胰岛素对糖尿病小鼠小肠功能的影响

Objective To investigate the effects of regular insulin (RI)on duodenal smooth muscle in diabetic mice. Methods Diabetes mellitus (DM) model was established by intraperitoneal injection of 150 mg/kg streptozotocin (STZ) in male BALB/c mice. The model mice were divided into DM group and DM treated with RI group with 6 each. Meanwhile, 6 normal mice were served as controls. The mice in treatment group were intraperitoneally injected with 40 U/kg of RI daily.Whereas the mice in DM and control groups were intraperitoneally injected with phosphate buffer solution (pH = 7. 40). After 6 weeks, the small intestinal transit rate of mice was determined by lavage of Indian ink. Interstitial cells of cajal (ICC) in duodenal myenteric plexus were counted using immunohistochemical staining. Slow waves of duodenal smooth muscle cells were recorded with intracellular recordings. Data were analysed by SPSS 17.0 software, and comparisons among three groups were done using LSD test. Results After intervention for 6 months, the clinical presentations,such as more water and food intake and polyuria, were improved in treatment group. The body weight was increased in treatment group [(23.33±3.13) g] compared with DM group [(15.42±1.40) g,P＜0.01] ,but dereased compared with control group [(26.78 ± 2.09) g, P＜0.05]. The level of blood glucose in DM group was significantly higher than that in control and treatment groups(P＜0.01). Small intestine transmission rate was significantly reduced in DM group than that in control and treatment groups (P＜0.01), but it was slower in treatment group than that in control group (P＜ 0. 01 ). Immunohistochemical study showed that the number of c-kit positive cells reduced obviously in DM group than that in control group and treatment group (P＜0.05), whereas it was lower in treatment group than that in control group (P ＜ 0.05). The slow wave frequency and amplitude of duodenal smooth muscle cells in DM group were reduced when compared with control and treatment groups (P＜0.01) and both were lower in treatment group than that in control group (P＜0. 01 ). Conclusion The findings indicate that DM mice have gastrointestinal dysmotility and exogenous insulin may improve small intestinal dysmotility in DM mice.     

胰岛素对糖尿病小鼠小肠功能的影响

目的 探讨胰岛素(regular insulin,RI)对糖尿病(DM)小鼠小肠功能的影响.方法 一次性给予雄性BALB/c小鼠链脲佐菌素(STZ,150 mg/kg)腹腔注射造模.将造模成功小鼠分为DM组6只,DM+RI组6只,另选血糖正常小鼠6只为对照组.DM+RI组给予RI 40U·kg-1·d-1皮下注射,对照组和DM组每天给予等量的磷酸盐缓冲液(pH=7.4)腹腔注射.所有小鼠干预6周结束后,给予印度墨水灌胃测定胃肠传输速率.免疫组织化学法检测各组小鼠十二指肠组织的c-kit阳性细胞数目.最后处死所有小鼠,用细胞内记录技术记录各组小鼠十二指肠平滑肌细胞内慢波的变化.采用SPSS 17.0软件分析,多组资料间比较采用LSD检验.结果 干预6周后,DM+RI组的多饮、多食、多尿症状比DM组减轻,体重较DM组明显增加[(23.33±3.13)g比(15.42±1.40)g,P＜0.01],但比对照组降低[(26.78±2.09)g,P＜0.05].第6周DM组血糖值显著高于对照组(P＜0.01),但比DM+RI组显著升高(P＜0.01).DM组小肠推进率比DM+RI组和对照组明显降低(P值均＜0.01);DM+RI组也较对照组降低(P＜0.01).光镜下观察到DM组c-kit阳性细胞数明显低于DM+RI组和对照组(P值均＜0.05),DM+RI组比正常组减少(P＜0.05).DM组慢波频率比DM+RI组和对照组显著降低(P值均＜0.01),而DM+RI组慢波频率比对照组降低(P＜0.01).DM组慢波波幅比DM+RI组和对照组明显降低(P值均＜0.01),而DM+RI组慢波波幅比对照组降低(P＜0.01).结论 DM小鼠存在胃肠动力障碍,外源性RI可能对DM小鼠小肠动力障碍有一定的改善作用.

Abstract：

Objective To investigate the effects of regular insulin (RI)on duodenal smooth muscle in diabetic mice. Methods Diabetes mellitus (DM) model was established by intraperitoneal injection of 150 mg/kg streptozotocin (STZ) in male BALB/c mice. The model mice were divided into DM group and DM treated with RI group with 6 each. Meanwhile, 6 normal mice were served as controls. The mice in treatment group were intraperitoneally injected with 40 U/kg of RI daily.Whereas the mice in DM and control groups were intraperitoneally injected with phosphate buffer solution (pH = 7. 40). After 6 weeks, the small intestinal transit rate of mice was determined by lavage of Indian ink. Interstitial cells of cajal (ICC) in duodenal myenteric plexus were counted using immunohistochemical staining. Slow waves of duodenal smooth muscle cells were recorded with intracellular recordings. Data were analysed by SPSS 17.0 software, and comparisons among three groups were done using LSD test. Results After intervention for 6 months, the clinical presentations,such as more water and food intake and polyuria, were improved in treatment group. The body weight was increased in treatment group [(23.33±3.13) g] compared with DM group [(15.42±1.40) g,P＜0.01] ,but dereased compared with control group [(26.78 ± 2.09) g, P＜0.05]. The level of blood glucose in DM group was significantly higher than that in control and treatment groups(P＜0.01). Small intestine transmission rate was significantly reduced in DM group than that in control and treatment groups (P＜0.01), but it was slower in treatment group than that in control group (P＜ 0. 01 ). Immunohistochemical study showed that the number of c-kit positive cells reduced obviously in DM group than that in control group and treatment group (P＜0.05), whereas it was lower in treatment group than that in control group (P ＜ 0.05). The slow wave frequency and amplitude of duodenal smooth muscle cells in DM group were reduced when compared with control and treatment groups (P＜0.01) and both were lower in treatment group than that in control group (P＜0. 01 ). Conclusion The findings indicate that DM mice have gastrointestinal dysmotility and exogenous insulin may improve small intestinal dysmotility in DM mice.     

Study on the therapeutic mechanisms of pseudolaric acid in mice with allergic contact dermatitis

Objective:To study the therapeutic mechanisms of pseudolaric acid on allergic contact dermatitis in mice.Methods:A total of 50 BALB/C mice were selected and randomly divided into control group,model group,and treatment A,B,C groups with 10 rats in each group.ACD model was established in model group,and treatment A,B,C groups but not in control group.Model group received no treatment,but treatment A,B,C groups were treated with external application of the concentration of 0.1%,0.2% and 0.4% of the pseudolaric acid for the lesions of ear skin.And the weight gain and the swelling degree of the mice' ear were recorded,weight of thymus and spleen were measured.Spleen suspension was prepared to test T lymphocyte and B lymphocyte levels of mice in five groups.Changes in serum IFN-ed through the enzyme linked immunosorbent assay(ELISAγ,IL-4 and IL-10 levels were test).Results:The weight gain of mice in model group were significant lower than those of mice in the control group and the treatment A,B,C groups(P0.05).Weight gain of mice in treatment A,B groups were significant lower than that of control group(P0.05),but the difference in weight gain between treatment C group and control group showed no significant difference(P0.05).The swelling degree and the weight of mice ears in model group were significant higher than those of mice in control group and treatment A,B,C groups(P0.05).Swelling degree and the weight of mice ears of treatment A,B,C groups were obviously higher than that of control group(P0.05).The swelling degree and weight of mice' ears in treatment A,B,C groups were decreased with the increase of the drug dosage,but comparison between A,B and C group showed statistically differences(P0.05).The thymus and spleen index of mice in model group were significant higher than those of the other four groups(P0.05),among the four groups,thymus and spleen index of treatment A and B group were higher than control group and treatment C group(P0.05).The stimulation index of T and B cells of mice in model group was significantly higher than the rest four groups(P0.05).The serum IFN-γ level of mice in control group and treatment A,B and C group was obviously lower than that of mice in model group(P0.05).The serum IFN-γ level of mice in treatment A,B and C group were decreased with the increasing of the drug dosage,and the level of C group was obviously lower than that of A and B group(P0.05).Conclusion:The pseudolaric acid has anti-inflammation and immune adjustment the effects showing a remarkable therapeutic effects for the ACD mice.     

Anti-tumor activity of tanshinone ⅡA in combined with cyclophosphamide against Lewis mice with lung cancer

Objective:To explore the anti-tumor activity of tanshinone ⅡA in combined with cyclophosphamide against Lewis mice with lung cancer and the effect on cellular immune function.Methods:Lewis tumor cells were inoculated suhcutaneously into the right armpit of mice in each group(n=20) to establish Lewis lung cancer mice model.After model establishment,mice in the model group were given normal saline by lavage,qd.Mice in treatment Ⅰ group were given intraperitoneal injection of TanIIA,15 mg/kg,qd.Mice in treatment Ⅱ group were given intraperitoneal injection of CTX,25 mg/kg,qd.Mice in treatment Ⅲ group were given intraperitoneal injections of TanIIA and CTX,in which the administration method of TanIIA was the same as in treatment Ⅰ group,continuously for 2 weeks,and the dosage of CTX was the same as in treatment Ⅱ group,24 h after model establishment,every other day.Mice were sacrificed 2 weeks after establishment.The tumor tissues were collected to calculate the anti-tumor rate.Immunohistochemistry was used to detect the expressions of Bcl-2,Bax,VEGF,Angiostatin,and Endostatin.FCM was used to detect T lymphocyte subsets in spleen and liver of mice.Results:The tumor weight in treatment Ⅰ,Ⅱ,and Ⅲ groups was significantly lower than that in the model group(P0.05).The tumor weight in treatment Ⅲ group was significantly lower than that in treatment Ⅰ and Ⅱ groups(P0.05).The anti-tumor rate in treatment Ⅱ and Ⅲ groups was significantly higher than that in treatment Ⅰ group(P0.05).Bcl-2 expression in the tumor tissues of treatment Ⅰ,Ⅱ,and Ⅲgroups was significantly lower than that in the model group(P0.05),while Bax expression was significantly higher than that in the model group(P0.05).Bcl-2 expression in the tumor tissues of treatment Ⅰ and Ⅱ groups was significantly higher than that in treatment Ⅲ group(P0.05),while Bax expression was significantly lower than that in treatment Ⅲ group(P0.05).CD4~+ and CD4~+/CD8~+ in treatment Ⅰ,Ⅱ,and Ⅲ groups were significantly higher than those in the model group(P0.05).CD4~+ in treatment Ⅲ group was significantly higher than that in treatment Ⅰ and Ⅱ groups(P0.05),while CD4~+/CD8~+ was significantly higher than that in treatment Ⅱ group(P0.05).The comparison of CD8~+ among each group was not statistically significant(P0.05).NK cell activity in treatment Ⅰ,Ⅱ,and Ⅲ groups was significantly higher than that in the model group(P0.05).NK cell activity in treatment Ⅲ group was significantly higher than that in treatment Ⅰ and Ⅱ groups(P0.05).Conclusions:TannA in combined with CTX can down regulate Bcl-2 expression in lung cancer tissues,up regulate Bax expression,inhibit the neovascularization of tumor tissues,and enhance the immunological function,with a significant anti-tumor activity.     

Long-pulse gastric electrical stimulation protects interstitial cells of Cajal in diabetic rats via IGF-1 signaling pathway

AIM: To investigate the effects of different parameters of gastric electrical stimulation(GES) on interstitial cells of Cajal(ICCs) and changes in the insulin-like growth factor 1(IGF-1) signal pathway in streptozotocininduced diabetic rats.METHODS: Male rats were randomized into control, diabetic(DM), diabetic with sham GES(DM + SGES), diabetic with GES1(5.5 cpm, 100 ms, 4 m A)(DM + GES1), diabetic with GES2(5.5 cpm, 300 ms, 4 m A)(DM + GES2) and diabetic with GES3(5.5 cpm, 550 ms, 2 m A)(DM + GES3) groups. The expression levels of c-kit, M-SCF and IGF-1 receptors were evaluated in the gastric antrum using Western blot analysis. The distribution of ICCs was observed using immunolabeling for c-kit, while smooth muscle cells and IGF-1 receptors were identified using α-SMA and IGF-1R antibodies. Serum level of IGF-1 was tested using enzyme-linked immunosorbent assay. RESULTS: Gastric emptying was delayed in the DM group but improved in all GES groups, especially in the GES2 group. The expression levels of c-kit, M-SCF and IGF-1R were decreased in the DM group but increased in all GES groups. More ICCs(c-kit+) and smooth muscle cells(α-SMA+/IGF-1R+) were observed in all GES groups than in the DM group. The average level of IGF-1 in the DM group was markedly decreased, but it was up-regulated in all GES groups, especially in the GES2 group. CONCLUSION: The results suggest that long-pulse GES promotes the regeneration of ICCs. The IGF-1 signaling pathway might be involved in the mechanism underlying this process, which results in improved gastric emptying.     

Inhibitory effect of interferon-α-2b on expression of cyclooxygenase-2 and vascular endothelial growth factor in human hepatocellular carcinoma inoculated in nude mice

AIM： To evaluate the effects of interferon-α-2b （IFN- α-2b） on expression of cyclooxygenase-2 （COX-2） and vascular endothelial growth factor （VEGF） in human hepatocellular carcinoma （HCC） inoculated in nude mice and to study the underlying mechanism of IFN-α- 2b against HCC growth. METHODS： Thirb/-two nude mice bearing human HCC were randomly divided into four groups （n = 8）. On the 10th day after implantation of HCC cells, the mice in test groups （groups A, B and C） received IFN-α- 2b at a serial dose （10000 IU for group A, 20000 IU for group B, 40000 IU for group C sc daily） for 35 d. The mice in control group received normal saline （NS）. The growth conditions of transplanted tumors were observed. Both genes and proteins of COX-2 and VEGF were detected by RT-PCR and Western blot. Apoptosis of tumor cells in nude mice was detected by TUNEL assay after treatment with IFN-α-2b. RESULTS： Tumors were significantly smaller and had a lower weight in the IFN-α-2b treatment groups than those in the control group （P 〈 0.01）, and the tumor growth inhibition rate in groups A, B and C was 27.78%, 65.22% and 49.64%, respectively. The expression levels of both genes and proteins of COX-2 and VEGF were much lower in the IFN-α-2b treatment groups than in the control group （P 〈 0.01）. The apoptosis index （AI） of tumor cells in the IFN-α-2b treatment groups was markedly higher than that in the control group （P 〈 0.01）. Group B had a higher inhibition rate of tumor growth, a lower expression level of COX-2 and VEGF and a higher AI than groups A and C （P 〈 0.05）, but there was no significant difference between groups A and C. CONCLUSION： The inhibitory effects of IFN-α-2b on implanted tumor growth and apoptosis may be associated with the down-regulation of COX-2 and VEGF expression. There is a dose-effect relationship. The medium dose of IFN-α-2b for inhibiting tumor growth is 20 000 IU/d.     

Characterization of smooth muscle,enteric nerve,interstitial cells of Cajal,and fibroblast-like cells in the gastric musculature of patients with diabetes mellitus

AIM To investigate histologic abnormalities in the gastric smooth muscle of patients with diabetes mellitus(DM).METHODS Full-thickness gastric specimens were obtained from patients undergoing surgery for gastric cancer. HE stain and Masson's Trichrome stain were performed to assess the degree of fibrosis. Immunohistochemical staining using various antibodies was also performed [antibodies against protein gene product 9.5(PGP9.5), neuronal nitric oxide synthase(n NOS), vasoactive intestinal peptide(VIP), neurokinin-1(NK1) receptor, c-Kit, and platelet-derived growth factor receptor-alpha,(PDGFRα)]. Immunofluorescent staining and evaluation with confocal microscopy were also conducted.RESULTS Twenty-six controls and 35 diabetic patients(21 shortduration patients and 14 long-duration patients) were included. There were no significant differences in basic demographics between the two groups except in mean body mass index(BMI)(higher in the DM group). Proportions of moderate-to-severe intercellular fibrosis in the muscle layer were significantly higher in the DM group than in the control group(P 0.01). On immunohistochemical staining, c-Kit- and PDGFRα-positive immunoreactivity were significantly decreased in the DM group compared with the control group(P 0.05). There were no statistically significant differences in PGP9.5, n NOS, VIP, and neurokinin 1 expression. On immunofluorescent staining, cellularity of interstitial cells of Cajal(ICC) was observed to decrease with increasing duration of DM.CONCLUSION Our study suggests that increased intercellular fibrosis, loss of ICC, and loss of fibroblast-like cells are found in the smooth muscle of DM patients. These abnormalities may contribute to changes in gastric motor activity in patients with DM.     

Neuron-protective effect of subanesthestic-dosage ketamine on mice of Parkinson's disease

Objective: To discuss the neuron-protective effect and possible mechanism of subanesthestic-dosage ketamine on Parkinson's disease mice induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.Methods: A total of 30 mice were divided equally into three groups, model control group(MC group), ketamine treatment group(KT group), and blank control group(BC group), respectively.The Parkinson's disease mice of MC group and KT groups were established by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(20 mg/kg/d), while mice in KT group were treated by intraperitoneal injection of subanesthestic-dosage ketamine(8 mg/kg).Differences on behaviors and the number of nigra dopaminergic neurons of mice in each group were compared through the behavioral test and tyrosine hydroxylase immunohistochemistry experiments after the treatments.Furthermore, Western blot was used to test the expression of autophagy-related gene LC3-Ⅱ, Beclin1, Parkin, PINK1,and mTOR.Results: Compared with the BC group, the neuroethology scores were lower and the amount of TH positive cells were less both in MC and MT groups; In KT group, the neuroethology scores were higher and the amount of tyrosine hydroxylase positive cells were significantly more than that in MC group(P 0.05).Moreover, expression levels of autophagy-related proteins LC3-II, Beclin1, Parkin, and PINK1 were higher, while the mTOR expression level was lower than that in MC group.Conclusions: The subanesthestic-dosage ketamine has some protective effects on the coordinating ability of movement and cognitive ability of Parkinson's disease mice induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.This is probably due to that the autophagy activity of cells is activated by subanesthestic-dosage ketamine and that the neurons are protected.     

Jianpi Qingchang decoction regulates intestinal motility of dextran sulfate sodium-induced colitis through reducing autophagy of interstitial cells of Cajal

AIM To investigate the underlying effect of Jianpi Qingchang decoction(JQD) regulating intestinal motility of dextran sulfate sodium(DSS)-induced colitis in mice. METHODS C57BL/6 mice were randomly divided into four groups: the control group, the DSS group, the JQD group, and the 5-aminosalicylic acid group. Except for the control group, colitis was induced in other groups by giving distilled water containing 5% DSS. Seven days after modeling, the mice were administered corresponding drugs intragastrically. The mice were sacrificed on the 15~(th) day. The disease activity index, macroscopic and histopathologic lesions, and ultrastructure of colon interstitial cells of Cajal(ICC) were observed. The levels of tumor necrosis factor-alpha(TNF-α), interleukin(IL)-1β, IL-10 and interferon gamma(IFN-γ), the expression of nuclear factor-kappa B(NF-κB) p65, c-kit, microtubule-associated protein 1 light chain 3(LC3-Ⅱ) and Beclin-l m RNA, and the colonic smooth muscle tension were assessed. RESULTS Acute inflammation occurred in the mice administered DSS. Compared with the control group, the levels of IL-1β, TNF-α, IL-10 and IFN-γ, the expression of LC3-Ⅱ, Beclin-1 and NF-κB p65 m RNA, and the contractile frequency increased(P 0.05), the expression of c-kit m RNA and the colonic smooth muscle contractile amplitude decreased in the DSS group(P 0.05). Compared with the DSS group, the levels of IL-10 and IFN-γ, the expression of c-kit m RNA, and the colonic smooth muscle contractile amplitude increased(P 0.05), the levels of TNF-α and IL-1β, the expression of LC3-Ⅱ, Beclin-1 and NF-κB p65 m RNA, and the contractile frequency decreased in the JQD group(P 0.05).CONCLUSION JQD can regulate the intestinal motility of DSS-induced colitis in mice through suppressing intestinal inflammatory cascade reaction, reducing autophagy of ICC, and regulating the network path of ICC/smooth muscle cells.     

Effect of operation-synchronizing transfusion of apoptotic spleen cells from donor rats on acute rejection of recipient rats after liver transplantation

AIM: To study effect of operation-synchronizing transfusion of apoptotic spleen cells from donor rats on acute rejection of recipient rats after liver transplantation. METHODS: Two of Wistar rats were chosen randomly for normal liver pathology control and ten of SD rats chosen randomly for liver function control as blank group (no operation). The rest of Wistar and SD rats were divided into four groups: control group (only liver transplantation), Dex group (donors receiving intraperitoneal injection of dexamethasone), SpC group (recipients receiving infusion of spleen cells of donors), Dex-SpC group (recipients receiving infusion of apoptotic spleen cells of donors), with each group except blank group, containing 10 SD rats and 10 Wistar rats, respectively. Wistar rats received liver transplantation from SD rats, in the meantime they received infusion of spleen cells of donors, which were induced by an intraperitoneal injection of dexamethasone (3 mg/(d.kg)·b.w) for three days before liver transplantation. The serum alanine transaminase (ALT), total bilirubin (T bili), liver pathological changes and survival time were analysed. Statistical analysis was carried out using SPSS 10.0 for Windows. Differences of the parametric data of ALT in means were examined by one-way ANOVA. Differences of ALT between two groups were examined by LSD. Differences of the nonparametric data of T bili in means and scores of pathology classification for acute rejection were examined by Kruskal-Willis H test. The correlations between ALT and T bili were analysed by Bivariate. Kaplan-Meier curves were used to demonstrate survival distribution. The log-rank test was used to compare the survival data. RESULTS: There were significant differences in ALT of the five groups (F= 23.164 P= 0.000), and ALT in Dex-SpC group was significantly higher than that in blank control, control, Dex, and SpC groups (P = 0.000), and ALT in SpC group was significantly higher than that in blank control (P= 0.000), control (P= 0.004), and Dex groups (P= 0.02). Results of nonparametric analysis of T bill showed that there were differences in T bill of the five groups (X2= 33.265 P= 0.000). T bili in Dex-SpC group was significantly higher than that in blank control, control, Dex, and SpC groups. T bili in SpC group was higher than that in blank control, control, and Dex groups. There were significant differences in scores of pathology classification for acute rejection in each of the groups (X2= 25.933, P= 0.000). The pathologically more serious acute rejection was found in Dex-SPC group than in other groups. No sign of acute rejection was observed in the blank control group. Slight acute rejection was observed in the control group. Slight-moderate acute rejection was observed in the Dex group. Moderate-acute rejection was observed in the SpC group. Severe-acute rejection was observed in the Dex-SpC group. The survival time in Dex-SpC group was shorter than in other groups (statistic = 11.13, P= 0.011). ALT and T bili were positively correlated (r= 0.747, P= 0.000, two-tailed). CONCLUSION: In order to reduce quantity of blood loss from rats after liver transplantation, only one of ALT or T bili is needed for liver function measurement of rats. Simultaneous injection of apoptotic spleen cells from donors induced by dexamethasone to liver transplantation rats aggravates acute rejection. One important mechanism of aggravation of acute rejection may be that apoptotic cells are not removed in time and that dead cells including apoptotic cells release inflammatory factors.