performed to determine the potential function of ELF3-AS1 on the proliferation and invasion of glioma cells. RESULTS: The ELF3-AS1 expression level was significantly higher in glioma specimens compared with adjacent nontumor specimens (p < 0.01). A high expression of ELF3-AS1 was shown to be associated with the WHO grade (p = 0.023) and KPS score (p = 0.012). ROC assays revealed that high ELF3-AS1 expression had an AUC value of 0.8073 (95% CI: 0.7610 to 0.8535) for glioma. Using the Kaplan-Meier analysis, we found that patients with a high ELF3-AS1 expression had significantly poor OS (p = 0.006) and DFS (p = 0.0002). In a multivariate Cox model, we confirmed that ELF3-AS1 expression was an independent poor prognostic factor for glioma patients. The functional assay revealed that knockdown of ELF3-AS1 suppressed the proliferation and invasion of glioma cells. CONCLUSIONS: Our findings confirmed that ELF3-AS1 functions as an oncogene in glioma and indicated that ELF3-AS1 is not only an important prognostic marker but also a potential therapy target for glioma.

Analysis of molecular networks and targets mining of Chinese herbal medicines on anti-aging.

BACKGROUND: Many kidney-tonifying Chinese herbal medicines exert effects on anti-aging by comprehensive interactions of multiple targets. However, the interactions of multi-targets targeted by effective ingredients of kidney-tonifying Chinese herbal medicines are unknown. In this study, to explore the systems pharmacology mechanisms of kidney-tonifying Chinese medicines on anti-aging, we establish the molecular networks with the interactions of multi-targets, analyze bio-functions and pathways with IPA, and calculated the mutual interaction pairs of targets (target pairs) with data mining, respectively. METHODS: Kidney-tonifying Chinese medicines with anti-aging effects were screened from the Chinese Pharmacopoeia and the literatures. Target proteins of these herbal medicines were obtained from bioinformatics databases. Comparisons of molecular networks, bio-functions and pathways given by Ingenuity Pathway Analysis system showed the similarities and the differences between kidney Yin-tonifying herbal medicines and kidney Yang-tonifying herbal medicines. Target pairs with high correlation related to anti-aging were also discovered by data mining algorithm. And regulatory networks of targets were built based on the target pairs. RESULTS: Twenty-eight kidney-tonifying herbal medicines with anti-aging effects and 717 related target proteins were collected. The main bio-functions that all targets enriched in were "Cell Death and Survival", "Free Radical Scavenging" and "Cellular Movement", etc. The results of comparison analysis showed that kidney Yin-tonifying herbal medicines focused more on "Cancer related signaling", "Apoptosis related signaling" and "Cardiovascular related signaling". And kidney Yang-tonifying herbal medicines focused more on "Cellular stress and injury related signaling" and "Cellular growth, proliferation and development related signaling". Moreover, the results of regulatory network showed that the anti-aging related target pairs with high correlated degrees of Kidney Yin-tonifying herbal medicines included TNF-PTGS2, TNF-CASP3, PTGS2-CASP3, CASP3-NOS2 and TNF-NOS2, and that of kidney Yang-tonifying herbal medicines included REAL-TNF, REAL-NFKBIA, REAL-JUN, PTGS2-SOD1 and TNF-IL6. CONCLUSIONS: In this study, we achieved some important targets, target pairs and regulatory networks with bioinformatics and data mining, to discuss the systems pharmacology mechanisms of kidney-tonifying herbal medicines acting on anti-aging. Mutual target pairs related to anti-aging found in this study included TNF-PTGS2, TNF-CASP3, PTGS2-CASP3, CASP3-NOS2, TNF-NOS2, REAL-TNF, REAL-NFKBIA, REAL-JUN, PTGS2-SOD1 and TNF-IL6. Target pairs and regulatory networks of targets could reflect more potential interactions between targets and comprehensive effects on anti-aging. Compared with the existing researches, it was found that the kidney-tonifying herbal medicines may exert anti-aging effects in multiple pathways in this study.

MTSS1 gene regulated by miR-96 inhibits cell proliferation and metastasis in tongue squamous cellular carcinoma Tca8113 cell line.

BACKGROUND: Metastasis suppressor-1 (MTSS1) is a novel potential metastasis suppressor gene in several types of human cancers. However, the exact function and regulatory mechanism of MTSS1 in Tongue squamous cellular carcinoma (TSCC) have not been elucidated. MATERIAL/METHODS: We first confirmed the MTSS1 gene expression by using quantitative real time-PCR (qRT-PCR) and immunohistochemical staining. Then we detected the effect of MTSS1 gene on Tca8113 cells proliferation and invasion ability by using MTT, wound healing and invasion assay. Finally by using bioinformatics analysis, luciferase reporter assay and a serial method, we analyzed the targeting of miR-96 on MTSS1 and the ability of miR-96 on MTSS1 gene mediated biological alterations in Tca8113 cells. RESULTS: Our findings showed that the expression of MTSS1 was down-regulated in both TSCC tissues and Tca8113 cells. Forced expression of MTSS1 led to inhibited cell proliferation ability, retarded wound closing and reduced trans-membrane cell numbers. MiR-96 is confirmed to be a direct target of MTSS1 gene and could regulate MTSS1 mediated Tca8113 cells proliferation and metastasis. But miR-96 could not completely restore the invasion ability of Tca8113 cells. CONCLUSIONS: MiR-96 targeting and promoting MTSS1 repression may precipitate in the TSCC tumorigenesis through bypassing cell proliferation and metastasis control.

Single nucleotide polymorphisms in premature ovarian failure-associated genes in a Chinese Hui population.

Premature ovarian failure (POF) is an ovarian defect characterized by the premature depletion of ovarian follicles in individuals <40 years old, and is a major cause of infertility in females. Genetic factors are considered to be responsible for the development of POF, however, the exact pathogenesis remains to be elucidated in the majority of cases. In the present study, the single nucleotide polymorphisms (SNPs) of growth differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15), inhibin ßB (INHBB) and follicle stimulating hormone receptor (FSHR) genes were investigated, and their association with POF in a Chinese Hui population of the Ningxia Hui Autonomous Region in western China was evaluated. Peripheral blood samples were collected from 63 patients diagnosed with POF (POF group) and 58 normal control individuals (control group), from which the genomic DNA was isolated. The GDF9, BMP15, INHBB and FSHR genes were amplified using polymerase chain reaction assays, and their SNPs were determined by sequencing. In the four SNPs identified across the GDF9 loci, D57Y (169G>T), rs1049127 (546G>A), rs254286 (447C>T) and rs254285 (969C>G), the frequencies of the 546G>A genotype and allele A were significantly higher in the POF group, compared with the normal control group (34.92, vs. 6.90%; P<0.05 and 19.05, vs. 3.23%; P<0.05, respectively), while no significant differences were observed in the occurrence of the c.447C>T and c.969C>G mutations between the two groups (60.32, vs. 50% and 50.79, vs. 55.17%, respectively). The c.169G>T mutation within the GDF9 gene was only detected in two patients with POF, and the mutation did not occur in the normal control group. A total of three SNPs were detected within the BMP15 gene, including rs3810682 (-9C>G), rs79377927 (788_789insTCT) and rs17003221 (852C>T), and no significant differences were observed in the frequencies of the -9C>G and 852C>T genotypes between the POF and control groups (7.94, vs. 6.90% and 4.76, vs. 3.45%, respectively). The 788_789insTCT genotype was detected in only two patients with POF. A novel mutation, c.1095C>A, was identified in exon 2 of the INHBB gene, however, no significant difference was found in the occurrence of the mutation between the two groups (30.16, vs. 22.41%; P>0.05). The rs6165 (919G>A) and rs6166 (2039G>A) SNPs were detected in exon 10 of the FSHR gene; however, no significant difference was observed in the genotype frequencies between the two groups (92.06, vs. 91.38% and 96.83, vs. 93.10%, respectively). These results demonstrated that GDF9 c.169G>T (D57Y), c.546G>A (rs1049127), and BMP15 rs79377927 (788_789insTCT) were associated with POF in the Chinese Hui population.

Association of insulin gene variable number of tandem repeats regulatory polymorphism with polycystic ovary syndrome.

The present meta-analysis aimed to investigate the association between insulin gene variable number of tandem repeats (INS VNTR) and polycystic ovary syndrome (PCOS). Systematic searches of electronic databases, reference lists of included articles, and the abstracts presented at related scientific societies meetings were performed. Statistical analyses were conducted using software Stata 11.0. The pooled odds ratios (ORs) with 95% confidence intervals (95% CIs) were applied. Publication bias was tested by Begg's funnel plot and Egger's regression test. A total of 9 studies including 1075 PCOS patients and 2878 controls were included in the meta-analysis. There were evidence of statistical significant association between INS VNTR and PCOS in allelic model (OR=1.25, 95% CI=1.08-1.43, P=0.002) and dominant model (OR=1.34, 95% CI=1.11-1.63, P=0.003) but not in additive model (OR=1.38, 95% CI=0.93-2.04, P=0.11) and recessive model (OR=1.26, 95% CI=0.96-1.65, P=0.09). No significant publication bias was shown by funnel plots and Egger's regression tests. In conclusion, our meta-analysis suggests that the III allele of INS VNTR is associated with increased risk of PCOS.

Identification of interneurons activated at different inclines during treadmill locomotion in adult rats.

By using c-fos as an activity-dependent marker, we identified the cholinergic interneurons around the central canal and lumbar interneurons throughout the gray matter that were activated after a 30-min bout of quadrupedal treadmill stepping at a 0° or 25° incline in adult rats. Increased loading (elevated treadmill incline) imposed during treadmill stepping activated more cholinergic interneurons in the proximity of the central canal, i.e., central canal cluster cells and partition neurons. Since cholinergic central canal cells are thought to modulate motoneuron excitability, these data suggest that increased load during stepping may increase motoneuronal activity through activating more cholinergic central canal cells. We identified the muscle-specific motoneurons and afferent terminals in the spinal cord by injecting cholera toxin subunit B in the soleus and tibialis anterior muscles. The number of interneurons in lumbar segments L4 (tibialis anterior) and L5 (soleus) was higher in both groups that stepped on the treadmill compared with control and was highest in rats that stepped at a 25° incline. In a majority of laminae, the distribution of total and muscle-specific activated interneurons was highest in the 25° incline group and lowest in the control group for both muscles. These data could reflect increased peripheral (proprioceptive) input as well as supraspinal drive associated with stepping and demonstrate the differences in 1) the activation of cholinergic interneurons near the central canal and 2) the laminar and segmental location of interneurons throughout the gray matter that play a role in generating stepping under different loading conditions in adult rats.

[Leiomyoma of tongue: a case report].

Oral leiomyoma is a benign smooth muscle tumor. Leiomyoma occurs most frequently in the uterine myometrium, gastrointestinal tract, and skin. Occurrence in the oral cavity is considered rare, probably because of the paucity of smooth muscle tissue at this area. Smooth muscle tumors can occur at any age and usually appear as a slow growing, firm submucosal nodule. Most lesions are asymptomatic, although occasional tumors can be painful. The most common sites are lips, palate and tongue. The diagnosis of leiomyoma in the oral cavity is mainly determined by histological studies. The purpose of this article is to present a case of a 48 year-old woman with a leiomyoma of tongue.

[Expression and clinical significance of tumor-associated macrophages and microvessel density in ameloblastomas].

PURPOSE: To investigate the expression and clinical significance of tumor-associated macrophages (TAMs) and microvessel density (MVD) in ameloblastomas (ABs). METHODS: The expression of TAMs (CD68) and MVD (CD34) was examined in 101 ABs and 26 oral mucosa by streptavidin-peroxidase (SP) immunohistochemical technique and then quantified. The data were analyzed by Student's t test and Pearson correlation test using SPSS 11.5 software package. RESULTS: TAMs and MVD were mainly distributed within the stroma of ABS. TAMs count and MVD count were significantly higher in ABs compared with normal oral mucosa (P<0.05). Linear correlation analysis revealed a positive correlation between TAMs count and MVD count in ABs (r=0.428, P<0.05). CONCLUSION: Increased TAMs is associated with angiogenesis and may play an important role in invasive growth of ABs.

[Speciation of aluminum in natural waters by fluorimetry].

Fluorimetric method has been proposed for the speciation of Al in natural waters, based on the different competitive complexation behavior of analytical reagent and natural organic matter to Al. With eriochrome blue black R (EBBR) or morin, the monomeric inorganic Al (Al(i)) was determined, while with 8-hydroxylquinoline, the total monomeric Al (Al(a)) was obtained. The analytical results of Al speciation in natural water are close to those by Driscoll's method. The method is simple and reliable. Fractionation of Al was realized by selecting appropriate reagents under the pH of natural water determined. The merits of this novel strategy are that the disturbance to original equilibrium of Al species is minimized and the results conform to the actual situation of natural waters.