General view in animal model of inflammatory bowel disease

The etiology and pathogenesis of inflammatory bowel disease are up to now still not clear and definite. Establishing the ideal animal model to study its cause and pathogenesis of this disease if very important. The ideal animal model should have the same manifestation with human inflammatory bowel disease on clinical and pathologic feature etc. In this article, the method, the pathologic character istics and concerning pathogenesis, of a few common useful experiment animal models are discussed.     

The effect and mechanism of H. polygyrus infection in T-cell-mediated inflammatory bowel disease in mice

AIM: To investigate the effect and mechanism of Heligmosomoides polygyrus (H. polygyrus) infection in mouse inflammatory bowel disease (IBD) mediated by CD4⁺ helper T-cells. METHODS: Ovalbumin (OVA) -specific CD4⁺ helper T-cells were transferred into SCID (severe combined immunodeficiency) mice to establish an IBD model. The IBD mice were infected by H. polygyrus and sacrificed 14 days later. The histological changes of the colon were observed, and the expression of interferon-gamma (IFN-γ) and interleukin-4 (IL-4) in mesenteric lymph nodes was detected by ELISA and flow cytometry. Additionally, IL-4 monoclonal antibody was intraperitoneally injected into the H. polygyrus-infected IBD mice to block the secretion of IL-4. The IL-4-blocking IBD mice were sacrificed 9 days later and the above indexes were also determined.RESULTS: Compared with the non-infection group, the H. polygyrus-infected IBD mice had more severe colonic lesions, higher level of IL-4 and lower level of IFN-γ in mesenteric lymph nodes (all P<0.05). Compared with the non-blocking group, the H. polygyrus-infected IBD mice with IL-4 blockage had less colonic lesions, lower IL-4 level and higher IFN-γ level (all P<0.05).CONCLUSION: H. polygyrus infection in CD4⁺ T-cell-mediated IBD model promotes inflammation in the early stage probably by inducing the secretion of Th2 cytokine and inhibiting the secretion of Th1 cytokine. The finding suggests that using worms for treatment of IBD needs to be cautious.     

Mechanism of Fuzilizhong decoction alleviating inflammatory damage of rats with non-alcoholic fatty liver disease

AIM To observe the effect of Fuzilizhong decoction on the inflammatory damage of non-alcoholic fatty liver disease （NAFLD） rats and to explore its mechanism. METHODS SPF male SD rats were randomly divided into 6 groups： control group, model group, high dose （20 mg·kg^-1·d^-1）, middle dose （10 mg·kg^-1·d^-1）, low dose （5 mg·kg^-1·d^-1） Fuzilizhong decoction group and Yishanfu （30 mg·kg^-1·d^-1）group, 8 rats in each group. A NAFLD rat modelwas established by intragastric administration of fat emulsion for 4 weeks. Then the drug was given for 4 weeks in each treatment group. HE staining was performed to observe the histopathological changes of the rat liver.The serum levels of interleukin-2（IL-2）, IL-6 and tumor necrosis factor-α（TNF-α） were measured by ELISA. The expression of toll like receptor 4（TLR4） and NF-κB p65 in liver tissues at mRNA and protein levels was determined by RT-qPCR and Western bolt,respectively. RESULTS Compared with control group, the inflammatory damage of liver tissue was more serious, the serum levels of IL-2, IL-6 and TNF-α, the mRNA expression TLR4 and NF-κB p65 in liver tissues were significantly increased in model group（P<0.05）. However, compared with model group, the liver pathological changes in each treatment group were significantly relieved, the serum levels of IL-2, IL-6 and TNF-α, the mRNA expression of TLR4 and NF-κB p65 in liver tissues were significantly reduced（P<0.05）.In addition, the changes of TLR4 and p-NF-κB p65 protein levels in liver tissue were consistent with the changes of TLR4 and NF-κB p65 mRNA. CONCLUSION Fuzilizhong decoction attenuates the inflammatory damages of NAFLD in rats by inhibiting TLR4/NF-κB p65 signaling pathway.     

Genetic variants of IL-33 are associated with clinical phenotypes of inflammatory bowel disease in Han population of southern China

AIM：To investigate whether the single nucleotide polymorphisms (SNPs) of interleukin-33 (IL-33) gene are associated with inflammatory bowel disease (IBD) in the Han population of southern China. METHODS：Eight tag-SNPs were selected from the IL-33 gene using the HapMap database. These tag-SNPs were genotyped in 250 Crohn disease (CD) patients, 115 ulcerative colitis (UC) cases and 622 healthy controls by MALDI-TOF MS assay. RESULTS：No difference of the distribution frequencies of genotypes and alleles between the cases and the controls was observed (P>0.05). Genotype-phenotype analysis suggested that several sites were associated with clinical phenotypes of CD.The T allele of SNP rs10118795 was a protective factor for extra-intestinal manifestation (EIM; P<0.05, OR=0.513, 95% CI： 0.281~0.938), while the CC genotype of SNP rs7025417 (P<0.05, OR=1.363, 95% CI： 1.006~1.846) was a risk factor for EIM. The C allele of rs10118795 decreased the risk for developing perianal lesions (P<0.05, OR=0.480, 95% CI： 0.232~0.994), while the CC genotype of rs10975519 was a risk factor for perianal lesions (P<0.05, OR=2.054, 95% CI： 1.053~4.009). The G allele of rs10975509 increased the risk of upper gastrointestinal CD (P<0.05, OR=3.570, 95% CI： 1.328～9.600), and the A allele of it increased the risk for developing ileocolonic CD (P<0.05, OR=0.613, 95% CI： 0.377~0.996). In the aspect of treatment, the genotypes of rs10118795, rs10975509 and rs7025417 were associated with mucosal healing after infliximab treatment for 30 weeks (P<0.05, P<0.01 and P<0.05). In the UC patients, no significant effect of the selected 8 tag-SNPs on the UC phenotypes was observed. CONCLUSION：Eight polymorphisms of IL-33 do not increase the risk of CD and UC in the Han population of southern China, but some of them have an effect on the clinical phenotypes of CD, and 3 SNPs may be potential markers for prediction of effectiveness of infliximab treatment.     

A meta-analysis of 5-aminosalicylic acid preventing the development of intestinal neoplasia in inflammatory bowel disease

AIM:We conducted an evaluation of clinical data with meta analysis to investigate the preventive effect of 5-aminosalicylic acid (5-ASA) on inflammatory bowel disease (IBD)-associated colorectal cancer (CRC) or dysplasia (Dys) (IBD-CRC/Dys). METHODS:The information was retrieved from the main databases such as PubMed, Web of Science, the Cochrane Library, etc. All full-text articles about the prevention of IBD-CRC/Dys by 5-ASA were included if they conformed to the standards. The odds ratio (OR) and its 95% confidence interval (CI) were calculated. According to the types of IBD and the treatment course, the subgroup analysis was conducted, respectively. RESULTS:Fifteen articles were selected, including 5 038 IBD patients. Pooled analysis showed a protective association between 5-ASA and IBD-CRC/Dys (OR=0.53, 95% CI: 0.37～0.76). Among them, both ulcerative colitis patients (OR=0.45, 95% CI: 0.27～0.77) and Crohn disease patients (OR=0.39, 95% CI: 0.16～0.97) with 5-ASA therapy were less likely to develop CRC/Dys compared with those without 5-ASA treatment. 5-ASA treatment for 1～20 years shows a preventive benefit (OR=0.43, 95% CI: 0.25～0.74). However, a minimum 5-ASA exposure of 2～6 months did not show a preventive benefit (OR=0.59, 95% CI: 0.26～1.34). CONCLUSION: 5-ASA protects against CRC/Dys in IBD patients. Additionally, the protective effect is treatment time dependent. Treatment course for 1～20 years shows an evident preventive benefit.     

Glu216Lys polymorphism of bactericidal/permeability-increasing protein gene has no association with inflammatory bowel disease in Chinese Han population

AIM：To investigate the association between Glu216Lys polymorphism of bactericidal/permeability-increasing protein (BPI) gene and inflammatory bowel disease (IBD) in Chinese Han population and to elucidate the potential interactions between genotypes and clinical features. METHODS：The single nucleotide polymorphism (SNP) of Glu216Lys was genotyped in 286 IBD patients, including 173 Crohn disease (CD) and 113 ulcerative colitis (UC) cases, and 332 age- and sex-matched healthy controls by primer-introduced restriction analysis PCR (PIRA-PCR). Univariate analysis and Logistic regression model were used to evaluate the influences of Glu216Lys polymorphism on IBD clinical features. RESULTS：No significant difference in the frequency of the genotypes and alleles between cases and controls (CD group vs control group, P>0.05; UC group vs control group, P>0.05) was observed. Glu216Lys polymorphism had no relationship with the clinical types of UC and CD (P>0.05). CONCLUSION：The SNP of Glu216Lys in BPI is not associated with IBD in Chinese Han population. The contribution of genetic determinants is significantly different among ethnicities．     

Expression and function of TNF-α in dorsal root ganglion of rats with chronically compressed dorsal root ganglion

AIM: To investigate the role of TNF-α and NF-κB in the mechanism of neuropathic pain due to chronically compressed dorsal root ganglion (CCD).METHODS: Based on the CCD model, von Frey filaments were used to quantify behavior test. The expression changes of TNF-α and NF-κB were determined by Western blotting, and the correlation between the expression of TNF-α and the 50% paw withdrawal threshold was also analyzed. Moreover, the location of TNF-α in dorsal root ganglion (DRG) was observed with immunofluorescence double staining.RESULTS: We found 50% paw withdrawal threshold of CCD decreased at the first day after operation. The mechanical allodynia was the most obvious at postoperative 7~14 d and lasted longer than 35 d. The expression of TNF-α and NF-κB increased significantly in DRG after operation (P<0.01), especially at 7~14 d, and then restored gradually. Moreover, there was a correlation between the protein expression of TNF-α and the changes of neuropathic behavior (P<0.05).CONCLUSION: TNF-α and NF-κB are involved in the mechanism of mechanical allodynia after chronically compressed DRG.     

Effects of electrical stimulation of vagus nerve on gut injury following intestinal ischemia-reperfusion in rats

AIM:To investigate the effects of electrical stimulation of vagus nerve on gut injury following intestinal ischemia-reperfusion in rats. METHODS:30 adult male Wistar rats subjected to bilateral cervical vagotomy were randomly divided into three groups (n=10 per group):(1) Intestinal ischemia-reperfusion group (group I/R):laparotomy and I/R induced by clamping arteria mesenterica superior for 1 h followed by reperfusion for 2 h. (2) Vagus nerve stimulation group (group VNS):laparotomy, I/R and electric stimulation with pulse train of constant amplitude 5V, pulse width 2 ms and frequency 1 Hz at the left caudal vagus ends for 20 minutes before and after occlusion. (3) Sham control group (group SC):sham operation and sham stimulation. Carotid artery was cannulated for mean arterial pressure (MAP) monitoring. A strip of small intestine was taken from distal end of ileum for light microscopic (LM) and transient electron microscopic (TEM) examination at the time of 2 h after reperfusion. Improved Chiu’s scale was used to quantitatively assay the damage degree. The levels of malondialdehyde (MDA) and TNF-α in plasma were detected. RESULTS:MAP in every group kept steady during ischemia, but decreased gradually with the prolongation in the time of reperfusion. MAP decreased more dramaticly in group I/R than that in group VNS (P<0.05). Histological changes by LM and TEM were more significant in group I/R than those in group VNS and SC. The improved Chiu’s scale in group VNS was obviously slighter than that in group I/R (P<0.05). In group I/R, the levels of MDA and TNF-α in plasma were significantly increased compared with group VNS and SC (P<0.01, P<0.05). MDA level in group VNS was significantly higher than that in group SC, but there was no significant difference in TNF-α between the two groups. CONCLUSION:Electrical stimulation of vagus nerves can lessen pathological changes of intestinal mucosa induced by I/R and improve BP during reperfusion, which may be related to reducing lipid peroxidation, and down-regulating TNF-α synthesis.     

Expression of BMP-7 and its receptors in rats with unilateral ureteral obstruction

AIM:To explore the expression changes of b one morphogenetic protein-7 (BMP-7) and its receptors (BMPR-Ⅱ,ALK-2,ALK-3,A LK-6) in the renal tubulo-interstitial lesions induced by unilateral ureteral ob struction (UUO).METHODS:Rats were divided into normal control,sham operation a nd UUO groups,and sacrificed at postoperative day 1,3,7 and 14.The mRNA leve ls of BMP-7,BMPR-Ⅱ,ALK-2,ALK-3 and ALK-6 were examined by RT-PCR.The protei n expression site and level of BMP-7 was detected by immunohistochemistry staini ng.RESULTS:Compared to sham operation group,the mRNA levels of BM P-7,BMPR-Ⅱ,ALK-2 and ALK-3 were significantly decreased,but the change of AL K-6 mRNA was not marked in UUO rats.The reduction of mRNA expression levels of BMP-7,BMPR-Ⅱ,ALK-2 and ALK-3 in the kidney tissue aggravated along with the d elayed post-operated days.The results of immunohistochemistry staining indicate d that BMP-7 mainly expressed in renal tubular and interstitial,rarely in glome ruli.In UUO rats,the protein expression of BMP-7 decreased in varying degrees according to the post-operated days.CONCLUSION:The loss of BMP-7 and its receptors (BMPR-Ⅱ,ALK-2,ALK-3) were observed in the early phase of fibrotic process and this may play a very important role in mediating the renal tubulointerstital fibrosis.     

Protective effects of sequoyitol on type 2 diabetic rats with liver inflammatory lesions

AIM：To investigate the possible protective effect of sequoyitol on type 2 diabetic rats with liver inflammatory lesions. METHODS：Type 2 diabetic rats were induced by feeding high-fat/high-sugar diet and injecting with a low dose of streptozotocin. Sequoyitol at doses of 12.5, 25 and 50 mg·kg-1·d-1 was orally administered in the model rats. At the end of the experiment, the rats were sacrificed. Serum levels of fasting blood glucose, alanine aminotransferase(ALT), aspartate aminotransferase(AST) and albumin(ALB) were determined. Liver wet was recorded and liver index was calculated. The levels of C-reactive protein(CRP),tumor necrosis factor α(TNF-α) and interleukin 6(IL-6) in the liver tissues were also measured. Real-time PCR was used to determine the mRNA expression of TNF-α. In addition, the pathological changes of the liver were observed with HE staining. RESULTS：Compared with the model rats, treatment with sequoyitol obviously decreased the levels of fasting blood glucose, ALT, AST, ALB, CRP, TNF-α and IL-6, reduced the liver index, down-regulated the mRNA expression of TNF-α in the liver, and ameliorated the pathologic changes of the liver. CONCLUSION：Sequoyitol attenuates liver lesions in type 2 diabetic rats through down-regulation of TNF-α and IL-6 expression.     

Effect of arachidonylethanolamide in paraventricular nucleus on cardiac function and sympathetic nerve activity in rats with heart failure

AIM:To determine the roles of the arachidonylethanolamide (AEA) in the paraventricular nucleus (PVN) in cardiac function and sympathetic activity in the rats with chronic heart failure (CHF). METHODS:Chronic heart failure was induced by left coronary ligation in Wistar rats and was confirmed using echocardiography. The rats with CHF and the sham-operated controls (sham group) were treated for 4 weeks with a continuous PVN infusion of AEA, cal-cium-calmodulin-dependent protein kinase Ⅱ (CaMKⅡ) selective inhibitor KN-93, transient receptor potential vanilloid type 1 (TRPV1) channel blocker capsazepine (CPZ), intracellular calcium chelator BAPTA-AM, small-conductance calcium-activated potassium channel (SK channel) blocker apamin and artificial cerebrospinal fluid (vehicle). Sympathetic drive indexes and cardiac function were detected. NG108 cells were incubated with AEA, and then the intracellular cal-cium concentration was measured by fluorometry. The protein expression levels of CaMKⅡ, SK₂ and phosphorylated TRPV1 were determined by Western blot. RESULTS:Compared with sham group, the left ventricular end-diastolic pressure (LVEDP) increased significantly, while peak rate of rise/decline of left ventricular pressure (±dp/dt_max) and ejection fraction (EF) decreased significantly in the CHF group. The concentrations of AEA and intracellular calcium, and the protein levels of CaMKⅡ, SK₂ and phosphorylated TRPV1 in PVN were significantly lower in CHF rats. Compared with the vehicle group, the mortality and sympathetic drive were decreased significantly and cardiac function was improved after treatment with AEA in CHF group. However, PVN perfusion of KN-93, CPZ, BAPTA-AM or apamin contributed to the sympathetic drive and deteriorated the cardiac function. AEA dose-dependently increased intracellular calcium ion concentration, and the protein levels of CaMKⅡ, SK₂ and phosphorylated TRPV1 in NG108 cells. CONCLUSION:AEA in the PVN may be involved in the improvement of cardiac function and sympathetic overdrive via CaMKⅡ/TRPV1/Ca²⁺/SK₂ pathway in rats with CHF.     

Comparison of immune-related pain induced by antigen-special complex with inflammatory pain induced by formalin in rats

AIM: To investigate the difference between immune-related pain induced by antigen-special complex and inflammatory pain induced by formalin, and to observe the differential expression of p38 mitogen-activated protein kinase in spinal cord. METHODS: Thirty adult health SD rats were randomly divided into control group, formalin group and immune complex group (10 rats in each group). After the baseline tests were finished, 5 rats in each group underwent intrathecal administration of p38 MAPK inhibitor SB203580. The right hindpaw of the rats were injected with PBS, formalin or rat IgG immune complex. The thickness of hindpaw and pain behaviors were observed at time points of 0 min, 30 min, 1 h, 2 h, 4 h, 8 h and 12 h after injection. The expression levels of total and activated p38 MAPK in spinal cord were determined by Western blotting analysis. RESULTS: The rats in formalin group showed significant nociceptive behaviors immediately, such as licking foot, and limping with highly swollen foot which could touch the ground. The pain threshold was decreased rapidly 30 min after injection and alleviated after then. The pain threshold of the rats in immune complex group obviously decreased 4 h after injection without red swollen hindpaw. The expression of activated p-p38 MAPK in spinal cord in formalin group was significantly higher than that in immune complex group and control group (P<0.01). No statistic difference of p-p38 expression between immune complex group and control group, also no significant effects of SB203580 on pain behaviors in immune complex group were observed. CONCLUSION: Activated p38 MAPK contributes to the pathogenesis of inflammatory pain, but not to the pathogenesis of immune-related pain. The mechanism of immune-related pain is different from inflammatory pain induced by formalin.     

Effects of hesperidin on inflammatory cytokine levels in chronic bronchitic rats

AIM: To observe the effects of hesperidin on the inflammatory cytokine levels in chronic bronchitic rats.METHODS: The rats were randomly divided into control group, model group and hesperidin treatment group. The rat model of chronic bronchitis was established by smoking. The pathological changes of the bronchial and lung tissues were observed by HE staining. The levels of TNF-α, IL-6 and IL-10 in the bronchoalveolar lavage fluid were analyzed by ELISA. The protein expression levels of ICAM-1 and VCAM-1 were detected by Western blot.RESULTS: Compared with model group, hesperidin treatment significantly reduced inflammatory infiltration in the bronchial and lung tissues, improved the integrity of the alveolar structure, and decreased the levels of TNF-α and IL-6 in the bronchoalveolar lavage fluid, but had no effect on the IL-10 level. Moreover, the protein expression of ICAM-1 and VCAM-1 were dramatically inhibited after hesperidin treatment.CONCLUSION: Hesperidin decreases the levels of TNF-α and IL-6 in the bronchoalveolar lavage fluid in the rats with chronic bronchitis, which may be associated with the inhibition of ICAM-1 and VCAM-1 expression.     

Effect of HO-1 expression on neuronal apoptosis in spinal cord of rats with formaldehyde inflammatory pain

AIM: To observe the change of heme oxygenase(HO-1)expression in the spinal cord during formaldehyde-induced pathological pain and investigate the effect of HO-1 on neuronal apoptosis in the spinal cord induced by formaldehyde inflammatory pain.METHODS: The protein expression of HO-1 in the left and the right spinal dorsal horn was detected by Western blotting. The neuronal apoptosis rate of the spinal cord was determined by flow cytometry.RESULTS: Compared to control group, the protein expression of HO-1 was significantly up-regulated at different time points after the injection of formaldehyde, which was most obviously 24 h after the injection of formaldehyde and was still higher than that in control group at 72 h. Compared to control group, the neuronal apoptosis rate of spinal cord increased in the rats with formaldehyde inflammatory pain. No significant difference of the neuronal apoptosis rate was observed between formaldehyde group and solvent control group . Intrathecal injection of 100 μg ZnppIX, an inhibitor of HO-1, attenuated the degree of spontaneous pain response, but induced an increase in the rate of neuronal apoptosis in spinal cord in the rats with formaldehyde inflammatory pain. CONCLUSION: Formaldehyde inflammatory pain induces the increases in HO-1 expression and neuronal apoptosis in the rat spinal cord. HO-1 promotes the response of spontaneous pain and inhibits the process of neuronal apoptosis in spinal cord induced by formaldehyde inflammatory pain.     

Activation of corticotrophin releasing hormone-containing neurons in hypothalamic paraventricular nucleus contributes to sympathoexcitation in rats with congestive heart failure

AIM:To observe the expression of corticotropin releasing hormone (CRH) within the paraventricular nucleus of hypothalamus (PVN) and to explore the relationship between the activated CRH-containing neurons and sympathetic activity in rats with heart failure (HF).METHODS:Healthy male Sprague-Dawley (SD) rats were subjected to coronary artery ligation to induce HF,and chronic intracerebroventricular (ICV) infusion was performed by osmotic pump for 4 weeks.The rats in sham group and HF group were given vehicle (VEH;artificial cerebrospinal fluid 0.25 μL/h).The rats in HF plus treatment group were treated with CRH competitive inhibitor αh-CRH (15 mg/h).Meanwhile,the Lewis rats and Fischer 344 rats for control study also underwent coronary ligation to induce HF or sham surgery.After 4 weeks,left ventricular end-diastolic pressure (LVEDP) and maximum positive/negative change in pressure over time (±dp/dt_max) were determined.The right ventricular-to-body weight (RV/BW) and lung-to-body weight (lung/BW) ratios were calculated.The renal sympathetic nerve activity (RSNA) was recorded and the plasma norepinephrine (NE) level was measured.The expression of CRH in the PVN combined with the plasma adrenocorticotrophic hormone (ACTH) levels were measured.RESULTS:Compared with the sham-SD rats,the HF-SD rats had a greater number of CRH positive neurons in the PVN (accordingly the plasma ACTH levels were increased),accompanied by decreased±dp/dt_max and increased RSNA,plasma NE,LVEDP,lung/BW and RV/BW.However,ICV treatment with αh-CRH attenuated these changes in the HF-SD rats (P<0.05).Compared with the sham-Fisher 344 rats,the HF-Fisher 344 rats also had a greater number of CRH positive neurons in the PVN (accordingly the plasma ACTH levels were increased).In addition,they had significantly increased RSNA and plasma NE level,higher LVEDP,RV/BW and lung/BW,and lower±dp/dt_max(P<0.05).Compared with the SHAM-Lewis rats,the HF-Lewis rats had not significantly changed in the above parameters.CONCLUSION:In CHF,the CRH-containing neurons in PVN are activated,thus aggravating cardiac function by increasing sympathoexcitation.     

Effect of cinobufagin on L-type voltage-gated calcium channel currents in dorsal root ganglion cells of rats with bone cancer pain

AIM: To investigate the modulation effect of cinobufagin on L-type voltage-gated calcium channel (L-VGCC) currents in dorsal root ganglion (DRG) cells of rats with bone cancer pain. METHODS: A rat model of bone cancer pain was constructed by intratibial injection of Walker 256 breast cancer cells. The rat DRG cells were isolated and the DRG neurons were identified by cellular immunofluorescence technique. The effect of cinobufagin on the L-VGCC current of DRG neurons in the model rats was recorded by the technique of whole-cell patch clamp. RESULTS: The size of DRG neurons obtained from primary culture was basically the same, and the purity was over 95%. Compared with the normal group, the L-VGCC current density of DRG cells in the model rats was increased (P<0.05), while cinobufagin significantly inhibited the L-VGCC current density of DRG cells in the model rats and shifted the inactivation curve to the right. CONCLUSION: In the rat model of bone cancer pain, the intrinsic electrophysiological characteristics of the membrane of DRG neurons are changed, the excitability is increased, and the calcium current is increased. Cinobufagin may exert pharmacological effects by regulating the current characteristics of L-VGCC in rat DRG cells.     

Effect of ghrelin in septal nucleus on gastric motility of diabetic gastroparesis rats and its potential mechanism regulated by hypothalamic arcuate nucleus

AIM：To study the effect of ghrelin in septal nucleus on the gastric motility of the rats with diabetes mellitus (DM) and to investigate the regulation of ghrelin pathway between arcuate and septal nucleus nuclei on gastric motility. METHODS：Streptozotocin was injected intraperitoneally to establish a DM rat model. Fluorescence immunohistochemistry and real-time PCR were used to detect the expression of ghrelin receptor GHS-R1a. The gastric motility was evaluated by implantation of a force transducer on the surface of rats stomachs and the motility index was also calculated. The neural connections between arcuate and septal nuclei were analyzed by the technique of fluorogold tracing. The neural control pathway of gastric motility was determined by central drug injection, nucleus lesion or nucleus electrical stimulation. RESULTS：The expression of GHS-R1a in the septal nucleus of DM rats was lower than that in normal rats (P<0.05). The amplitude and frequency of gastric motility in the DM rats were lower than those in the normal rats (P<0.05). The gastric motility of normal and DM rats were increased by injection of ghrelin into the septal nucleus in a dose-dependent manner. Seven days after injection of fluorogold into the septal nucleus, some neurons in arcuate nucleus were labeled by fluorogold and part of the labeled neurons were ghrelin immunopositive. No effect of nucleus lesion or nucleus electrical stimulation on the gastric motility in the normal rats was observed. In DM rats, the lesion of septal nucleus decreased the gastric motility (P<0.05). In the normal rats, the change of gastric motility caused by electrical stimulation in arcuate nucleus was not affected by the lesion of septal nucleus (P>0.05), while the change was attenuated in DM rats (P<0.05). The ghrelin receptor antagonist ［D-Lys-3］-GHRP-6 had no significant effect on the gastric motility induced by electrical stimulation in arcuate nucleus of the normal rats (P>0.05), but it reduced the change in the DM rats (P<0.05). CONCLUSION:Ghrelin in septal nucleus and the ghrelinergic pathway between arcuate and septal nuclei play an important role in the modulation of gastric motility in DM rats.     

Dual effects of antithrombin Ⅲ on inflammatory factor and bloo d coagulatory factor in rats with hemorrhagic shock

AIM:To observe the changes of inflammatory factors and blood coagulatory factors and effects of antithrombin Ⅲ (ATⅢ) on activated inflammatory factors and blood coagulatory factors in rats with hemorrhagic sho ck .METHODS:The rat model of hemorrhagic shock was set up.40 SD rats were randomized into four groups:sham operation,shock,routine dose ATⅢ and high dose ATⅢ groups,each group was composed of 10 SD rats.Shock group w as administered common resuscitation fluid,routine dose ATⅢ group was administ ered ATⅢ 20 U/kg,high dose ATⅢ group was administered ATⅢ 100 U/kg everyday for successive three days.Plasma NF-κB,6-Keto-prostaglandinF1α,E-selecti n,sICAM-1,thrombin-ATⅢ complexes,thrombinogen fragment F1+2 (PF F1+2),D-dimer and TMD levels were detected.RESULTS:Plasma NF-κB,sICAM-1,E -selectin levels were signifi cantly lower in high dose ATⅢ group than those in shock group and routine dose ATⅢ group (P<0.01).No significant difference between shock group and rou tine dose ATⅢ group was observed.Plasma 6-Keto-prostaglandinF1α levels were h igher in routine dose ATⅢ and high dose ATⅢ groups than that in shock group.P lasma thrombinogen F1+2,D-dimer,TMD,TAT levels were higher in shock rats ( P<0.01).CONCLUSION:Hemorrhagic shock activates coagulatory and inflamma tory reactions.High dose ATⅢ inhibits inflammatory reactions.     

Effect of JNK expression in dorsal root ganglia on histological change of foot skin in diabetic rats

AIM: To observe the change of skin histology in diabetic rats and to investigate the possible me-chanism of c-Jun N-terminal kinase (JNK) protein in the dorsal root ganglion (DRG) during the process. METHODS: Diabetic animal model was established in the male SD rats by intraperitoneal injection of streptozotocin. Plantar skin specimens of the rats were collected from control group, DM 2-week group (DM2), DM 4-week group (DM4), and DM 8-week group (DM8). Immunohistochemical staining and HE staining were used to observe the change of PGP 9.5 immunoreactive nerve terminals and the structures of the skin tissues. The protein expression of PGP 9.5 in the plantar skin tissues, and JNK and p-JNK protein in the DRG within lumbar 5, 6 (L5, 6), and sacral 1 (S1) spinal cord segments were detected by Western blotting. RESULTS: PGP 9.5 immunoreactive nerve terminals of the plantar skin of the rats mainly distributed in the basal layer of the epidermis and papillary dermis. Compared with control group, PGP 9.5 positive nerve terminals in DM4 group showed reduced density and sparse distribution. PGP 9.5 positive nerve terminals in DM8 group showed significantly reduced distribution, thinner nerve diameter, shorter length and distorted shape. Histological changes of the thinner epidermal tissue, reduced epidermal cell layers, uneven cell distribution and arrangement in DM4 group, and significantly reduced epidermal cell layers, swollen and blurred cells, increasing cell gap, lack of stratified epidermis arrangement for part of epidermis, atropal and degenerated dermal collagen fiber, significantly decreased subcutaneous fat in DM8 group were observed. The results of Western blotting showed that the protein expression of PGP 9.5 in the plantar skin tissue of DM rats was progressively decreased along with the disease, while the protein level of p-JNK in L5, 6-DRG or S1-DRG showed a gradual increasing trend. PGP 9.5 immunoreactive positive nerve terminal density of plantar skin in DM rats had a negative correlation with the protein level of p-JNK in L5, 6-DRG and S1-DRG (P<0.01), but showed a significant positive correlation with the plantar skin thickness (P<0.01). CONCLUSION: The protein level of p-JNK within L5, 6-DRG or S1-DRG in DM rats shows a progressive enhancement. At the same time, there is a significant change in the skin tissue density and structure. The changes of skin tissue and nerve morphology in DM rat may be related to the activation of JNK/SAPK pathway in L5, 6-DRG or S1-DRG cells. Blocking or inhibiting JNK/SAPK pathway may delay the diabetic peripheral neuropathy and reduce the risk of skin lesions.     

Effects of N-acetylcysteine combined with azithromycin on oxidative stress in rats with chronic obstructive pulmonary disease

AIM: To investigate the effects of N-acetylcysteine (NAC) combined with azithromycin (AZI) on oxidative stress in the rats with chronic obstructive pulmonary disease (COPD). METHODS: Male Wistar rats (n=60) were randomly divided into control group, model group, AZI intervention group,NAC intervention group and AZI+NAC group. The COPD model was established by passive smoking and intratracheal instillation of lipopolysaccharide. Each day 30 min prior to smoking, intragastric administration with AZI, NAC or combination of the 2 drugs was given for AZI, NAC, and AZI+NAC groups, respectively. On the 31st day, all rats were killed following lung function test. Cell counts of bronchoalveolar lavage fluid (BALF) were performed, and the contents of interleukin-8 (IL-8), interleukin-17 (IL-17) and tumor necrosis factor alpha (TNF-α) in BALF were measured by ELISA. The histopathology of the lung tissues was observed under light microscope, and the levels of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and malondialdehyde (MDA) in the lung homogenate were measured. RESULTS: Compared with control group, the other 4 groups showed decreased pulmonary function, and inflammatory cell infiltration and alveolar destruction in histopathology. Compared with control group, the other groups showed higher white blood cells, monocyte-macrophages, neutrophils and lymphocytes in the BALF (P<0.05). Compared with model group, AZI group and NAC group, lower white blood cells, neutrophils and lymphocytes in the BALF were observed in AZI+NAC group (P<0.05). Compared with model group, IL-8, IL-17, TNF-α and MDA in AZI group, NAC group and AZI+NAC group significantly decreased (P<0.05), while SOD and GSH-Px significantly increased (P<0.05). Compared with AZI or NAC group, IL-8, IL-17, TNF-α and MDA in AZI+NAC group significantly decreased (P<0.05), while SOD and GSH-Px increased significantly (P<0.05). CONCLUSION: Both NAC and AZI attenuate the lung inflammation and oxidative damage in COPD model rats. Combined medication exerts preferable anti-oxidation effects, which might be more suitable for the treatment of COPD.