Anti-colorectal cancer targets of resveratrol and biological molecular mechanism: Analyses of network pharmacology, human and experimental data.

In this study, colorectal cancer (CRC)-diseased targets and resveratrol (Res)-associated targets were combined and constructed by the use of grouped databases for identification of the predicted targets. After production of target-functional protein interaction network of Res anti-CRC, the topological analysis was used to create the core targets of Res anti-CRC. All core targets performed the analyses of biological function and pathway enrichment to optimize the biological processes and key signaling pathways of Res anti-CRC. The resultant five core therapeutic targets of Res anti-CRC were identified as protein kinase B1 (AKT1), interleukin 6 (IL6), Tumor protein p53 (TP53), vascular endothelial growth factor, and mitogen-activated protein kinase 1, respectively. Biological processes of Res anti-CRC were predominantly associated with regulating apoptosis, immune response, cellular communication, signal transduction, and metabolism of the nuclide. In addition, the top 10 key signaling pathways were identified, respectively. In human CRC sample assays, CRC histologic sections showed elevated expression of AKT1 and IL6 proteins, accompanied with abnormal changes in blood molecules. In pharmacological experiments of Res anti-CRC in vitro, Res-treated HCT116 cells showed inhibited cell growth, induced cell death. In addition, downregulation of intracellular AKT1 and IL6 expression were checked in Res-treated HCT116 cells. Taken together, these bioinformatic findings and preliminary validated data uncovered pharmacological molecular mechanisms associated with Res anti-CRC, and further identified top five core therapeutic targets. Beneficially, these five predicted targets might serve as potential biomolecules for anti-CRC treatment.

Protective Effects of Rhubarb in Rats with Acute Pancreatitis and the Role of Its Active Compound Rhein on Mitochondria of Exocrine Cells.

Da-Cheng-Qi-Decoction (DCQD) has been used in the treatment of acute pancreatitis (AP) in China for many years. The aim of the current study was to examine the principal ingredient rhubarb of DCQD and its potential link to the pancreatic repair effects in rats with AP. The pancreatitis was induced in SD rats by intraperitoneal injections of cerulein. The results showed that rhubarb significantly increased blood perfusion of pancreatic tissue, reversed mitochondrial damage, and promoted pancreatic acinar and stellate cell proliferation. In addition, the rhein (from rhubarb) had high distribution in pancreas tissue and protected mitochondria in AR42J cells via the activation of PI3K/AKT/mTOR signaling pathway and activity inhibition of AMPK (P < 0.05). The results provide some preclinical evidence on the protective effects of DCQD for the treatment of acute pancreatitis. Rhein is regarded to be the active compound of rhubarb and can be expected to be a new compound for the treatment of AP.

Oxyresveratrol prevents murine H22 hepatocellular carcinoma growth and lymph node metastasis via inhibiting tumor angiogenesis and lymphangiogenesis.

The purpose of this study was to investigate the effects and mechanisms of oxyresveratrol (Oxyres) on hepatocellular carcinoma (HCC) in vitro and in vivo. The MTT and Transwell assays were performed to investigate the effects of Oxyres on cell proliferation and migration of two HCC cell lines, QGY-7701 and SMMC-7721 cells. H22 cells were subcutaneously injected into hind foot pads of 70 male mice to establish a lymph node metastasis model. These mice were randomly divided into seven groups as follows, control group, HCC group, Oxyres 20 mg/kg group, Oxyres 40 mg/kg group, Oxyres 60 mg/kg group, Resveratrol (Res) group, and Adriamycin (ADM) group. Oxyres, Res, and ADM were intraperitoneally injected daily for consecutive 21 days. Tumors and popliteal lymph node were isolated and embedded for histology analysis. Expressions of CD31 and vascular endothelial growth factor receptor-3 (VEGFR3) in tumors were detected by immunohistocehmistry. Expressions of vascular endothelial growth factor C (VEGF-C) were measured by Western blot. Oxyres significantly inhibited the proliferation and migration of QGY-7701 and SMMC-7721 cells. Oxyres significantly inhibited tumor growth (p < 0.001) and metastasis to sentinel lymph nodes (70%) in a dose-dependent manner. Oxyres showed a similar inhibition rate as Res. Oxyres also significantly decreased micro-blood vessel density and micro-lymphatic vessel density in tumors (p < 0.05). Expressions of CD31, VEGFR3, and VEGF-C of tumors were also inhibited by Oxyres (p < 0.05). Oxyres exerts anti-tumor effects against HCC through inhibiting both angiogenesis and lymph node metastasis, which suggests Oxyres be a potential therapeutic agent.

Dehydroandrographolide enhances innate immunity of intestinal tract through up-regulation the expression of hBD-2.

BACKGROUND: Dehydroandrographolide (DA) is one of major active components in the well-known oriental herbal medicine Andrographis paniculata (Burm.f) Nees which belongs to the Acanthaceae family. DA is used for the treatment of infections in China. However, DA has not been found to significantly inhibit bacterial and viral growth directly. The current study investigates the effect of DA on the expression of human ß -defensin-2 (hBD-2) in human intestinal epithelial cells and the possible signaling pathways. METHODS: Human intestinal epithelial HCT-116 cells were incubated with 1-100 µM DA for 2-24 h. RT-PCR and Western blot were used to assess the expression of hBD-2. The specific inhibitors were used and the levels of phosphorylation of signaling molecules were detected for dissecting the signaling pathways leading to the induction of hBD-2. RESULTS: MTT assay showed there was no obvious cytotoxicity for HCT-116 cells by 1-100 µM DA treatment. RT-PCR and Western blot assays showed that DA (1-100 µM) could up-regulate the expression of hBD-2, and the effect lasted longer than 24 h. By using SB203580 and SB202190 (inhibitors of p38), the enhancement of hBD-2 expression were significantly attenuated. However, inhibitor of ERK and inhibitor of JNK could not block the effect of DA. Furthermore, Western blot found activation of p38 but not ERK and JNK in DA-treated HCT-116 cells. CONCLUSION: The results suggested that DA enhanced innate immunity of intestinal tract by up-regulating the expression of hBD-2 through the p38 MAPK pathways.

[HIV-1gp120 injures neurons by alteration of neuronal expressions of NR2B and PSD-95].

OBJECTIVE: To investigate the mechanisms underlying HIV-1 envelope glycoprotein 120 (gp120)-associated neurotoxicity on rat cortical neuronal cultures. METHODS: Experiments were carried out on primary cortical neuronal cultures prepared from fetal Sprague-Dawley fetal rats. Immunocytochemistry and Western blot techniques were employed to examine the changes of the expressions of N-methyl-D-asparate receptors 2B(NR2B) and postsynaptic density 95 (PSD-95) in cultured neurons in the presence and absence of gp120 in the culture media. Gp120-induced neurotoxicity was determined by MTT assay. RESULTS: MTT assay showed gp120 produced dose-dependent neuronal injury when added to the culture media and the gp120-induced neuronal injury was blocked by memantine, a specific NR2B receptor antagonist. Further studies revealed that gp120 induced neuronal injury by up-regulation of NR2B and down-regulation of PSD-95 expressions in rat cortical neurons. CONCLUSION: These results demonstrated that gp120 injures neurons via an increase of NR2B and a decrease of PSD-95 expressions. The gp120-induced neuronal injury can be blocked by a specific NR2B receptor antagonist, suggesting NR2B may function as a potential target for the development of therapeutic strategies.

Protective effects of Carthamus tinctorius injection on isoprenaline-induced myocardial injury in rats.

CONTEXT: Carthamus tinctorius injection (CTI) is a traditional Chinese medicine (TCM) specifically used for the treatment of cerebral ischemia and myocardial ischemia. OBJECTIVE: This study evaluated the protective effects of CTI on isoprenaline-induced acute myocardial ischemia (AMI) in rats and explored the underlying mechanisms. MATERIALS AND METHODS: (i) Sprague-Dawley rats were randomly divided into 5 groups: control, myocardial ischemia model, and high-, low-dose of CTI groups (2.5 and 0.625 g/kg, respectively, i.p. for 5 days), and Xiang-Dan (20 g/kg) group (n = 10 in each group). AMI was induced by isoproterenol (5 mg/kg) by intraperitoneal injection. Assessment of electrocardiograms (ECG) was carried out. (ii) Another 40 rats were randomly divided into 5 groups, the concentration of IL-6 and TNF-α in serum were measured by radioimmunological assay; Bcl-2 and Bax protein expression were measured by immunohistochemistry. RESULTS: CTI (2.5 and 0.625 g/kg) significantly inhibited the typical ECG S-T segment elevation, reduced concentration of IL-6 and TNF-α in serum, suppressed overexpression of Bax protein and also inhibited the reduction of Bcl-2 expression and markedly depressed the Bax/Bcl-2 ratio. DISCUSSION AND CONCLUSION: These findings demonstrate that CTI is cardioprotective against AMI in rats and is likely to related to decrease inflammatory response mediated by TNF-α and IL-6, down-regulate protein level of Bax and up-regulate that of Bcl-2 in the heart tissue.

Calycosin stimulates proliferation of estrogen receptor-positive human breast cancer cells through downregulation of Bax gene expression and upregulation of Bcl-2 gene expression at low concentrations.

BACKGROUND: Calycosin is one of main components in the herb radix astragali and is considered a typical phytoestrogen. It has either estrogenic or antiestrogenic effects that mainly depend on estrogen levels in vivo. This study investigated the effects and mechanisms of calycosin on estrogen receptor (ER)-positive human breast cancer (MCF-7) cells in vitro. METHODS: ER-positive MCF-7 cells were treated with different concentrations of calycosin. Effects of calycosin on the proliferation of ER-positive MCF-7 cells were determined by the MTT assay. Apoptosis in these treated cells was examined by flow cytometry. The mRNA and protein levels of Bcl-2 and Bax in these treated cells were also determined by reverse-transcription polymerase chain reaction and immunohistochemical staining, respectively. RESULTS: Compared with the vehicle control, calycosin stimulated proliferation of ER-positive MCF-7 cells at low concentrations (2, 4, and 8 µmol/L). Furthermore, at these concentrations, calycosin decreased the percentage of early apoptosis in MCF-7 cells, downregulated mRNA and protein levels of Bax, and upregulated those of Bcl-2 at low concentrations. On the other hand, calycosin at higher concentrations (16 and 32 µmol/L) inhibited cell proliferation. CONCLUSION: At relatively low concentrations, calycosin has stimulatory effects on the proliferation of MCF-7 cells, with the estrogenic effect the mechanism.

Effect of Da-Cheng-Qi decoction on pancreatitis-associated lung injury in patients and anti-inflammatory responses in rat models.

BACKGROUND: The traditional herbal medicinal formula Da-Cheng-Qi decoction (DCQD) has long been used to treat pancreatitis in China; however, the underlying mechanisms remain unclear. AIM: To investigate whether DCQD is beneficial to the patients with lung injury in severe acute pancreatitis (SAP); if it is, then to explore the lung protective effect of DCQD and the mechanism involved in rats. METHODS: DCQD was enema administered to 70 patients for 7 days. Mortality, (multi)organ failure during admission were observed, blood samples for laboratory analysis were drawn on admission, on Days 3, 7, and 14 of the treatment. We also experimentally examined the function of two doses of DCQD in SAP rat models. IL-1ß, IL-6, and IL-10 mRNA expression in rat lungs was measured quantitatively by the RT-PCR method and confirmed by morphometric studies of the lungs. RESULTS: It was demonstrated that the administration of DCQD did shorten the average time that patients suffered acute respiratory distress syndrome (ARDS). Compared with untreated rats, the lungs of rats treated with DCQD showed significantly lower levels of proinflammatory cytokine IL-1ß and IL-6 mRNA. Rats treated with DCQD had lower mean pathological lung lesion scores than those in SAP rats. CONCLUSION: DCQD has good prospects in the treatment for SAP because it did shorten the average time that patients suffered ARDS in the clinic. It exerts therapeutic effects on this disease through inhibiting the production of inflammatory mediators, decreasing the anti-inflammatory factors, and mitigating the pathological damage of the lung injury in SAP model.

In vitro and in vivo antitumor effects of acetylshikonin isolated from Arnebia euchroma (Royle) Johnst (Ruanzicao) cell suspension cultures.

BACKGROUND: Shikonin derivatives have cytotoxic and antitumor effects. This study aims to investigate the antitumor effects of acetylshikonin isolated from a Chinese medicinal herb Arnebia euchroma (Royle) Johnst. METHODS: The 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to determine the in vitro antitumor effects of acetylshikonin on human lung adenocarcinoma cell line A549, human hepatocellular carcinoma cell line Bel-7402, human breast adenocarcinoma cell line MCF-7 and mouse Lewis lung carcinoma (LLC) cell line. C57BL/6 mice with LLC model were used to study the in vivo antitumor effects of acetylshikonin. The expression of bax, bcl-2 and caspase-3 proteins in LLC tissue was determined with immunohistochemical staining. RESULTS: In A549, Bel-7402, MCF-7 and LLC cell lines, acetylshikonin inhibited cell growth in a dose-dependent manner. IC50 (means +/- SD) were 5.6 +/- 0.86 microg/ml, 6.82 +/- 1.5 microg/ml, 3.04 +/- 0.44 microg/ml and 2.72 +/- 0.38 microg/ml respectively. Acetylshikonin suppressed tumor growth in C57BL/6 mice with LLC. The inhibition rate of acetylshikonin (2 mg/kg) was 42.85%. Immunohistochemical staining revealed that in the acetylshikonin groups the expression of bax and caspase-3 increased, whereas the expression of bcl-2 decreased, suggesting that acetylshikonin induced tumor cell apoptosis through activating the pro-apoptotic bcl-2 family and caspase-3. CONCLUSION: Acetylshikonin isolated from Arnebia euchroma (Royle) Johnst cell suspension cultures exhibits specific in vivo and in vitro antitumor effects.

[Effect of human beta defensin 2 on Staphylococcus aureus infection: in vivo study with transgenic mice].

OBJECTIVE: To investigate the effect of human beta defensin 2 (HBD-2) on Staphylococcus aureus infection. METHODS: A minigene of HBD-2 containing pCMV promoter, full length of HBD-2 cDNA, and BGH polyA tail was generated by PCR amplification and introduced into the fertilized oocytes of C57/ICR hybridized mouse by microinjection. After gestation of 3 - 4 weeks, immunohistochemistry was used to detect the expression of HBD-2 peptide in different tissues of the transgenic young mice. Staphylococcus aureus was cultured and injected intraperitoneally to wild type mice and transgenic mice to observe their surviving status. RESULTS: PCR showed that the HBD-2 fragment had been successfully integrated into the chromosome of the mice. A widespread expression of HBD-2 gene was found in many tissues of the transgenic mice: trachea, lung, intestine, esophagus, testis, spleen, skin, endothelium, brain, etc. Four of the 7 transgenic mice survived the Staphylococcus aureus infection, and 10 wild type mice all died within 24 hours. CONCLUSION: HBD-2 may play an important role ion the host defense against Staphylococcus aureus infection.

[Construction and identification of HBD-2 transgenic mice].

Human beta defensin 2 (HBD-2) may play an important role in human defense against infection. Its antimicrobial capacity has been fully documented in in vitro study. In order to evaluae its in vivo effects, we developed an HBD-2 transgenic mouse model. The HBD-2 minigene containing CMV promoter, full length of HBD-2 cDNA and BGH polyA tail was generated by PCR amplification and introduced into the fertilized oocytes of C57 X ICR hybridized mouse by microinjection, and offspring were produced. DNA was isolated from the tails of the mouse pups, and the HBD-2 minigene incorporation was analyzed by PCR using HBD-2 specific primers. The HBD-2 gene expression in the multi-tissues of transgenic mice was determined at mRNA level by RT-PCR and at peptide level by immunohistological staining with the use of HBD-2 monoclonal antibody. The results showed that among 17 F0 transgenic mice, HBD-2 positive signal was determined by PCR in 4 mice, suggesting that HBD-2 minigene has been incorporated into the offspring mice. Meanwhile, a widespread expression of HBD-2 mRNA and peptide was detected in the F1 transgenic mice's multi-tissues such as trachea, lung, intestine, esophagus, testis, spleen, skin, endothelium and brain.

[Application of HMGN2-tag constructs to analysis of HMGN2 distribution in HeLa cells].

This study sought to clarify the distribution of HMGN2 in HeLa cells. The recombinant eukaryotic expression vectors pcDNA3. 1-myc-his-HMGN2 and pEGFP-N1-HMGN2 were constructed, and then were transfected into HeLa cells. immunocytochemistry staining indicated that HMGN2 were present not only in HeLa nucleus but also in the cytoplasm. The presence of HMGN2 was also detected in the culture supernatant by ELISA with rabbit anti-serum against HMGN2 and mouse anti-His6 monoclonal antibodies. The confocal microscope observation showed the same subcellular localization as that of immunocytochemistry staining. There results suggested that HMGN2 could be present in the nucleus and cytoplasm of HeLa cell as well as in the extracellular environment.

[Production of HMGN2 polyclonal antibody by immunization with recombinant GST-HMGN2 fusion protein and its application to analysis of HMGN2 distribution in human monocytes].

OBJECTIVE: To prepare high mobility group chromosomal protein N2 (HMGN2) polyclonal antibodies and determine the subcellular localization of HMGN2 in human monocytes. METHODS: The recombinant prokaryotic expression vector pGEX-1lambdaT-HMGN2 was constructed and E. coli-based product of GST-HMGN2 fusion protein was prepared and used to immunize rabbit for producing the anti-serum against HMGN2. The polyclonal antibodies were partially purified by caprylic acid and ammonium sulfate precipitation. The titter of specific polyclonal antibodies against HMGN2 was detected by ELISA. The immunocytochemical staining was performed to determine the distribution of HMGN2 in THP-1 cells. RESULTS: Gel electrophoresis of the enzyme-digested recombinant plasmid and the DNA sequencing confirmed that the recombinant prokaryotic expression vector pGEX-1lambdaT-HMGN2 was correctly constructed. After IPTG induction, the recombinant-transformed E. coli produced a bulk of GST-HMGN2 fusion protein. The polyclonal antibodies to HMGN2 was obtained from the serum of rabbit immunized with GST-HMGN2 fusion protein and its ELISA titer was 1:2000. The immunocytochemistry staining indicated that when stimulated with LPS, HMGN2 was present not only in THP-1 nucleus but also in the cytoplasm. The presence of HMGN2 was also detected in the culture supernatant. CONCLUSION: This result suggests that recombinant peptide fusion protein could be used to produce peptide antibody. HMGN2 could be present in the cytoplasm of monocytes and release to the extracellular environment when stimulated with lipopolysaccharide (LPS).