Estrogen protects against liver damage in sepsis through inhibiting oxidative stress mediated activation of pyroptosis signaling pathway.

Sepsis was characterized by systemic inflammatory response and multisystem organ dysfunction, refering to the activation of inflammatory and oxidative stress pathways. Estrogen has been shown to have anti-inflammatory and antioxidant effects as well as extensive organ protective role. However, whether estrogen alleviates sepsis-induced liver injury and the mechanisms involved remain unknown. Septic mice were constructed by intraperitoneal injection lipopolysaccharide, and the effect of estrogen on liver injury was investigated. Furthermore, the roles of NLRP3 inhibitor MCC950 and mitochondrial ROS specific scavenger Mito-tempo, on the liver injury were explored in septic mice. Female septic mice exhibited liver damage with increased serum AST and ALT level as well as the existence of extensive necrosis, and which was more serious in male septic mice. Moreover, Ovariectomy (OVX) aggravated sepsis-induced liver damage and activation of pyroptosis signaling pathway, which was alleviated by estrogen as evidenced by decreased serum AST, ALT level and number of infiltrating inflammatory cell, as well as protein expression related to pyroptosis. OVX aggravated mitochondrial dysfunction and liver injury in septic mice was also partly reversed by Mito-tempo and MCC950. These results demonstrated that estrogen protected against sepsis-induced liver damage through alteration of mitochondrial function and activation of inflammatory-mediated pyroptosis signaling pathway.

LOXL2 upregulates hypoxia­inducible factor­1α signaling through Snail­FBP1 axis in hepatocellular carcinoma cells.

Lysyl oxidase­like 2 (LOXL2), a member of the lysyl oxidase gene family, is involved in the progression of hepatocellular carcinoma progression and metastasis. Increased expression of LOXL2 has been identified in several types of cancer, including hepatocellular carcinoma. Recently, LOXL2 has been reported to promote epithelial­mesenchymal transition by reducing E­cadherin expression via the upregulation of Snail expression. The present study provided evidence demonstrating that LOXL2 inhibited the expression of fructose­1, 6­biphosphatase (FBP1) and enhanced the glycolysis of Huh7 and Hep3B hepatocellular carcinoma cell lines in a Snail­dependent manner. Overexpression of the point­mutated form of LOXL2 [LOXL2(Y689F)], which lacks enzymatic activity, does not affect the expression of Snail1 or FBP1. Notably, targeting extracellular LOXL2 of Huh7 cells with a therapeutic antibody was unable to abolish its regulation on the expression of Snail and FBP1. Knockdown of LOXL2 also interrupted the angiogenesis of Huh7 and Hep3B cells, and this effect could be rescued by the overexpression of Snail. Furthermore, upregulation of hypoxia­inducible factor 1α (HIF­1α) and vascular endothelial growth factor (VEGF) expression was observed in Huh7 and Hep3B cells expressing wild­type LOXL2. Notably, the selective LOXL2 inhibitor LOXL2­IN­1 could upregulate the expression of FBP1 and inhibit the expression of Snail, HIF­1α and VEGF in HCC cells, but not in FBP1­knockdown cells. The results of the present study indicated that the intracellular activity of LOXL2 upregulated HIF­1α/VEGF signaling pathways via the Snail­FBP1 axis, and this phenomenon could be inhibited by LOXL2 inhibition. Collectively, these findings further support that LOXL2 exhibits an important role in the progression of hepatocellular carcinoma and implicates LOXL2 as a potential therapeutic agent for the treatment of this disease.

Notch signaling pathway regulates T cell dysfunction in septic patients.

Sepsis disrupts innate and adaptive immune response, and immune disorders may also impact clinical course of sepsis. Notch signaling pathway plays a vital role in T cell modulation and differentiation. The aim of current study was to investigate the immunoregulatory function of Notch signaling pathway to T cells in patients with sepsis and septic shock. Twenty-seven sepsis patients, twenty-five septic shock patients, and twenty-one normal controls (NCs) were enrolled. Notch receptors mRNA levels were semi-quantified by real-time PCR. The absolute numbers of CD3+, CD4+, and CD8+ T cells were measured by flow cytometry. Key transcriptional factors of CD4+ T cells, cytotoxic molecules in CD8+ T cells, and cytotoxicity of CD8+ T cells were investigated. The regulatory activities of Notch signaling inhibition by γ-secretase inhibitor (GSI) on purified CD4+ and CD8+ T cells from sepsis and septic shock patients were also assessed. Notch1 mRNA relative level was significantly elevated in sepsis and septic shock patients when compared with NCs. CD4+ and CD8+ T cells were dysfunctional in sepsis and septic shock, which presented as decreased cell accounts, down-regulation of Th1/Th17 transcriptional factors and cytotoxic molecules (perforin, granzyme B, and FasL), and reduced cytotoxicity of CD8+ T cells. Notch signaling inhibition by GSI increased Th1 and Th17 differentiation of CD4+ T cells. Moreover, GSI stimulation not only promoted perforin, granzyme B, and FasL mRNA expression in CD8+ T cells, but also elevated CD8+ T cell-induced target cell death and IFN-γ/TNF-α production in sepsis and septic shock. The current data suggest that Notch signaling pathway contributes to T cell dysfunction and limited immune response in sepsis.

Heparanase expression in blood is sensitive to monitor response to anticancer treatment in pancreatic cancer, a pilot study.

BACKGROUND: /Objectives: High heparanase level was shown in maliganant tumor; however, whether or not heparanase may serve as a sensitive marker to monitor response to anticancer treatment is still unknown. METHODS: In the pilot study, heparanase mRNA expression in peripheral blood mononuclear cell fraction (PBMC) and activity in plasma and urine were detected by quantitative real time RT-PCR and heparan-degrading enzyme assay in 31 pancreatic cancer patients. RESULTS: Heparanase mRNA and activity in samples from cancer patients were significantly higher than that in healthy donors. Both heparanase mRNA and activity in plasma and urine decreased significantly in 17 patients who underwent R0 resection, but increased remarkably in 6 patients when recurrence or metastasis occurred (P < 0.05). However, those who underwent R1 or R2 resection in 6 patients kept stable. For 8 patients who received chemotherapy, heparanase mRNA and activity in plasma and urine decreased in each of the samples (P < 0.05). Patients with high heparanase mRNA (≥a cutoff value of 1.84) in PBMC and activity in plasma (≥1.30U/ml) were associated with a poor postoperative survival (P = 0.02 and P = 0.04). CONCLUSIONS: Heparanase mRNA in PBMC and activity in plasma are closely correlated with therapeutic responsiveness and survival time, indicating that heparanase level in blood might be a sensitive but non-specific marker to monitor patients' response to anticancer treatment and to predict survival.

Investigation of the effect of nocodazole on the PI3K/Akt signaling pathway in COS7 cells.

A number of studies have demonstrated that nocodazole suppresses Akt phosphorylation; however, the underlying molecular mechanism remains unclear. In the current study, the mechanism of nocodazole­induced suppression of Akt phosphorylation was investigated. The cell cycle was analyzed using flow cytometry and EGF receptor dimerization was evaluated through a cross-linking assay. Immunoprecipitation experiments were performed to investigate the interaction between P85 and EGFR and cell migration was evaluated through a wound healing assay. COS7 cells were observed to be rounded following a 24­h treatment with nocodazole, and the results revealed that ~45% of COS7 cells were arrested at the G2/M phase and that the cyclin B1 expression level was greatly increased. EGF­mediated Akt phosphorylation was markedly inhibited in nocodazole­treated cells. In addition, the levels of internalized EGF­EGFR complexes in nocodazole­treated cells were reduced, and EGF­EGFR dimerization was found to be affected by nocodazole. Akt phosphorylation in COS7 cells was demonstrated to be overridden by AG1478 and wortmannin. The results also showed that p85 did not bind to activated EGFR in nocodazole­treated cells, and that nocodazole and protein inhibitors reduced cell migration. In summary, these results indicate that nocodazole inhibits the PI3K/Akt pathway by interfering with the binding of p85 binding to activated EGFR and further affects the growth of cells.

Effect of the GLP-1 analog exendin-4 and oxaliplatin on intrahepatic cholangiocarcinoma cell line and mouse model.

The influence of Glucagon-like peptide-1 (GLP-1) and Exendin-4 on development of intrahepatic cholangiocarcinoma (ICC) is evaluated in the study. In vitro tests, including acute toxicity test, cell colony formation assays, cells proliferation and apoptosis, transwell assay, were performed. An ICC in situ tumor animal model was established. Then, animals were randomly divided into four groups (n = 6): control, Exendin-4 treatment, oxaliplatin treatment and Exendin-4-oxaliplatin treatment. Animals in the Exendin-4 treatment and Exendin-4-oxaliplatin treatment groups received a subcutaneous injection of Exendin-4 (100 µg/kg/day) for 1 week, and then received oxaliplatin (10 mg/kg/week) by tail vein injection. Animals in the control group received PBS. Immunohistochemistry tests were used for PCNA, Ki67, Caspase 3 expression in tumor tissue. Results show that that, after incubation of human cholangiocarcinoma cell lines, HuCCTI and GLP-1, or HuCCTI and Exendin-4, colony formation number was sharply decreased. However, GLP-1, HuCCTI or Exendin-4 did not affect the colony of normal cells. Combination treatment with oxaliplatin and Exendin-4 can significantly inhibit tumor cells' proliferation and promote apoptosis. The combined effect is stronger than that of oxaliplatin or Exendin-4. Combination treatment with oxaliplatin and Exendin4 can significantly decrease Ki67 and PCNA proteins' expression in subcutaneous tumors of nude mice. The inhibitory effect of Combination treatment with oxaliplatin and Exendin4 is clearly stronger than that of oxaliplatin. In addition, Combination treatment with oxaliplatin and Exendin4 can significantly increase Caspase3 protein positive expression. In short, these results show that combination treatment with oxaliplatin and Exendin4 can inhibit tumor cells' proliferation, and promote apoptosis.

Effects of modified splenocaval shunt plus devascularization on esophagogastric variceal bleeding: a comparative study of this treatment and devascularization only in cirrhotic portal hypertension.

BACKGROUND: Pericardial devascularization (PCDV) and portosystemic shunt were reported to have favorable results for the management of portal hypertension in cirrhotic patients in China and the West, respectively. This study was undertaken to investigate the effects of a modified proximal splenocaval shunt plus PCDV on variceal bleeding in patients with portal hypertension. METHODS: From January 1997 to December 2007, 168 patients with portal hypertension of cirrhotic origin received an operation for gastroesophageal variceal bleeding. Of these, 90 patients received a splenocaval shunt plus a PCDV procedure (Combined Group) and the other 78 patients received a PCDV procedure only (PCDV Group). The procedure-related morbidity and mortality, rebleeding, encephalopathy, and survival rates were analyzed. RESULTS: Postoperative mortality was 3.3% in the combined group and 5.1% in the PCDV group (P > 0.05). Overall morbidity was 13.3% in the combined group and 15.4% in the PCDV group (P > 0.05). The rate for rebleeding, including variceal bleeding and gastropathy, was 5.1% in the combined group, which was significantly lower than that in the PCDV group, at 16.7% (P < 0.05). The incidence of encephalopathy was 6.63% in the combined group and 6.67% in the PCDV group (P > 0.05). The 1-, 3-, 5- and 10-year survival rates were 97.4, 91.7, 80.0, and 60.0% in the combined group and 96.7, 83.3, 73.3, and 53.3% in the PCDV group (P > 0.05). CONCLUSIONS: The modified splenocaval shunt plus PCDV is a safe and effective procedure for the long-term control of variceal bleeding; the procedure may not only maintain the portal flow to the liver, but may also protect the liver function in cirrhotic patients. The better clinical outcome means that the procedure may be one of the best choices for treating portal hypertension of cirrhotic origin.

Hypoxia activates heparanase expression in an NF-kappaB dependent manner.

Hypoxia was shown to increase tumor cell invasion into the extracellular matrix in vitro. This result suggests that heparanase (Hpa), one of the key enzymes involved in tumor invasion and metastasis, may be regulated by hypoxia. RT-PCR, Western blot and Matrigel invasive assays were used to study the regulation of Hpa under hypoxia in human pancreatic MIA PaCa-2 cancer cells. Compared with those in normoxia (20% O2), Hpa mRNA, protein and enzymatic activity levels, were up-regulated by a reduction in the oxygen level (1% O2). Invasion by tumor cells into the extracellular matrix was found to be significantly enhanced. A reduction in Hpa protein levels was observed when nuclear factor kappaB (NF-kappaB) activation was blocked by pyrrolidine dithiocarbamate. The levels of Hpa were also reduced when Hpa was inhibited by an Hpa-specific antisense oligonucleotide. The MMP-9 mRNA, protein and gelatinase B activity levels in supernatants decreased significantly when Hpa was inhibited. We conclude that up-regulation of Hpa by hypoxia is NF-kappaB-dependent in MIA PaCa-2 cells and inhibition of Hpa reduces MMP-9 activity. This reduction in MMP-9 activity may be an important mechanism in tumor metastasis.

[Effects of resveratrol on matrix metalloproteinase-9 expression in hepatoma cells].

OBJECTIVE: To observe the effects of resveratrol on proliferation of human hepatocellular carcinoma cell line SMMC-7721 cells and expression of matrix metalloproteinase-9 (MMP-9) in vitro. METHODS: SMMC-7721 cells were treated with different concentrations of resveratrol for 24, 48 and 72 h, respectively. The effect of resveratrol on proliferation of SMMC-7721 cells was assessed with methyl thiazolyl tetrazolium (MTT). The expression of MMP-9 mRNA was determined by reverse transcription polymerase chain reaction (RT-PCR). MMP-9 protein was identified by Western blot analysis. RESULTS: Resveratrol could inhibit the proliferation of SMMC-7721 cells with dose- and time-dependent effects. Moreover, both MMP-9 mRNA expression and MMP-9 protein production were markedly reduced after resveratrol treatment. CONCLUSION: Resveratrol can inhibit the proliferation of SMMC-7721 cells and down-regulate MMP-9 expression. It is presumed that resveratrol may suppress the invasion and metastasis of hepatocellular carcinoma.

Resveratrol inhibits tumor necrosis factor-alpha-mediated matrix metalloproteinase-9 expression and invasion of human hepatocellular carcinoma cells.

Resveratrol is an active polyphenol found in red wine that has anti-cancer effects, but the molecular mechanisms of resveratrol on tumor invasion inhibition have not been well documented. The aim of this study was to elucidate the effects of resveratrol on invasion ability of human hepatocellular carcinoma cells and TNF-alpha-mediated MMP-9 expression. The expression activity of MMP-9 was measured by zymography, RT-PCR and western blot analysis. The expression of NF-kappa B was measured by EMSA and western blot analysis. TNF-alpha induced the MMP-9 expression in HepG2 cells. Resveratrol significantly inhibited TNF-alpha-mediated MMP-9 expression in HepG2 cells. NF-kappa B inhibitor induced a marked reduction in MMP-9 expression, and it suggested that NF-kappa B could play an important role in TNF-alpha-mediated MMP-9 expression. Furthermore, resveratrol significantly suppressed TNF-alpha-mediated NF-kappa B expression and invasion of HepG2 cells. Our results showed that resveratrol inhibited TNF-alpha-mediated MMP-9 expression and invasion of human hepatocellular carcinoma cells. The inhibitory effects are partly associated with the downregulation of the NF-kappa B signaling pathway.

Heparanase expression in gallbladder carcinoma and its correlation to prognosis.

BACKGROUND AND AIM: Heparanase is an endo-beta-glucuronidase that cleaves heparan sulfate and has been implicated in tumor angiogenesis and metastasis. The present study was to analyze the expression of and explore the prognostic value of heparanase and two important transcriptional factors, namely hypoxia-inducible factor-1alpha (HIF-1alpha) and nuclear transcriptional factor kappa B p65 (NF-kappaB p65) in gallbladder cancer. METHODS: Heparanase, HIF-1alpha and NF-kappaB p65 protein levels in 38 patients with gallbladder carcinoma were detected by immunohistochemistry and analyzed for clinicopathological significance. RESULTS: The heparanase, HIF-1alpha and NF-kappaB p65 proteins were found in 24 (63.2%), 13 (34.2%) and 22 (57.9%) specimens, respectively. High heparanase expression was closely related to advanced TNM stage (P = 0.007), depth of tumor invasion (P = 0.016), lymph node metastasis (P = 0.040) and decreased postoperative survival at 3 years (50.0% vs 20.8%, P = 0.001). Both HIF-1alpha and NF-kappaB p65 proteins were correlated with tumor size (P = 0.039 and P = 0.027, respectively) and patients positive for HIF-1alpha expression had a decreased survival rate compared with those negative for HIF-1alpha expression (40.0% vs 15.4%, P = 0.035). In addition, heparanase-positive cases had high expression of NF-kappaB p65 compared with the heparanase-negative cases (P = 0.047). CONCLUSION: Heparanase and HIF-1alpha are frequently expressed in gallbladder carcinoma and are associated with decreased survival. High expression of heparanase, combined with NF-kappaB p65, may contribute to the highly invasive and metastatic behavior of gallbladder carcinoma.

[Expression of heparanase and nuclear factor kappa B in pancreatic adenocarcinoma].

OBJECTIVE: To detect the expressions of heparanase and nuclear factor kappa B p65 (NF-kappaB p65) in pancreatic adenocarcinomas and analyze their relation to patients' prognosis and the regulatory mechanism of NF-kappaB on heparanase expression. METHODS: Heparanase and NF-kappaB p65 proteins in the tumor and adjacent tissues were detected by immunohistochemistry in 48 patients with pancreatic adenocarcinoma and analyzed for their clinicopathological significance. RESULTS: Heparanase and NF-kappaB p65 proteins were found in 30 (62.5%) and 22 (45.9%) tumor specimens, respectively, a rate significantly higher than that in the adjacent tissues. High heparanase expression was closely related to advanced TNM stage (P=0.031), lymph node metastasis (P=0.003) and decreased 3-year postoperative survival (20.0% vs 0%, P=0.001). NF-kappaB p65 expression was associated with lymph node metastasis (P=0.017) and distant metastasis (P=0.031), but had a higher positive rate in heparanase-positive cases than in heparanase-negative cases (P=0.018). Multivariate analysis showed that neither heparanase nor NF-kappaB p65 was the independent prognostic factors. CONCLUSION: Heparanase is overexpressed in pancreatic adenocarcinomas in association with decreased postoperative survival. NF-kappaB may up-regulate heparanase expression and promote heparanase-dependent tumor invasion and metastasis.

[Efficacy of modified acupotome combined with blocking therapy in patients with carpal tunnel syndrome].

OBJECTIVE: To observe the efficacy of modified acupotome combined with blocking therapy in patients with carpal tunnel syndrome (CTS). METHODS: Fifty-five patients with CTS were divided into three groups, which were modified acupotome group including 26 CTS patients with 28 lesions treated by modified acupotome combined with blocking therapy, traditional acupotome group including 14 CTS patients with 16 lesions treated by traditional acupotome combined with blocking therapy, and blocking therapy group including 15 CTS patients with 15 lesions only treated by local blocking. The treatment outcome and one-year recurrence rate were observed. RESULTS: The response rate and one-year recurrence rate after operation in the modified acupotome group were 85.7% (24/28) and 20.8% (5/24) respectively, which had no significant differences as compared with 81.3% (13/16) and 38.5% (5/13) in the traditional acupotome group. The response rate and one-year recurrence rate after operation in the above two groups were both improved significantly as compared with those in the blocking therapy group which were 46.7% (7/15) and 85.7% (6/7) respectively. There were no acupotome-related adverse effects and injuries observed in the modified acupotome group. CONCLUSION: The modified acupotome is a considerable treatment method for CTS with respect to its simple manipulation and high effectiveness.

[Efficacy of activated charcoal-epirubicin suspension for treatment of breast cancer with axillary metastasis].

OBJECTIVE: To investigate the effect of activated charcoal-epirubicin suspension (Epi-CH) for treatment of breast cancer and clearance of axillary lymph node metastasis. METHODS: Sixty patients with breast cancer of stages II-III were randomized into Epi-CH group (n=40) receiving injection with 10 mg Epi-CH in the tissue around the primary tumor 72 h before modified radical resection and control group (n=20) with 10 mg of aqueous epirubicin injection in the same region. The dissected axillary lymph nodes and the staining lymph nodes were counted. The concentration of epirubicin in the lymph nodes was detected by high-performance liquid chorography, and the specimens of lymph nodes were observed microscopically. RESULTS: In comparison with the control group, Epi-CH injection allowed dissection of 4.04 more lymph nodes (P<0.01) and resulted in the staining rate of the axillary lymph nodes of 86.9% (565/650). The proportion of the staining lymph nodes with diameter>1.0 cm was significantly lower than that with diameter 0.05). In Epi-CH group, the concentration of epirubicin in the stained lymph nodes was significantly higher than that in non-stained ones (443.0-/+123.1 vs 31.8-/+11.9 ng/g, P<0.01). Remarkable degeneration and necrosis could be observed microscopically in the stained lymph nodes. CONCLUSION: Epi-CH allows more effective treatment of breast cancer and clearance of axiliary metastasis by increasing the dissected lymph nodes and raising the concentration of epirubicin in the axillary lymph nodes.

[Impact of preoperative lymphatic chemotherapy on relapse and metastasis of breast cancer and its mechanism].

BACKGROUND & OBJECTIVE: Although it is reported that lymphatic chemotherapy could raise drug concentrations in local lymph nodes and prolong survival time of patients with gastrointestinal tumors, its effect on breast cancer has not been explored. This study was to explore the impact of lymphatic chemotherapy on relapse and metastasis of breast cancer, and to investigate the mechanism. METHODS: Sixty patients with breast cancer of stage II-III were randomized into 2 groups: 40 patients in Epi-CH (carbon activated absorbing epirubicin) group were injected with 10 mg of Epi-CH in the tissue around primary tumor 72 h before modified radical resection; 20 patients in control group were injected with 10 mg of aqueous epirubicin in the same region. The stained nodes full of tumor cells in Epi-CH group and non-stained nodes in control group were selected. The apoptotic index (AI) of cancer cells in metastatic axillary lymph node was calculated by TUNEL method; the expression of Fas/Fas-L proteins was examined by SP immunohistochemistry; the relapse and metastatic rate was compared. RESULTS: The AI of cancer cells in metastatic axillary lymph node was significantly higher in Epi-CH group than in control group [(9.5+/-2.7)% vs. (3.8+/-1.4)%, P<0.01]. The expression of Fas protein was significantly higher in Epi-CH group than in control group (P<0.05), but the expression of Fas-L protein had no difference between the 2 groups (P>0.05). No chemotherapy-related local and whole body reaction occurred in both groups. The relapse and metastatic rate was significantly lower in Epi-CH group than in control group (P<0.05). CONCLUSION: Preoperative Epi-CH lymphatic chemotherapy could suppress relapse and metastasis of breast cancer, which might through up-regulating expression of Fas protein and inducing apoptosis of axillary metastasis cells.

[Effect of preoperative lymphatic chemotherapy on Bcl-2 and Bax expression in axillary metastasis of breast cancer].

OBJECTIVE: To observe the expression of Bcl-2 and Bax proteins and cell apoptosis induced by preoperative lymphatic chemotherapy with epirubicin-activated carbon suspension (Epi-CH) in the cells of axillary metastatic lymph node of breast cancer and investigate the mechanism. METHODS: Sixty patients with breast cancer of stages II-III were randomly divided into two groups. Forty patients in Epi-CH group were injected with 10 mg Epi-CH in the tissue around the primary tumor or biopsy excision 72 h before operation. Twenty patients in the control group were injected with 10 mg epirubicin solution in the same region. The stained lymph nodes full of tumor cells in Epi-CH group and the non-stained nodes in the control group were selected for apoptotic detection by TUNEL method. The expression of Bcl-2 and Bax proteins were examined by SP immunohistochemistry. RESULTS: The apoptotic index of the metastatic cancer cells in Epi-CH group was increased remarkably in comparison with that in the control group [(9.5+/-2.7) % vs (3.8+/-1.4) %, P<0.01). Compared with the control group, the expression of Bcl-2 and Bax proteins were up-regulated significantly in Epi-CH group (P<0.05 and P<0.01, respectively), resulting in decreased ratio of Bcl-2/Bax. CONCLUSION: Lymphatic chemotherapy can promote cell apotosis in axillary metastasis of breast cancer, which may result from decreased ratio of Bcl-2/Bax.