Effect of metabolites of Clostridium butyricum,butyric acid and hydrogen,on acute gastric mucosal lesion

AIM: To observe the antiulcer effect of butyric acid and hydrogen, the main metabolites of Clostridium butyricum (C. butyricum), and to explore the underlying mechanism. METHODS: The mouse model of acute gastric mucosal lesion was prepared by gavage with ethanol. The mice were randomly divided into 4 groups: normal group, model group, butyric acid group and hydrogen group. The mice in butyric acid group and hydrogen group were given butyrate and hydrogen prior to model establishment, respectively. Macroscopic observation of the pathological changes in gastric tissues was performed to evaluate the effect of the 2 metabolites of C. butyricum. Meanwhile, the mRNA expression levels of inflammatory factors, such as IL-12, RAN1 and MCP-1, were determined by RT-qPCR. The expression levels of apoptosis-related proteins Bcl-2 and Bax were detected by immunohistochemical staining. RESULTS: The macroscopic observation found that butyrate, not hydrogen, protected gastric mucosa. HE staining also showed that butyrate significantly attenuated the pathological damage of the gastric mucosa induced by ethanol. Compared with model group, the mRNA levels of inflammatory factors IL-12, RAN1 and MCP-1 in butyrate group significantly decreased (P<0.01). In butyrate group, the protein level of Bax was obviously decreased compared with model group (P<0.01), while the protein level of Bcl-2 was significantly increased (P<0.01). CONCLUSION: The gastric mucosa protective metabolite of C. butyricum may be butyric acid, not hydrogen. Butyric acid protects the gastric mucosa against ethanol-induced lesion by inhibiting the inflammation and reducing the expression ratio of Bax/Bcl-2.     

Protective effect of MUC2 in mice with colitis and the expression of anti-CBir1 antibody in serum

AIM: To investigate the protective effect of mucin 2 (MUC2) on intestinal mucosa of colitis model mice, and to explore the correlation between the expression of anti-CBir1 flagellin antibody and MUC2. METHODS: The mice were randomly divided into normal control group, 2,4,6-trinitrobenzenesulfonic acid (TNBS) group, lipopolysaccharide (LPS)+ovalalbumin (OVA)+TNBS group and ketotifen+TNBS group. The expression of MUC2 in colon tissue was determined by PAS staining and immunohistochemistry, and the anti-CBir1 antibody level in the serum of mice in each group was measured by ELISA. RESULTS: The scores of disease activity index and histological index in TNBS group were higher than those in normal control group (P<0.05). The scores in LPS+OVA+TNBS group were much higher than those in TNBS group (P<0.05). However, the values in ketotifen+TNBS group were lower than those in TNBS group (P<0.05). PAS staining showed a decrease in goblet cells in TNBS group. Compared with TNBS group, the colonic mucosa integrity in LPS+OVA+TNBS group was destroyed, and the number of goblet cells in ketotifen+TNBS group increased significantly. Immunohistochemical staining showed that the expression of MUC2 in the intestinal tract of each mo-del group was basically consistent with the results of PAS staining. The serum anti-CBir1 antibody level in TNBS group was higher than that in normal control group (P<0.05), and that in LPS+OVA+TNBS group was significantly higher than that in TNBS group (P<0.05), whereas that in ketotifen+TNBS group was decreased slightly (P<0.05). CONCLUSION: MUC2 plays a protective role in the pathogenesis of colitis in mice, and there is a negative correlation between the expression of MUC2 and the bacterial flagellin in the intestinal mucosa of mice with colitis.     

Effect of cumin total flavonoids on experimental gastric ulcer and its mechanism

AIM:To discuss the effects of cumin total flavonoids on experimental gastric ulcer and the mechanisms.METHODS:Three rat gastric ulcer models induced by pylorus ligation,ethanol or reserpine were selected.Based on these models,the gastric juice,pH value,ulcer index and suppressive rate were recorded to determine the inhibitory effects of cumin flavonoids on rat gastric ulcers.In addition,the concentrations of gastrin (GAS),prostaglandin E₂(PGE₂) and epidermal growth factor (EGF) were detected to further uncover the mechanisms of these effects.RESULTS:Cumin flavonoids exhibited significant anti-gastric ulcer effects on the three models.Low-dose treatment group (P<0.01) demonstrated higher inhibitory effects than high-dose group (P<0.01),and both low-and high-dose groups had lower effects than cimetidine treatment.Furthermore,cumin flavonoids were able to profoundly decrease the amount of gastric juice,free acidity,total acidity and output of total acid,and enhance the expression of PGE₂ and EGF,but down-regulate the level of GAS in rat serum in a dose-dependent manner.CONCLUSION:Cumin flavonoids might inhibit experimental gastric ulcers by affecting and regulating the gastric juice,PEG₂,EGF and GAS.     

Effects of PFOA exposure on inflammatory mediators IL-4, IFN-γ and glucocorticoid receptor in asthmatic mice

AIM: To investigate the effects of perfluorooctanoic acid (PFOA) exposure on the changes of asthmatic mouse airway inflammation, inflammatory mediators interleukin-4 (IL-4) and interferon-γ (IFN-γ) in serum, and glucocorticoid receptor (GR) expression in the lung tissue.METHODS: BALB/c mice (n=30) were randomly divided into 5 groups:normal control (C) group, asthma (A) group, asthma+low-dose PFOA (AP10) group, asthma+ mode-rate-dose PFOA (AP50) group and asthma+high-dose PFOA (AP100) group. Asthma model and PFOA exposure model of mice were established according to the grouping. The animals were sacrificed and their lungs were collected for HE staining, transmission electron microscopy, Western blot and immunohistochemical staining. ELISA was applied to detect the levels of IL-4 and IFN-γ in the serum.RESULTS: HE staining of the lungs showed that the asthmatic mice, compared with the normal control mice, had obvious mucus secretion around the airways and infiltration of inflammatory cells around airways and blood vessels, and the effects were much more marked in AP groups. Ultrastructural alteration of the lung tissues in the asthmatic mice were indicated by transmission electron microscopy. Compared with C group, the results of ELISA in A group and AP groups proved that IL-4 in the serum was increased and IFN-γ was decreased significantly (P<0.05). Compare with A group, IL-4 was significantly increased and IFN-γ was decreased in AP100 group (P<0.05), and no difference of those between AP10 group and AP50 group was found. The results of Western blot indicated that GR protein expression in the asthmatic mice were decreased compare with the normal mice (P<0.05), and no difference of that among A group and AP groups was observed. Immunohistochemical staining manifested that GR protein was mainly located in the cytoplasm of bronchial columnar epithelial cells, airway smooth muscle cells and vascular smooth muscle cells.CONCLUSION: Acute airway PFOA exposure in asthmatic mice dose-dependently exacebates lung inflammation by inducing Th2 type immune responses, promotes infiltration of inflammatory cells and mucus secretion around the airways and blood vessels, and destroys the ultrastructure of the lung tissues.     

Repair effect of purple sweet potato anthocyanin on intestinal barrier injury in mice with ulcerative colitis induced by DSS

AIM To explore the repair effect of purple sweet potato anthocyanin on intestinal barrier injury of ulcerative colitis mice induced by dextrin sulfate sodium （DSS）. METHODS The mice were randomly divided into normal drinking group， DSS model group， different doses of purple sweet potato anthocyanin （12.5， 25， 50 and 75 mg/kg） groups， and 5-aminosalicylic acid （5-ASA） positive drug control group. Except using normal drinking water for control group， the mice in the other groups were treated with 2.5% DSS in drinking water for 7 days to induce the ulcerative colitis model. The mice in purple sweet potato anthocyanin treatment group and the 5-ASA positive drug control group were given the drug by intragastric gavage on the first day of modeling. The body weight of the mice and the hematocheziawere recorded every day. After continuous administration for 8 days， the mice in each group were killed and colon tissue was retained. Immunohistochemical technique （IHC） was used to detect the expression of tight junction protein ZO-1 and occludin and inflammatory factors tumor necrosis factor-α （TNF-α） and interleukin-6 （IL-6） in the colon of mice. The expression of mucin in goblet cells of colon tissue was observed by glycogen PAS staining. The protein expression of ZO-1， occludin， TNF-α were determined by Western blot， Sirius red staining was used to detect colonic fibrosis in mice. RESULTS Compared with control group， the disease activity index and histological injury of DSS model mice were significantly increased（P<0.01）. Compared with model group， the disease activity index scores of the mice in different dose groups of purple sweet potato anthocyanin were decreased. The expression and distribution of ZO-1 and occludin in colon tissues were increased， and the expression and distribution of TNF-α and IL-6 in colon tissues were decreased. Glycogen PAS staining showed a significant increase in the distribution and expression of mucin in goblet cells of colon tissues in the purple sweet potato anthocyanin treatment group. Sirius red staining also showed that the degree of fibrosis in the purple sweet potato anthocyanin treatment groups was lower than that in model group. CONCLUSION Purple sweet potato anthocyanins has therapeutic effect on ulcerative colitis in mice induced by DSS， mainly through up-regulating the expression of tight junction proteins ZO-1 and occludin， to protect the integrity of intestinal barrier， inhibiting the expression of inflammatory cytokines TNF-α and IL-6 and intestinal fibrosis， to suppress the development of colonic inflammation.     

Activity of H+-K+-ATPase in gastric parietal cells under stress in rats and analysis of electron microscopic enzyme cytochemistry

AIM: To demonstrate the changes of activity and electron microscopic enzyme cytochemistry staining of H⁺-K⁺-ATPase of gastric parietal cells under stress in rats. METHODS: Twenty-four male SD rats were randomly divided into normal group, stress group and stress+omeprazole (OM) group. Water immersion-restraint stress (WRS) model in SD rats was performed. The ulcer index (UI) of gastric mucosa and H⁺-K⁺-ATPase activity of gastric parietal cells were measured. The changes of ultrastructure and electron microscopic enzyme cytochemistry staining of parietal cells were observed under transmission electron microscope (TEM). RESULTS: Compared with control group, the UI of gastric mucosa and H⁺-K⁺-ATPase activity of gastric parietal cells increased (P<0.01 and P<0.05) in stress group. In stress+OM group, both UI and H⁺-K⁺-ATPase activity decreased (P<0.01) compared with stress group. Parietal cells were in a resting state in control group, and became active in stress group, where plenty of intracellular canaliculi were observed under the TEM. In stress+OM group, the dilated intracellular canaliculi lined with rare microvilli were founded. Enzyme cytochemistry staining showed that there was little black punctate enzyme reactive product scatted in intracellular canaliculi and the apical plasma membrane of parietal cells in control group, and there were large amounts of black enzyme reactive product accumulated at the intracellular canaliculi in stress group. Scarcely deposition of enzyme reactive product in intracellular canaliculi was observed in stress+OM group. CONCLUSION: The results indicate that the H⁺-K⁺-ATPase activity of gastric parietal cells increases under WRS, and is in accordance with ultrastructure changes. These findings suggeste that gastric acid might be one of the most important factors that result in stress ulcer.     

Inhalation of inactivated Mycobacterium phlei down-regulates expression of nuclear factor-kappa B and intercellular adhesion molecule-1 in asthmatic mice

AIM:To investigate the effect of inhalation of inactivated Mycobacte-rium phlei on the expression of nuclear factor-kappa B (NF-κB), intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 in the lung tissues of asthmatic mice. METHODS:Male BALB/c mice (n=24) were randomly divided into normal control group (A), asthmatic model group (B), and inactivated Mycobacterium phlei inhalation group (C). Asthmatic model was made by inhalation of chicken ovalbumin. The mice in group C were treated with inactivated Mycobacterium phlei for 5 d. The lung tissues and bronchoalveolar lavage fluid (BALF) were harvested. HE and AB-PAS staining were used to measure the lung inflammation and mucus production. The inflammatory cells in the BALF were counted. The mRNA expression of NF-κB, ICAM-1 and VCAM-1 was detected by real-time fluorescence quantitative PCR. RESULTS:Mycobacterium phlei treatment alleviated lung inflammation, attenuated mucus production, and reduced the percentage of eosinophils in the BALF. The mRNA levels of NF-κB and ICAM-1 were significantly decreased after treated with Mycobacterium phlei. However, no significant difference of VCAM-1 mRNA expression was found before and after treatment. The correlation between NF-κB mRNA and ICAM-1 mRNA, and between NF-κB mRNA and VCAM-1 mRNA was not found. CONCLUSION:Inhalation of inactivated Mycobacterium phlei attenuates asthmatic airway inflammation. NF-κB participates in the pathogenesis of asthma. NF-κB signal pathway may be associated with the therapeutic mechanism. Another important mechanism is the reduction of adhesion molecule expression.     

Effects of gastrin on expression of cyclooxygenase and growth factors in rat gastric mucosa

AIM: To clarify the effects of gastrin on the expression of cyclooxygenase (COX) and several growth factors in rat gastric mucosa. METHODS: Male Sprague Dawley rats were fasted for 24 hours and subcutaneously injected with saline or gastrin 17 at doses of 1 μg/kg, 10 μg/kg and 100 μg/kg, respectively. The expression of COX-1, COX-2, heparin-binding epidermal growth factor-like growth factor (HB-EGF) and hepatocyte growth factor (HGF) in the gastric mucosa were examined using Western blotting and immunohistochemical staining. Effects of a potent gastrin receptor antagonist YM022 on the expression of COX-1, COX-2, HB-EGF and HGF in gastric mucosa were also evaluated. RESULTS: Gastrin dose-dependently increased the expression of COX-2 and HB-EGF in rat gastric mucosa while the expression of COX-1 and HGF did not change significantly after treatment with gastrin. However, pretreatment with YM022 dose-dependently abolished the up-regulation of COX-2 and HB-EGF expression induced by gastrin. CONCLUSIONS: This study demonstrates that gastrin up-regulates COX-2 and HB-EGF expression in rat gastric mucosa, indicating that COX-2 and HB-EGF are involved in pathogenesis of the gastrin-related gastric mucosal hyperplasia and carcinoma of stomach.     

Expression of Wnt1 and LGR5 in gastric mucosa with stress ulcer after TBI in rats

AIM To investigate the expression of Wnt1 and LGR5 in gastric mucosa with stress ulcer after traumatic brain injury （TBI） in rats, and to study the relationship between Wnt1/LGR5 expression and stress ulcer after TBI. METHODS Healthy 7-week-old male SD rats （n=30） were randomly divided into 3 groups： sham operation （sham） group, mild TBI （mTBI） group, and severe TBI （sTBI） group. The mTBI and sTBI were induced by electronic cortical contusion impactor. Neurological severity score （NSS） was calculated 24 and 48 h after modeling. Mucosal blood flow in gastric fundus, greater curvature, pylorus and cardia of anesthetized rats 48 h after injury was measured by Doppler flowmeter. All rats were sacrificed, and their gastric tissues were harvested after 48 h and then stained with hematoxylin and eosin. The protein expression of Wnt1 and LGR5 was analyzed by Western blot and immunohistochemistry. RESULTS Compared with sham group, the NSS in mTBI group and sTBI group was significantly increased （P<0.01）. Compared with sham group, the gastric mucosal blood flow of the rats after TBI was significantly decreased （P<0.01）, and the decrease in sTBI group was more significant than that in mTBI group （P<0.05）. The protein expression of Wnt1 and LGR5 in mTBI group was significantly higher than that in sham group （P<0.05）, and that in sTBI group was significantly higher than that in mTBI group （P<0.05）. Normal glandular gastric tissue was observed, and no abnormal change was found in sham group, while the infiltration of inflammatory cells in gastric lamina propria, mucosa and submucosa was obvious in mTBI group and sTBI group. CONCLUSION Traumatic brain injury activates Wnt1 and LGR5 expression to induce inflammatory changes in gastric mucosa, and then induces stress ulcer. This results provides experimental basis for the pathogenesis and treatment of stress ulcer after trauma.     

Isorhamnetin inhibits ovalbumin-induced pulmonary inflammation in asthmatic mice

AIM To investigate the effect of isorhamnetin on pulmonary inflammation in asthmatic mice and to analyze its primary mechanism. METHODS BALB/c mice were randomly divided into normal control group, asthma model group, isorhamnetin low-dose （50 mg/kg） treatment group and isorhamnetin high-dose （150 mg/kg） treatment group. Ovalbumin （OVA） was used to establish a mouse asthma model. HE staining and PAS staining were used to observe the pathological changes of lung tissue. The contents of cysteinyl leukotriene1 （CysLT1）, cystelinyl leukotriene receptor 1 （CysLTR1）, interleukin-4 （IL-4）, IL-5, IL-13 and tumor necrosis factor-α （TNF-α） in bronchoalveolar lavage fluid （BALF） were measured by ELISA. The expression of nuclear factor of activated T cells 4（NFATc4） in lung tissue was detected by immunohistochemical staining. The protein expression of NFATc4, intercellular cell adhesion molecule-1 （ICAM-1） and vascular cell adhesion molecule-1 （VCAM-1） was determined by Western blot. RESULTS Isorhamnetin improved histopathological changes in OVA-induced asthma model mice, reduced the contents of CysLT1, CysLTR1, IL-4, IL-5, IL-13 and TNF-α in BALF, and reduced NFATc4, ICAM-1 and VCAM-1 expression in lung tissue （P<0.05）. CONCLUSION Isorhamnetin inhibits the inflammatory response of lung tissue in asthmatic model mice, and the mechanism may be related to the inhibition of the calcineurin/NFATc4 signaling pathway.     

朱顶红无菌苗叶片高效再生体系

以朱顶红（Hippeastrum vittatum）叶片为外植体进行离体培养，具有取材方便、试材充足、成本低等优势，但叶片诱导再生率极低，是朱顶红离体培养的一大难题。本试验中分别以‘花孔雀’和‘黑天鹅’朱顶红无菌苗叶片为外植体，探究了不同植物生长调节剂和不同取材部位对不定芽诱导和继代增殖的影响。结果表明：最佳外植体为 MS 培养基中培养 10 d 形成的幼嫩叶片基部（0.5 cm），在光照 16 h · d-1（光照强度 36 μmol · m-2 · s-1）下，不定芽诱导的最适培养基为 MS + 2 mg · L-1 6-BA + 1 mg · L-1 NAA + 2 mg · L-1 TDZ，两个品种的不定芽均以间接途径发生，其中‘花孔雀’在培养 40 d 后形成愈伤组织，55 d形成不定芽，诱导率可达 69.44%；‘黑天鹅’在培养 45 d 后形成愈伤组织，65 d 形成不定芽，诱导率达到 66.67%；最适体细胞胚诱导培养基为 MS + 2 mg · L-1 6-BA + 1 mg · L-1 PIC，‘花孔雀’和‘黑天鹅’的诱导率分别达到 66.67%和 63.89%；最佳不定芽增殖培养基为 MS + 2 mg · L-1 6-BA + 1 mg · L-1 NAA + 1 mg · L-1 TDZ，‘花孔雀’和‘黑天鹅’的增殖系数分别达到 4.67 和 3.46；在不添加植物生长调节剂的 MS培养基中进行生根培养，30 d 后两个品种的生根率均达到 100%；将生根培养 30 d 的小植株转移至室温条件下放置 3 d，摘去封口膜再驯化 3 d 后，移栽至经高温消毒的草炭︰蛭石（体积比）为 1︰1 的基质中，成活率达到 100%     

Effects of Huanglian-jiedu decoction on TLR4/NF-κB signaling pathway and goblet cells of mucosa in rats with allergic rhinitis

AIM To investigate the effect of Huanglian-jiedu decoction （HLJD） on goblet cells and Toll-like receptor 4 （TLR4）/nuclear factor （NF）-κB signaling pathway in allergic rhinitis rats. METHODS The rat model of allergic rhinitis was made by ovalbumin. The model rats were divided into model group, low-, medium- and high-dose HLJD groups, and positive control drug group. The rats in low-, medium- and high-dose HLJD groups were given different doses（5, 10 and 20 g/kg, respectively） of crude drug by intragastric administration, the rats in positive control group was given fluticasone propionate nasal spray （50 μg per side）, and the rats in control group and model group were given normal saline, once per day for 10 days. The behaviors were observed and scored after modeling and treatment, the weight of nasal secretion was measured after the treatment. The goblet cells in nasal mucosa were observed by periodic acid-Schiff （PAS） staining. The morphological changes of nasal mucosa were observed by hematoxylin-eosin （HE） staining. The interleukin （IL）-4 and IL-5 levels in nasal mucosa were measured by ELISA. The mRNA levels of mouse calcium-activated chloride channel 3 （mCLCA3） and mucin 5AC （MUC5AC） were detected by RT-qPCR. The protein expression of TLR4 and NF-κB p65 was determined by Western blot. RESULTS After modeling, compared with control group, the behavioral scores in model group, low-, medium- and high-dose HLJD groups and positive control group were increased （P<0.05）. The eosinophils, neutrophils and lymphocytes in nasal mucosa were seriously infiltrated, inflammatory infiltration was obvious, and obvious small vessel dilatation and interstitial edema in model group were observed. With the increase in the dosage of HLJD, the lymphatic infiltration was obviously relieved, but the eosinophil and neutrophil infiltration still existed, and the inflammatory infiltration was relieved. Compared with control group, the behavioral score, nasal secretion, the relative proportion of goblet cells in the mucosa, the IL-4 and IL-5 levels, the mRNA levels of mCLCA3 and MUC5AC, and the protein expression of TLR4 and NF-κB p65 in the mucosa of model group were increased （P<0.05）. Compared with model group, the behavioral score, nasal secretion, the relative proportion of goblet cells in the mucosa, the IL-4 and IL-5 levels, the mRNA levels of mCLCA3 and MUC5AC, and the protein expression of TLR4 in the mucosa of low-, medium- and high-dose HLJD groups and positive control group were increased （P<0.05）, and the protein expression of NF-κB p65 in the mucosa of medium- and high-dose HLJD groups and positive control group was decreased （P<0.05）. CONCLUSION Huanglian-jiedu decoction reduces the relative proportion of goblet cells in the nasal mucosa of rats with allergic rhinitis, which may be achieved by inhibiting TLR4/NF-κB signaling pathway.     

Effects of glucocorticoid on miRNA-155 expression in CD4+T cells of asthmatic mice

AIM:To investigate the effects of glucocorticoid on the regulation of microRNA-155 (miRNA-155) expression in the CD4⁺ T cells of asthmatic mice. METHODS:The ovalbumin (OVA)-induced asthma mouse model was established and the mice were treated with glucocorticoid. The effects of glucocorticoid on the pulmpnary histopathological changes, the expression of miRNA-155 in the lung tissues and CD4⁺T cells, and the levels of cytokines in the bronchoal-veolar lavage fluid (BALF) were evaluated. RESULTS:The results of RT-qPCR showed that the expressions of miRNA-155 in the lung tissues and CD4⁺T cells from the spleen of asthmatic mice were significantly increased, and the level of miRNA-155 in the CD4⁺T cells was significantly increased with the increase in the allergen exposure time (P<0.01). HE and PAS staining showed that OVA significantly increased inflammatory cell infiltration as compared with control group, and the peribronchial and perivascular inflammation and mucus secretion of proliferative goblet cells were significantly reduced after glucocorticoid treatment. Glucocorticoid treatment inhibited the increase in the proportion of CD4⁺ CD8^- cells in the spleen and decreased the accumulation of CD4⁺ T cells in the lung tissues of asthmatic mice (P<0.01). After glucocorticoid treatment, the levels of interleukin-4 (IL-4), IL-5 and IL-13 in BALF were decreased, while the level of interferon-γ was increased significantly (P<0.01). CONCLUSION:Glucocorticoid reduces the accumulation of CD4⁺ T cells and inhibits the expression of miRNA-155 in the lung tissues and spleen CD4⁺ T cells of asthmatic mice.     

Apoptosis of implanted tumor of human primary gastric cancer cells in nude mice induced by resveratrol

AIM: To investigate the molecular mechanism of the apoptosis of implanted tumor of human primary gastric cancer cells in nude mice induced by resveratrol. METHODS: Human primary gastric cancer cells were planted into nude mice to establish the cancer model. Resveratrol at different doses were injected near the carcinoma on the nude mice. After treatment, transmission electron microscope and TUNEL staining method were used to detect the apoptosis of implanted tumor cells. Immunohistochemical staining and RT-PCR were used to detect the expression of apoptosis-related genes bcl-2 and bax in implanted tumor. RESULTS: Resveratrol significantly inhibited carcinoma growth when it was injected near the carcinoma. The apoptotic cells in implanted tumor induced by resveratrol were detected by transmission electron microscope and TUNEL staining, immunohistochemical staining and RT-PCR showed resveratrol inhibited bcl-2 expression and increased bax expression in human primary gastric cancer cells. CONCLUSION: Resveratrol inhibits implanted tumor of human primary gastric cancer cells in nude mice through inducing apoptosis. This apoptosis may be mediated by down-regulation of bcl-2 expression and up-regulation of bax expression.     

Effect of captopril on AGS nude mouse model of gastric cancer

AIM: To observe the effect of captopril on the genesis and development of gastric cancer, and to explore its clinical treatment feasibility for gastric cancer. METHODS: The human gastric cancer cell line AGS was used to establish a tumor model in nude mice, and the model mice were randomly divided into 3 groups: positive control (5-fluorouracil) group, normal control (saline) group and experimental (captopril) group. After intraperitoneal injection or intragastric administration of the drugs, the tumor growth curve was determined, and the tumor tissues were also sampled to detect the expression of Ki-67, STAT3, Bax and Bcl-2 by real-time quantitative PCR and immunohistochemistry. The apoptosis was detected by TUNEL+DAPI staining. RESULTS: The tumor growth curve showed that the tumor model in the nude mice was successfully established. The tumor volumes among groups showed significantly different after 14 d growth. The increase in the tumor volume in normal control group was significantly faster than that in the other two groups, and that in positive control group was the slowest. The expression of Bax in captopril group increased, and the expression of STAT3, Ki-67 and Bcl-2 was reduced as compared with normal control group and positive control group. Compared with normal control group, the apoptotic rate increased significantly, and the protein expression of p-STAT3 and STAT3 decreased obviously in positive control group and captopril group. CONCLUSION: With better feasibility, angiotensin-converting enzyme inhibitor captopril has a significant effect on treating gastric cancer in the AGS nude mouse model by regulating the expression of STAT3, Bax, Bcl-2 and Ki-67 to accelerate the apoptosis of cancer cells, thus inhibiting tumor growth.     

Role of resistin in hepatic insulin resistance and the underlying mechanism

AIM: To investigate the role of resistin in hepatic insulin resistance and its mechanism. METHODS: A mouse model of hyperresistinemia in C57BL/6 mice was established by intravenous administration of the recombinant adenovirus encoding mouse resistin. Using periodic acid-Schiff staining we observed the effects of resistin on hepatic glycogen storage. Western blotting was used to measure AMPK-α protein and phosphrylated AMPK-α (Thr172) protein. mRNA levels of phosphoenolpyruvate carboxykinase gene and glucose-6-phosphatase gene were measured by real-time PCR. RESULTS: On day 5 after Adv injection, the concentration of plasma resistin was much higher in Adv-resistin-EGFP-treated mice than that in saline- or Adv-EGFP-treated mice. Semiquantitation of hepatic glucogen storage by PAS showed that the mice with hyperresistinemia had decreased glycogen particles compared to normal control and Adv-EGFP groups. The ratio of phosphorylated AMPK (Thr172)-α to total AMPK-α was used to evaluate hepatic AMPK activation. Compared with normal control and Adv-EGFP groups, the Adv-resistin-EGFP-treated mice had significantly lower ratio of p-AMPK/AMPK, and higher expression levels of G6Pase and PEPCK mRNA in liver. CONCLUSION: Resistin may decrease AMPK activation with downregulating expression of gluconeogenic enzymes, resulting in increased glucose production and decreased hepatic glycogen storage. Resistin may play an important role in hepatic insulin resistance.     

Exogenous hydrogen sulfide attenuates obstructive kidney injury in mice

AIM:To investigate the protective effect of exogenous hydrogen sulfide (H₂S) on obstructive renal injury in mice, and to explore the possible potential mechanisms involved in this animal model. METHODS:Male C57BL/6 mice (8 weeks old) were randomly divided into sham group, operation group and H₂S group, with 5 rats in each group. The model of obstructive renal injury was induced by unilateral ureteral obstruction (UUO). The mice in H₂S group were intraperitoneally injected with NaHS daily, while the mice in sham group and operation group were administered with the same volume of saline intraperitoneally. After 7 d, the mice were executed and the renal tissues were taken out for experiments. RNA was extracted to detect the mRNA expression of H₂S catalytic enzymes in the mice of 3 groups. HE staining was performed to observe the structural changes of renal tissues in the mice. Renal fibrosis in the mice of 3 groups was evaluated by Masson staining. The content of cystatin C in the plasma was detected to reflect glomerular filtration ability. The protein expression of LC3, beclin-1 and fibronectin (FN) in the mice of 3 groups was determined by Western blot. RESULTS:Compared with sham group, the mRNA expression of cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3-MST) in operation group decreased significantly. The collagen fiber content in operation group was increased significantly, while collagen fiber content in H₂S group was decreased significantly as compared with operation group. Compared with sham group, the protein expression of FN in operation group was increased significantly, while the protein expression of FN in H₂S group was decreased significantly as compared with operation group. Compared with sham group, the protein expression of LC-Ⅱ and beclin-1 in operation group was increased significantly, while the protein expression of LC-Ⅱ and beclin-1 in H₂S group was increased significantly as compared with the operation group. CONCLUSION:Exogenous H₂S possibly mitigates renal fibrosis in UUO mice by up-regulating autophagy.     

Role of humoral immune response in the protection induced by H. pylori vaccine with chitosa as adjuvant

AIM:To study the immunological protection of H. pylori vaccine with chitosa as adjuvant. METHODS:One-grade female BALB/c mice were randomly divided into nine groups and immunized by ①PBS alone; ②chitosan solution alone; ③chitosan particles alone; ④H. pylori antigen alone; ⑤H. pylori antigen plus chitosan solution; ⑥H. pylori antigen plus chitosan particles; ⑦H. pylori antigen plus CT; ⑧H. pylori antigen plus chitosan solution and CT; ⑨H. pylori antigen plus chitosan particles and CT. At 4 weeks after the last immunization, these mice were challenged by alive H. pylori（1×1012CFU/L） twice at two-day intervals. At 4 weeks after the last challenge, these mice were all killed and gastric mucosa were embedded in paraffin, sectioned and assayed with Giemsa staining. The other gastric mucosa were used to quantitatively culture with H. pylori. ELISA was used to detect H.pylori IgA in saliva and gastric mucosa and anti-H.pylori IgG, IgG1, IgG2a in serum, and immunohistochemical method was used to examine sIgA in gastric mucosa. RESULTS:①In the groups with chitosan as adjuvant, 60% mice achieved immunological protection, which was according to that with CT as adjuvant (58.33%), and was significantly higher than H. pylori antigen alone and other groups without H. pylori antigen（P<0.01 or P<0.05）. While the rates of protection in the groups with chitosan plus CT as adjuvant were 84.62%,85.71% and the H. pylori colonization score in it was significantly lower than that in the groups with CT as an adjuvant and without adjuvants（P<0.01 or P<0.05）. ②The each isotype IgG levels induced by H. pylori vaccine with adjuvants were significantly higher than those in control group and non-adjuvant groups（P<0.01 or P<0.05），while the anti-H. pylori IgG levels in the groups with CT plus chitosan as adjuvant were significantly higher than those in the groups with CT or chitosan as an adjuvant alone（P<0.05）. ③ The labeling index for sIgA-positive lumen of glands and special anti-H. pylori IgA levels in gastric mucosa in the groups with chitosan as an adjuvant had no difference with those in the group with CT as an adjuvant（P>0.05）and were significantly higher than those in non-adjuvant groups, while those in the groups with chitosan plus CT were significantly higher than those in the group with CT as an adjuvant（P<0.01 or P<0.05）. CONCLUSION:H. pylori vaccine with chitosan as adjuvant could protect against H. pylori infection, suggested that chitosan is a mucosa adjuvant of H. pylori vaccine.     

Protective role of heat-shock protein 70 in gastric mucosal lesion induced by endotoxin in cirrhotic rats with portal hypertensive gastropathy

AIM: To investigate the protective role of heat-shock protein 70 (HSP70) in the pathogenesis of gastric mucosal damage in cirrhotic rats with portal hypertensive gastropathy (PHG).METHODS: The rat model of liver cirrhosis with PHG was established by injection with tetrachloride.The animals were divided into normal control group, PHG group, PHG+heat treatment group, PHG+BPI21 group and PHG+endotoxin groups.The endotoxin used in the experiment was at the dose of 3 mg/kg and endotoxin antagonist BPI21 was at the dose of 2 mg/kg.HSP70 was induced by pre-treating the animals with mild whole-body heating.The levels of HSP70 and tumor necrosis factor alpha (TNF-α) in the gastric mucosa were measured by ELISA.Furthermore, the pathological changes of the gastric mucosa were observed under microscope with HE staining.RESULTS: Compared with the normal control rats, the rats in PHG group showed obvious gastric pathological lesion, decrease in HSP70 production and increase in TNF-α level in the gastric mucosa, and increased endotoxin concentration in the plasma.Compared with PHG+endotoxin group, the gastric mucosal lesion in PHG+BPI21 group was significantly attenuated, accompanied by the increase in HSP70 production and decrease in TNF-α level in the gastric mucosa.Heat treatment increased HSP70 production and decreased TNF-α concentration in the PHG rats, thus attenuating the gastric mucosal damage.CONCLUSION: HSP70 alleviates the gastric mucosal lesion induced by endotoxin in cirrhotic rats with PHG and decreases the concentration of TNF-α in gastric mucosa, indicating a protective role of HSP70 in the pathogenesis of gastric mucosal damage in PHG.