Roles of novel cannabinoid chemicals O-1602 and cannabidiol in DSS-induced mouse colitis

AIM:To investigate the roles of O-1602 and cannabidiol(CBD) in dextran sulfate sodium(DSS)-induced mouse colitis. METHODS:The model of colitis was induced in C57BL/6 mice by drinking water containing 4% DSS for 7 days. The model mice were treated with O-1602(5 mg/kg), CBD(1 mg/kg) or SB203580. A colitis scoring system was used to evaluate the colon local lesion, and the systemic inflammatory responses were observed by detecting the plasma levels of tumor necrosis factor α(TNF-α), interleukin 6(IL-6) and cytokine-induced neutrophil chemoattractant 1(CINC-1), and the activity of myeloperoxidase(MPO) in the lung tissues. The expression of G protein-coupled receptor 55(GPR55) was detected by the method of immunohistochemistry. The expression of p38 and phosphorylated p38(p-p38) in colon tissues was determined by Western blotting. RESULTS:O-1602 and CBD improved the pathological changes in the mice with DSS-induced colitis and decreased the plasma levels of TNF-α, IL-6 and CINC-1, and the activity of MPO in the lung tissues(P<0.05). Lower expression of p-p38 was observed after treatment with O-1602, CBD and SB203580(P<0.05). The expression of GPR55 was mainly in the submucosa of mouse colon tissues. CONCLUSION:O-1602 and CBD show protective effect on the mice with experimental colitis, and the anti-inflammatory roles of O-1602 and CBD are related to the inhibition of p38 MAPK. The expression level of GPR55 in the submucosa of mouse colon tissue is low.     

Effects of new kinds of cannabis preparations O-1602 and cannabidiol on experimental acute pancreatitis

AIM: To investigate the therapeutic effects of O-1602 and cannabidiol (CBD), the new kinds of cannabis preparations, on caerulein (CAE)-induced acute pancreatitis (AP) in mice.METHODS: AP was induced by intraperitoneal injection (ip) of CAE in mice (50 μg/kg hourly with a total of 6 times), and the mice in control group were given normal saline (NS) ip in stead of CAE in the same way. The AP mice were administrated O-1602 or CBD for the therapeutic evaluation by observing the following parameters: pathological changes of pancreatic tissue, plasma activity of amylase and lipase (biochemical methods), the levels of TNF-α and IL-6 in the plasma (ELISA), and the activity of myeloperoxidase (MPO) in the lung (biochemical methods). Meanwhile, real-time PCR and Western blotting were used to evaluate the expression of heat shock protein 60 (HSP60) at mRNA and protein levels, respectively. RESULTS: The pancreatic tissues in AP group appeared obvious edema and neutrophil infiltration, which were significantly improved by treating with AP+O-1602 or AP+CBD. The activity of amylase, lipase and MPO, as well as the levels of TNF-α and IL-6 in AP group significantly increased compared with NS group (P<0.05), while these parameters were significantly lower in AP+O-1602 group and AP+CBD group than those in AP group. Meanwhile, the expression of HSP60 at mRNA and protein levels in pancreas tissues was reduced in AP group (P<0.05), and was improved to some extent after treating with O-1602 or CBD (P<0.05).CONCLUSION: The O-1602 and CBD show anti-inflammatory effects on CAE-induced AP in mice and the mechanisms might be related to the effects of cannabinoids on inhibiting inflammatory mediators and cytokines, and increasing the expression of cytoprotective factor HSP60.     

Protective effects of grape polyphenol on pancreatic tissues of mice with caerulein-induced acute pancreatitis

AIM:To investigate the effects of grape polyphenol (GP) on caerulein-induced acute pancreatitis (AP) in mice. METHODS:Two-month-old female mice of ICR strains (n=21) were randomly divided into 3 groups: normal control (NC) group, AP group, and GP-treated AP group. Before AP induction, the mice in GP-treated AP group were continuously administrated with 1.5 g/kg GP aqueous solution by gavage for 7 d, while those in NC group and AP group were treated with saline as a vehicle control. On the 7th day, the mice in AP group and GP-treated AP group were intraperitoneally injected with caerulein (50 μg/kg) in 1 h interval for 7 serial injections in total. The mice in NC group were treated with saline according to the same procedure in experimental group. All the mice were sacrificed 24 h after AP induction, and the pancreatic tissues and lung tissues were harvested for further investigation of the pathological changes, macrophages infiltration, myeloperoxidase (MPO) activity and expression of inflammatory and oxidative stress factors. RESULTS:Compared with AP group, the mice in GP-treated AP group showed milder morphological changes and lower pathological scores, including the scores of edema, inflammation and vacuolization (P<0.05), but the necrosis scores and total scores showed no statistical difference between these 2 groups. Besides, the mice in GP-treated AP group had fewer macrophage infiltration, lower lung MPO activity (P<0.01), and lower expression of inflammatory factors, tumor necrosis factor α (TNF-α)and monocyte chemotactic protein 1 (MCP-1) (P<0.05), and oxidative stress factors, superoxide dismutase (SOD)-1, SOD-2 and NADPH oxidase 2 (NOX-2) (P<0.01). CONCLUSION: Grape polyphenol has remarkable protective effect on pancreatic tissues of mice with caerulein-induced acute pancreatitis, and the mechanisms may be related to down-regulation of inflammatory and oxidative stress factors.     

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.     

Strain difference of susceptibility to caerulein-induced acute pancreatitis in mice

AIM: To study the difference of susceptibility to caerulein-induced acute pancreatitis (AP) among the mice of C57BL/6J, BALB/c and ICR strains.METHODS: Two-month-old female mice of C57BL/6J, BALB/c and ICR strains (12 mice for each strain) were divided into control group (n=6) and experimental group (n=6), respectively. The mice were intraperitoneally injected with caerulein (50 μg/ kg) in 1 h interval for 7 serial injections in total. The mice in control group were treated with saline according to the same procedure in experimental group. The blood samples were collected at 0 h, 3 h, 6 h, 9 h, 12 h and 24 h after the first injection of caerulein or saline for plasma α-amylase and lipase assays. The mice were sacrificed 24 h after AP induction, and the pancreatic tissues were harvested for further investigating the pathological changes and expression of inflammatory factors.RESULTS: After AP induction, the mice of BALB/c and ICR strains demonstrated more dramatic increase in plasma α-amylase activity and lipase activity than those of C57BL/6J mice. C57BL/6J mice showed milder morphological changes and lower expression of inflammatory factors in pancreata than those of BALB/c and ICR mice.CONCLUSION: The mice of C57BL/6J strain have less susceptibility to caerulein-induced AP than that of BALB/c and ICR mice.     

Expression and potential role of chaperonin 60 in experimental acute pancreatitis

AIM:To explore the expression and potential impacts of chaperonin 60 (Cpn60) in the hepatic and pancreatic tissues from animals endured experimental acute pancreatitis (AP) with various severities. METHODS:Induction of mild acute pancreatitis (MAP) in mice was made by intraperitoneal injection of caerulein, and sodium deoxycholate was used by injection through pancreato-biliary duct backward to induce severe acute pancreatitis (SAP) in rats. The liver and pancreas from sacrificed animals at 1 h, 5 h and 10 h time points post-induction of AP were harvested for pathological examination and observing the dynamic change of Cpn60 expression with techniques of immunoprecipitation (IP) and Western blotting. RESULTS:In the MAP and SAP models, pancreatic tissues showed swollen or hemorrhagic necrotic changes, respectively. The characteristic differences of Cpn60 expression were also observed. The Cpn60 protein was expressed as two distinctive bands in pancreatic and hepatic tissues, and relative densities of the two bands varied differently at these time points in both AP models. CONCLUSION:The results suggest that not only the quantitative, but also, probably, qualitative abnormalities of Cpn60 expression in AP exist. These abnormalities may play important roles in the pathogenesis and development of acute pancreatitis.     

Effects of new cannabis preparations O-1602 and cannabidiol on LPS-induced intestinal motility disorder in rodents

AIM: To investigate the therapeutic effects and related mechanisms of two new cannabis preparations, O-1602 and cannabidiol (CBD), on lipopolysaccharide (LPS)-induced rodent models of intestinal motility disorder in vivo and in vitro. METHODS: The animal model of intestinal motility disorder was induced by intraperitoneal injection of LPS in mice. The gastrointestinal transit was measured by gavaging charcoal marker. Western blotting was applied to evaluate the protein expression of G-protein-coupled receptor 55 (GPR55). Meanwhile, the levels of tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6) were tested by ELISA to assess the inflammatory degree. Smooth muscle strips from the rat and mouse ileum were incubated with LPS in vitro to establish motility disorder, and both the spontaneous contraction and electrically-evoked contraction were recorded using the organ bath technique. The traditional intracellular microelectrode technique was used to record the changes of membrane potential of smooth muscle cells. The method of determining phosphorus content was applied to assay the Ca²⁺-ATPase activity in smooth muscle tissues. RESULTS: In vivo, LPS resulted in significant inflammation and the disorder of gut movement (P<0.01). Pretreatment with CBD decreased both the level of IL-6 (P<0.01) and the expression of GPR55 (P<0.01), and further improved the motility of gut movement (P<0.05). O-1602 and CBD selectively normalized LPS-induced spontaneous and electrically-evoked contraction disorder of intestinal smooth muscle strips of rats and mice in vitro (P<0.05 or P<0.01), but they had no effect on the membrane potential of the smooth muscle cells both in normal and pathophysiological states. CBD also decreased the elevated Ca²⁺-ATPase activity in smooth muscle tissues induced by LPS (P<0.05). CONCLUSION: In vivo, CBD shows protective effect on LPS-induced intestinal motility disorder by reducing inflammation and down-regulating GPR55 expression. O-1602 and CBD counterbalance LPS-induced intestinal motility disorder to some extent in vitro, and the possible mechanism may be involved in regulating the Ca²⁺-ATPase activity of smooth muscle tissues, but not including the change of membrane potential.     

Escherichia coli promotes recovery from dextran sulfate sodium-induced colitis in mice

AIM: To investigate the important role of intestinal microflora in the pathogenesis of inflammatory bowel disease by studying the effects of E.coli on dextran sulfate sodium (DSS)-induced colitis in mice. METHODS: BALB/c mice were given 3.5% DSS in the drinking water for 5 days to induce acute colitis. The control mice (n=6) did not drink DSS water. The mice subject to drinking DSS were randomly divided into 3 groups: (1) simple DSS treatment group; (2) bacteria-depleted mice treated with DSS alone; (3) bacteria-depleted mice treated with DSS+E.coli. Four aspects of the treatment response were evaluated in each group: (1) general conditions, including body weight, disease activity index (DAI) score, colon length and weight; (2) histological score; (3) chemical colorimetric detection of myeloperoxidase (MPO) activity in the diseased tissues; (4) the activation of NF-κB detected by immune histochemistry. RESULTS: The bacteria-depleted mice that did not treat with E.coli were difficult to recover from DSS-induced colitis. Compared with non-treatment group, the general score, histological score and MPO activity in E.coli treatment group improved significantly (P<0.05). The activity of NF-κB in E.coli treatment group was significantly higher than that in non-E.coli treatment group (P<0.05). CONCLUSION: Intestinal microbiota is necessary during the recovery from DSS-induced colitis. E.coli promotes the recovery from DSS-induced colitis in mice. The mechanism may be associated with NF-κB activation.     

Effect of limb ischemic postconditioning on brain focal ischemia and reperfusion injury in mice

AIM: To establish the mouse model in which the limbic ischemic postconditionning (LIPostC) enhances the tolerance against brain ischemia, and to investigate the effects of LIPostC on the ischemic extent and roles of heat shock protein 70 (HSP70) in ischemia and reperfusion injury. METHODS: The male Kunming mice were used in the study. The brain ischemia reperfusion (I/R) model was made by middle cerebral artery occlusion (MCAO). In the first test, the male mice were randomly divided into 9 groups (n=10): sham group, ischemia/reperfusion (I/R) groups (with ischemia for 0.5 h, 1 h,1.5 h and 2 h) and LIPostC+I/R groups (0.5 h+LIPostC,1 h+LIPostC,1.5 h+LIPostC,2 h+LIPostC). The reperfusion was performed after LIPostC for 24 h. After the neurologic deficit scores were evaluated, the brains were taken out to measure the infarct volume with TTC staining and to observe the pathological changes of cerebral cortex with HE staining. The neuronal apoptosis was determined by TUNEL. In the second test, the male mice were randomized into 4 groups (n=6): sham group, I/R group, LIPostC+I/R group and LIPostC+I/R+quercetin group (2 h ischemia). The neurological deficit scores were evaluated at 24 h after operation. The expression of HSP70 was determined by Western blotting.RESULTS: The duration of brain ischemia was related to the motor behavior and degree of brain injury. The longer the ischemic duration of the brain was performed, the more severe the pathological and behavioral changes were observed. The brain injury in 2 h MCAO mice was more severe than that in 1 h and 1.5 h MCAO mice (P<0.05). Compared to I/R group, each LIPostC group showed lower neurological score, less infarct volume and TUNEL positive neuron. The expression of HSP70 protein was increased and neurological functions were improved significantly in the mice with LIPostC. However, the neuroprotective role of LIPostC was attenuated by treating with quercetin, an inhibitor of HSP70.CONCLUSION: LIPostC promotes the expression of HSP 70, improves the neurological functions and attenuates the ischemia and reperfusion injury in MCAO mice. HSP70 produces a marked effect on the ischemic tolerance induced by LIPostC in MCAO mice.     

Effect of heat shock protein on dendritic cell maturation

AIM: To study the effect of heat shock protein (HSP) on the maturation of dendritic cells (DCs) and observe the morphological changes of DCs dynamically. METHODS: The HSP (gp96)-peptide complex was purified from the tissues of hepatocellular carcinoma. Hepatoma cells were treated by heat shock for preparation of the HSP expressed on cell surface and then marked with DiI fluorescence. Immature DCs from peripheral blood mononuclear cells were cocultured with two types of HSPs. The morphological changes of DCs were observed dynamically and the effects of HSPs on the maturation of DCs analyzed by Flowcytometer. RESULTS: The morphological changes and the processes of antigen capture of DCs cocultured with DiI marked tumor cells were well showed. Four ways to capture antigens of DCs were observed, including direct contact, besieging, forming bubbles and extending pseudopodia with bubbles on the terminals. Results also indicated that both types of HSP could promote the maturation of DCs. CONCLUSION: DiI is a good fluorescent dye suitable for the morphological studies of DCs. Four ways for DCs to capture antigens were indicated in this paper. Different types of HSP, purifed from tumor tissues or expressed on tumor cells surface, promote DC maturation, and the purified HSP is more effective.     

Effect of new Qing Yi Tang decoction on expression of inhibitor kappa B-alpha in pancreas and liver tissues of rats with acute pancreatitis

AIM:To investigate the expression of inhibitor kappa B alpha (IκBα) in pancreas and liver tissues of rats with experimental acute pancreatitis (AP) and to explore the therapeutic mechanism of Chinese medicine new Qing Yi Tang (QYT) on AP. METHODS:70 SD rats were randomly divided into three groups:normal control group (n=10), AP+QYT group (n=30), and AP+normal saline (NS) group (n=30). AP model was induced by retrograded injection of 4% sodium deoxycholate into the pancreatic duct. QYT or NS was infused to the rats respectively by a gastric catheter repeatedly every five hours after AP induction. At 1 h, 4 h, 10 h after operation, rats were sacrificed, and the pancreas and liver were moved out individually. Real-time RT-PCR was performed to detect IκBα mRNA expression in the liver. Western blotting was applied to detect IκBα protein expression in the liver and pancreas. IκBα proteins including phosphorylated form (IκBα-p) and non-phosphorylated form (IκBα-n) were tested. Serum level of leukotriene C4 (LTC4) was detected by ELISA. The pathological changes of pancreas and lung tissues stained with HE were observed under light microscope. RESULTS:Compared with the normal control group, expression of IκBα mRNA in liver was higher in AP rats in the observation period (P<0.01, or P<0.05). QYT treated group had lower expression of IκBα mRNA as compared with AP+NS group (P<0.05). Expression of IκBα protein (including IκBα-p and IκBα-n) in liver and pancreas were also higher in AP group as compared with that in control group (P<0.05). IκBα-p displayed an increased tendency during the observation period in AP+ NS group. However, QYT treatment induced a decrease in IκBα-p protein expression and an increase in IκBα-n expression. The serum LTC4 level in AP group was increased in a time-dependent manner, and QYT attenuated the increased LTC4 level in certain degree (P<0.05). The pathological changes in pancreas and lung tissues of AP rats, such as edema, hemorrhage, and inflammatory cell infiltration were lightly attenuated by QYT. CONCLUSION:QYT alleviated the inflammatory reaction of AP by inhibiting IκBα-p expression and then reducing the inflammatory mediators.     

Effect of glutamine on heat shock protein-70 and tumor necrosis factor-α expession in endotoxemic rats

AIM: To study the protective effect of glutamine (Gln) against endotoxemia by observing the effect of glutamine on heat shock proteins (HSPs) and tumor necrosis factor-α (TNF-α) in endotoxemic rats. METHODS: The rats were randomly divided into 3 groups, lipopolysaccharide group (LPS), glutamine-treated group (Gln) and control group (C). The blood was drawn from lateral tail vein for analysis of cytokine levels at 0, 2, 4 and 6 h post-lipopolysaccharide (LPS) challenge. TNF-α was measured by radioimmunity assay. Multiple tissues were harvested from the rats, and HSP70 was detected by immunohistochemistry. At the same time, lung, liver, and ileum tissue section were stained with hematoxylin and eosin. RESULTS: Gln treatment resulted in marked attenuation of TNF-α expression at 2 h post-LPS injection (P＜0.01). Gray gradients of HSP70 in lungs, liver and ileum tissue in group Gln were much lower than those of group LPS (P＜0.05), This suggested that HSP70 content in these tissues of group Gln was higher than that of group LPS. Tissue sample from lung, liver and ileum revealed significantly less evidence of endotoxin-induced tissue damage in Gln-treated animals. CONCLUSION: Gln can significantly enhance HSP70 expression in multiple tissues of endotoxin-treated rats. A single dose of intravenous Gln given concomitantly with an endotoxin injury can markedly reduce organ histological damage, and attenuate pro-inflammatory cytokine release.     

Chinese herbal preparation Qing Yi Tang Ⅱ granule protects against experimental acute pancreatitis in mice

AIM: To investigate the protect effect of Chinese herbal preparation, Qing Yi TangⅡgranule (QYT), on acute pancreatitis (AP) mice and its mechanism. METHODS: Adult male and female C57BL/6 mice (n=24) were randomly divided into control group, AP group and AP+QYT group. Severe AP was induced by combined intra-peritoneal injection of caerulein (50 μg/kg) and lipopolysaccharide (LPS; 10 mg/kg). Drinking water or 24% QYT solution was given to the mice in AP group or AP+QYT group by oral gavage. The mice in control group were intraperitoneally injected with equivalent volume of normal saline and gavaged with water. The mice were sacrificed 3 h after the last injection. Severity of AP was assessed by biochemical markers and histology. The plasma level of IL-6 and MCP-1, and lung myeloperoxidase (MPO) levels were determined for assessing the extent of systemic inflammatory response. The intestinal microflora, T lymphocytes and T-lymphocyte subgroups were examined for assessing the function of the intestinal barrier. RESULTS: Compared with control group, the mice in AP group presented significant increases in pathological histological scores, plasma amylase activity and IL-6 and MCP-1 levels, as well as the MPO activity in the lung and pancreatic tissues. QYT attenuated these changes to some extent. Furthermore, the increased intestinal microflora was significantly reversed by QYT. No difference of the numbers of Peyer's patches in small intestine in the 3 groups was observed, but the percentage of CD3⁺ T lymphocytes decreased significantly in AP group, and increased percentage of CD4⁺ and CD4⁺/CD8⁺ ratio were found in AP group and AP+QYT group. CONCLUSION: QYT protects against cearulein and LPS-induced acute pancreatitis in mice. The mechanisms may be related to the suppression of the inflammatory response, promoting intestinal bacteria removal, and regulating the functions of T lymphocytes in the intestinal barrier.     

LPS and cerulein induce isolated rat pancreatic tissue injury and inhibit expression of HSP60 protein

AIM: To investigate the effects of lipopolysaccharides (LPS) or cerulein on the expression of HSP60 in isolated rat pancreatic tissues. METHODS: The tissue of rat pancreas was isolated by surgical operation and prepared into tissue snips. The isolated pancreatic tissue was cultured, and stimulated with low- and high-concentrations of cerulein (Cer, 10-11 mol/L, 10-5 mol/L) or lipopolysaccharides (LPS, 10 mg/L, 20 mg/L). Normal saline (NS) was used as control reagent. Before stimulation and 1 h or 4 h after stimulation, the following parameters for evaluating injury were detected: the viability and the level of trypsinogen activation peptide (TAP) in the pancreatic tissues, and the level of interleukin 6 (IL-6) in the culture supernatants. Meanwhile, real-time PCR and Western blotting were used to determine the HSP60 expression at mRNA and protein levels respectively. RESULTS: Under the stimulation with LPS or cerulein, the viability of the pancreatic tissues decreased slightly at 1 h and became much lower with prolonged treatment for 4 h (P<0.05). The TAP level in the pancreatic tissues increased obviously at both 1 h and 4 h (P<0.05) after treatment, except LPS at lower dose. The IL-6 level in the culture supernatant showed no significant change at 1 h stimulation, then increased remarkably at 4 h after stimulation, especially under the conditions of stimulating with LPS or cerulein at higher doses (P<0.05). The expression of HSP60 at mRNA and protein levels decreased sharply with the increase in the concentration of LPS and the prolonged stimulation time; In cerulein stimulation group, the expression of HSP60 mRNA showed an obvious increase (P<0.01), whereas HSP60 protein was reduced remarkably, especially stimulated with cerulein at higher dose and with longer stimulation time (P<0.05). CONCLUSION: LPS or cerulein induce injuries in isolated pancreatic tissues in dose and time dependent manners. Meanwhile, the protein expression of HSP60 is reduced significantly, indicating that the decrease in the cellular protection of HSP60 may involve in the injury of pancreatic tissues.     

Hereditary extremely hypertriglyceridemic mice are more susceptible to acute pancreatitis

AIM:To investigate the susceptibility to pancreatitis in LPL deficient hypertriglyceridemic (HTG) mice and to establish an experimental animal model for study on HTG pancreatitis. METHODS:LPL deficient HTG mice was rescued by somatic gene transfer. Plasma amylase and pathological changes in pancreas were analyzed for comparison between LPL deficient HTG and wild type mice to assess the incidence of spontaneous pancreatitis. In addition, acute pancreatitis(AP) was induced by L-arginine for further assessment. RESULTS:According to pancreatic pathological scores, incidence of spontaneous pancreatitis was 34.5% in LPL deficient mice. The affected LPL deficient HTG mice showed typical morphological changes of pancreatitis with inflammatory infiltration and acinar necrosis, accompanying fibrosis or hemorrhage occasionally, while there was no pancreatitis in wild type mice. The severity of pathological changes correlated positively with plasma TG levels （r=0.604，P<0.05）. However, the amylase levels did not increase significantly. When AP inducer, L-arginine, was injected at low dose (2 g/kg body weight, ip), LPL deficient mice showed more severe pathological damage than wild type mice （P<0.01）, though there was no significant change in amylase levels. CONCLUSION:LPL deficient HTG mice developed spontaneous pancreatitis, and the susceptibility to L-arginine-induced pancreatitis increased. These findings show that HTG results in pancreatitis on mice as on human. Therefore, LPL deficient HTG mice would be a useful experimental model for study of HTG pancreatitis.     

Expression of hepatocyte nuclear factor 4α in experimental colitis in mice

AIM: To investigate the role of hepatocyte nuclear factor 4α (HNF4α) in the pathogenesis of ulcerative colitis (UC) by measuring the expression of HNF4α in the colon tissues in experimental colitis mice. METHODS: BALB/c mice were exposed to 2% or 2.5% (W/V) dextran sulfate sodium (DSS) to induce acute colitis, and the severity of colitis was assessed by observation of disease activity index (DAI), histological injuries and inflammatory cytokines. The correlation between the expression of HNF4α and the severity of disease as well as E-cadherin (E-CAD), junctional adhesion molecule 1 (JAM-1) and desmocollin 2 (DSC-2) was analyzed. RESULTS: Compared with the normal controls, DAI, histological injuries and the mRNA expression of inflammatory cytokines in DSS-treated mice were significantly elevated (P<0.05). The expression of HNF4α at protein and mRNA levels was significantly decreased (P<0.01). The result of Pearson analysis indicated an inverse correlation between the protein expression of HNF4α and the severity of disease (P<0.01). The positive correlation between the mRNA expression of HNF4α and E-CAD/JAM-1/DSC-2 (P<0.01) was also observed. CONCLUSION: There is a close relationship between the expression of HNF4α and the severity of colitis as well as the intercellular linking proteins. The low expression of HNF4α in intestine might aggravate the function of intestinal mucosal barrier, thus promoting the development of UC.     

Relationship between CHIP and myocardial ischemia/reperfusion injury

Carboxyl terminus of the HSP70-interacting protein (CHIP) is a co-chaperone of HSPs as well as an E3 ubiquitin ligase, and it is the connexin between heat shock proteins and ubiquitin-proteasome system. Recent research discovered that CHIP also possesses an intrinsic chaperone activity that enables it to recognize and bind nonnative proteins independently. CHIP regulates the HSPs expression and activity, and facilitates the client proteins ubiquitination and subsequent proteasome-dependent degradation. CHIP also inhibits apoptosis through MAPKs signaling pathways, and affects eNOS specific activity by means of the effects on Akt. Moreover, CHIP plays an important role in mitochondrial oxidative stress protection. With these above points, this paper reviews the function of CHIP in myocardial ischemia/reperfusion injury.     

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.     

Effects of Astragalus polysaccharide on expression of HSP70, PKB and P53 in rat cerebral cortex with cerebral ischemia and reperfusion

AIM:To explore the molecular effects of Astragalus polysaccharide(AP) on improving nervous functions and preventing neuronal apoptosis in rat cerebral cortex with cerebral ischemia and reperfusion. METHODS:One hundred and twenty male Wister rats were randomly divided into sham operation group(SOG), model groups(MG-1 d, 3 d and 7 d), low-dose AP treatment groups(L-APTG-1 d, 3 d and 7 d), and high-dose AP treatment groups(H-APTG-1 d, 3 d and 7 d). The right middle cerebral artery of the rats in MG and AGTG was intercepted by operation to induce ischemic brain injury. The rats in L-APTG and H-APTG were treated with AP at the doses of 5 mg/kg and 15 mg/kg by intraperitoneal injection, respectively. On the 1st day, 3rd day and 7th day after operation, those animals were sacrificed to collect the brain specimens for the study after cerebral blood flow reperfusion and determination of neurological deficit scores. The structural changes of the neurons were observed under electron microscope. Apoptosis was analyzed by flow cytometry. The protein levels of heat-shock protein 70(HSP70), protein kinase B(PKB) and P53 in cerebral corical neurons were determined by immunohistochemical staining and Western blotting. RESULTS:The neurological deficit scores and the apoptotic rate of cerebral cortical neurons in H-APTG were significantly lower than those in MG and L-APTG(P<0.05). The structures of the neurons in H-APTG, such as ribosome endoplasmic reticulum, nucleolus, Golgi complex, mitochondria, etc, were better than those in MG and L-APTG. On the 1st day, 3rd day and 7th day, the protein levels of HSP70 and PKB in cerebral cortical neurons in H-APTG were significantly higher than those in L-APTG, which were significantly higher than those in MG(P<0.05). However, the P53 protein level in H-APTG was significantly lower than that in L-APTG, which was significantly lower than that in MG(P<0.05). CONCLUSION:AP improves nervous functions and inhibits neuronal apoptosis during ischemia and reperfusion. The molecular mechanisms are associated with variations of protein expression in cerebral cortical neurons, such as promotion of HSP70 and PKB and inhibition of P53.