[Effect and mechanism of free total rhubarb anthraquinones on pyroptosis of J774A.1 macrophages].

In this study, J774A.1 macrophages stimulated by lipopolysaccharide(LPS) and adenosine triphosphate(ATP) were used to establish an in vitro model of pyroptosis, and the intervention mechanism of free total rhubarb anthraquinones(FTRAs) on pyroptosis was investigated. J774A.1 macrophages were cultured in vitro, and the experiment was assigned to the control group and groups with different concentrations of LPS(0.25, 0.5, and 1 µg·mL~(-1)) and ATP(1.25, 2.5, and 5 mmol·L~(-1)). An in vitro model of macrophage pyroptosis was established by detecting cell viability through CCK-8, propidium iodide(PI) apoptotic cell staining, lactate dehydrogenase(LDH), interleukin(IL)-18, and tumor necrosis factor(TNF)-α release. Then, J774A.1 macrophages were randomly divided into six groups: blank control group, LPS+ATP group, high-dose FTRA group, and low, medium, and high-dose FTRA pre-protection group. The phenotypic characteristics and key indicators of pyroptosis were detected as the basis for evaluating the effect of FTRAs on pyroptosis induced by LPS and ATP. Western blot and RT-PCR were used to detect the expression levels of protein and mRNA related to the pyroptosis pathway in caspase-1/11 and elucidate the molecular mechanism of the anti-pyroptosis effect. The results showed that the stimulation condition of 0.50 µg·mL~(-1) LPS+5.00 mmol·L~(-1) ATP was the most effective in the in vitro model of macrophage pyroptosis. FTRAs pre-protected cells for 24 h and then can increase cell viability under pyroptosis conditions, alleviate cell damage, lower the positive rate of PI staining, and reduce the release of LDH, IL-18, and TNF-α. FTRAs were able to significantly inhibit the activation of GSDMD proteins and significantly down-regulate the protein expression of the pyroptosis pathway signature molecules, TLR4, NLRP3, cleaved-caspase-1, and cleaved-caspase-11, but they had no significant effect on ASC proteins. FTRAs were also able to significantly inhibit the mRNA expression of caspase-1, caspase-11, and GSDMD. These results indicate that FTRAs have an inhibitory effect on the pyroptosis model induced by LPS and ATP and play an anti-pyroptosis effect by regulating classical and non-classical pyroptosis signaling pathways and reducing the production of inflammatory cytokines.

Influence of Intestinal Lymphatic Ligation on Pulmonary Injury in Rats with Severe Acute Pancreatitis.

OBJECTIVE: The present study explored the mechanisms involved in intestinal lymphatic ligation in rats with severe acute pancreatitis (SAP). METHODS: Male Sprague Dawley rats were randomly divided into 4 groups: saline group, saline+ligation group, SAP group, and SAP+ligation group. Rats in the SAP group were administered sodium taurocholate solution. Isolated mesenteric lymph duct ligation was administered to the saline+ligation and SAP+ligation groups. Endotoxin (ET), nitric oxide (NO), tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1), myeloperoxidase (MPO), and superoxide dismutase (SOD) were detected. Nuclear factor-κB (NF-κB) and intercellular cell adhesion molecule-1 (ICAM-1) proteins were observed. The mRNA of inducible nitric oxide synthase(iNOS) and Toll-like receptor 4 (TLR4) was detected by PCR. RESULTS: Pathomorphological analysis showed that necrosis was present in the lung of rats in the SAP group, but only mild lesions in the SAP+ligation group. ET, NO, TNF-α, and IL-1 in the serum and lung tissue were significantly decreased and MPO was increased in the SAP+ligation group as compared with the SAP group. However, MPO was increased. The expression of NF-κB and ICAM-1, either iNOS or TLR4, was upregulated in the SAP group, but downregulated in the SAP+ligation group. Intestinal lymph duct ligation prevented ET translocation, the release of inflammation factors, and inflammation injury. CONCLUSION: The intestinal lymph duct ligation could alleviate SAP-induced pulmonary injury by suppressing NF-κB activation in rats.

[Effect of effective fractions and its compatibilities and proportions of xie-xin decoction on nitric oxide production in peritonea macrophages from rat].

OBJECTIVE: To observe the effect of effective fractions (Conjunct anthraquinone, free anthraquinone and total flavonoids) and its compatibilities and proportions of Xie-Xin decoction on NO production in peritonea macrophaes from rat. METHODS: Growth activity of macrophages cultured with different levels of active components were detected by MTT. NO concentrations in peritoneal macrophages induced by LPS were detected by Griess method. RESULTS: The NO production from macrophages induced by LPS was inhibited obviously by active components at the levels of 0.01-0.1 mg/ml. The best time of administration was 1 h induced by LPS. The inhibition of best proportions of compatibilities of Conjunct anthraquinone and total flavonoids, free anthraquinone and total flavonoids were stronger than effective fractions solo. CONCLUSION: The NO production is inhibited obviously by these effective fractions, especially by conjunct anthraquinone. Effective fractions can inhibit the function of activated macrophages.

[Role of cyclooxygenase-2 in pulmonary microcirculation disorder in rats with acute pancreatitis].

OBJECTIVE: To identify the expression of Cyclooxygenase-2 (Cox-2) in lung tissues and its potential role in pulmonary microcirculation disorder in rats with acute pancreatitis. METHODS: The acute hemorrhagic and necrotic pancreatitis (AHNP) model was induced by the standard retrograde infusion of bilio-pancreatic duct with 4% sterile sodium taurocholate solution in Sprague-Dawley rats. The rats were randomly allocated into sham surgery group, AHNP group, and prophylactic celecoxib treated AHNP (C+ AHNP) group. The HE, phosphotungstic acid hematain (PTAH) and immunohistochemistrical (IHC) staining were employed to assess the dynamical alterations and interrelations of the histopathology, density of micro-thrombus and Cox-2 expression of lung tissue respectively over a time course of 3, 6, 12 and 24 hours. RESULTS: A significant and progressing increase in histopathologic scoring and Cox-2 expression in the lung tissues were found. There was a positive correlation between the Cox-2 expression and the histopathological scoring. A significant increase of pulmonary micro-thrombosis was detected at the early stage of the rat AHNP induced by sodium taurocholate, and the density of micro-thrombus was positively associated with the histopathological scoring. The prophylactic treatment with celecoxib, a highly selective inhibitor of Cox-2, attenuated the changes in histopathology, pulmonary micro-thrombosis and Cox-2 expression (P < 0.05). The down-regulated intensity of the Cox-2 expression by celecoxib was positively correlated with the improvement of the histopathology injury, but not with the change of the pulmonary micro-thrombosis. CONCLUSION: The pulmonary microcirculation dysfunction caused by micro-thrombosis is an early event or even an enabler of the development of APALI. The over-expression of Cox-2 may have promoted the procoagulant activity which plays a key role in the development of pulmonary thrombus.

Inactivation of PTEN is associated with increased angiogenesis and VEGF overexpression in gastric cancer.

AIM: To investigate the expression of PTEN/MMAC(1)/TEP(1) and vascular endothelial growth factor (VEGF), their roles in biologic behavior and angiogenesis and their association in gastric cancer. METHODS: Immunohistochemical staining was used to evaluate the expression of PTEN, VEGF and microvascular density (MVD) on paraffin-embedded sections in 70 patients with primary gastric cancer and 24 patients with chronic superficial gastritis (CSG). Expression of PTEN, VEGF and MVD were compared with clinicopathological features of gastric cancer. The relationship between expression of PTEN, VEGF and MVD as well as the relationship between PTEN and VEGF expression in cancer cells were investigated. RESULTS: PTEN expression significantly decreased (t = 3.98, P<0.01) whereas both VEGF expression and MVD significant increased (t = 4.29 and 4.41, respectively, both P<0.01) in gastric cancer group compared with CSG group. PTEN expression was significantly down-regulated (t = 1.95,P<0.05) whereas VEGF expression (t = 2.37, P<0.05) and MVD (t = 3.28, P<0.01) was significantly up-regulated in advanced gastric cancer compared with early-stage gastric cancer. PTEN expression in gastric cancer showed a negative association with lymph node metastasis (t = 3.91, P<0.01), invasion depth (t = 1.95, P<0.05) and age (t = 4.69, P<0.01). MVD in PTEN-negative gastric cancer was significantly higher than that in PTEN-positive gastric cancer (t = 3.69, P<0.01), and there was a negative correlation between PTEN expression and MVD (gamma= -0.363, P<0.05). VEGF expression was positively associated with invasion depth (especially with serosa invasion, t = 4.69, P<0.01), lymph node metastasis (t = 2.31, P<0.05) and TNM stage (t = 3.04, P<0.01). MVD in VEGF-positive gastric cancer was significantly higher than that in VEGF-negative gastric cancer (t = 4.62, P<0.01), and there was a positive correlation between VEGF expression of and MVD (gamma= 0.512, P<0.05). VEGF expression in PTEN-negative gastric cancer was significantly stronger than that in PTEN-positive gastric cancer (t = 2.61, P<0.05), and there was a significantly negative correlation between the expression of VEGF and PTEN (gamma= -0.403, P<0.05). CONCLUSION: Our results imply that inactivation of PTEN gene and over-expression of VEGF contribute to the neovascularization and progression of gastric cancer. PTEN-related angiogenesis might be attributed to its up-regulation of VEGF expression. PTEN and VEGF could be used as the markers reflecting the biologic behaviors of tumor and viable targets in therapeutic approaches to inhibit angiogenesis of gastric cancers.