GHRP-6 Induces CREB Phosphorylation and Growth Hormone Secretion via a Protein Kinase Cσ-dependent Pathway in GH3 Cells

This study examined the effect of GHRP-6, a known GHSs receptor agonist, on the phosphorylation of cAMP-responsive element-binding protein （CREB） and the tmderly mechanism. GH3 cells were cultured and subjected to different treatments as follows： GHRP-6, GHRP-6 plus GHRH, phorbol ester （PMA）, an activator of PKC, alone or in combination with GHRP-6, G66983, a general inhibitor of PKCs, in the presence or absence of GHRP-6, rottlerin, an inhibitor of PKCs, alone or plus GHRP-6. The cells were transiently transfected with PKCσ-specific siRNA and then treated with GHRP-6. GH level was measured by enzyme-linked immunosorbent assay （ELISA）. The expression of phosphor-CREB, PKCσ, PKC0 and phosphor-PKCo was determined by Western blotting. The results showed that GHRP-6 stimulated GH secretion in both time- and dose-dependent manners and enhanced the effect of GHRH on GH secretion. GHRP-6 was also found to induce CREB phosphorylation. Moreover, GH secretion was enhanced by the PKC activator PMA and reduced by the PKC inhibitors （G66983, rottlerin） and knockdown of PKCσ. PKCσ could be activated by GHRP-6. It is concluded that PKC, especialiy PKCσ, mediates CREB phosphorylation and GHRP-6-induced GH secretion.     

Role of cellular immunity in halothane hepatitis： an in vitro study

Objective： To explore the effect of cellular immunity in halothane hepatitis. Methods： Hepatotoxicity model was established by exposing male Hartley guinea pigs to 1% halothane via inspiration for 4 h each time for 1 or 3 times within a 42-day interval. Then their hepatocytes and lymphocytes were collected and divided into 2 parts for different cultures. Hepatocytes were cultivated with or without 1% halothane for 4 h and lymphocytes were cultivated with or without 12.5 μg/ml trifluoroacetylated guinea pig serum albumin （TFA-GSA）. Then the 2 kinds of hepatocytes were co-cultivated with lymphocytes （1：100） with or without TFA-GSA induction respectively and the supernatant fluid Was taken after 24, 48 and 72 h to determine the concentration of alanine aminotransferase （ALT）. The halothane cultivated hepatocytes were co-cultivated with various proportion of TFA-GSA antigen induced lymphocytes and ALT was determined after 48 h to determine the proper proportion of hepatocytes and lymphocyte. Results： Lymphocytes of 3 times halothane induced guinea pigs caused a significant increase of ALT in hepatocytes with or without halothane induction. But the lymphocytes of 1 time halothane induced guinea pigs only caused a significant increase of ALT in hepatocytes with induction of halothane. The increase of ALT was only seen after 48- and 72-hour co-culture. The proper proportion of hepatocytes and lymphocytes was 1：100 for lymphocytes cytotoxicity. Conclusion： Lymphocytes is sensitized after inhalation of halothane and generates cytotoxicity to hepatocytes. The immune response of lymphocytes to hepatocytes will be enhanced by repeated inhalation of halothane. The cellular immunity may be one of the mechanisms of halothane induced hepatotoxicity.     

Effect of the Recipe of Zhishi Xiaopi Pill and its Ingredients on Gastric Emptying Rate and Plasma Motilin Level in Rats

To investigate the influence of the recipe of Zhishi Xiaopi Pill （ZSXPP） and its ingredients on gastric emptying and plasma motilin （MOT） level in rats. After the rats were douched by different herbs, cisapride or normal saline in different groups for 4 weeks, The gastric emptying rate and plasma MOT levels were measured respectively. The gastric emptying rate and plasma motilin levels were both increased significantly by using the total recipe of ZSXPP and some parts of the recipe. ZSXPP can increase the gastric emptying rate and plasma motilin levels in rats. The herbs can remove food and qi stagnancy and relieve flatulence; Zhishi, Houpu and Maiya were the main effective ingredients in this recipe.     

Downregulation of metabotropic glutamate receptors mGluR5 and glutamate transporter EAAC1 in the myenteric plexus of the diabetic rat ileum

Objective: To study the morphologic abnormalities of the myenteric plexus in diabetic rats and to explore the mechanism of their effect on gastrointestinal motility. Methods: Forty rats were randomly divided into a diabetic group and a control group, Gastric emptying and small intestine transit rates were measured and histologic and molecular changes in glutamatergic nerves in the ileal myenteric plexus were observed, mGluR5 receptor and EAAC1 transporter changes in the diabetic rats were studied using fluorescence immunohistochemistry and RT-PCR. Results:Eighteen weeks after the establishment of the diabetic rats model, gastric emptying and small intestine transit rates were found to be significantly delayed in the diabetic group when compared with the control group. The density of glutamatergic ganglia and neurons in the ileal myenterie plexus were significantly decreased in the diabetic group when compared with control group(P < 0.05) and the mGluR5 receptors and EAAC1 transporters were downregulated in the diabetic rats(P < 0.05). Conclusion: Decreased glutamatergic enteric ganglia and neurons and decreased mGluR5 receptors and EAAC1 transporters in the intestinal myenteric plexus is one of the mechanisms of diabetic gastroenteropathy in rats.     

A new anastomosis method for choledochojejunostomy by the way behind antrue pyloricum

Background Reflux cholangitis has been the most common In this study we intended to evaluate the perioperative and choledochojejunostomy. complication after Roux-en-Y choledochojejunostomy. long-term efficacy of a new anastomosis method for Methods Clinical data of 143 eligible patients who underwent choledochojejunostomy in the Eastern Hepatobiliary Surgery Hospital affiliated to the Second Military Medical University, China between January 2007 and December 2010 were retrospectively analyzed. Among the patients, 38 consecutive cases underwent this new anastomosis method for choledochojejunostomy （improved group, IG） and 105 underwent standard Roux-en-Y choledochojejunostomy （control group, CG）. Changes in the incidence of cholangitis, the time of beginning to eat liquid meals, post-operative delayed gastric emptying and liver function between the two groups were compared. Results There was no statistical difference in the levels of alanine transaminase, alkaline phosphomonoesterase and gamma-glutamy transferase between the two groups. The time of beginning to eat liquid meals was significantly shorter in IG than CG （P 〈0.05）. The incidence of delayed gastric emptying was lower in IG than CG, with statistical tendency between the two groups （P=0.052）. Among nine patients with different degrees of acute cholangitis in the two groups, one patient （2.6%） in IG and eight （7.6%） in CG suffered from acute cholangitis within six months of follow-up after discharge, but with no statistical difference between the two groups （P 〉0.05）. Of the nine patients with acute cholangitis, none in IG and four in CG were hospitalized for further treatment （P 〉0.05）. Conclusions Patients in IG had satisfactory perioperative and long-term prognosis with shorter time of beginning to eat liquid meals and lower incidence of delayed gastric emptying. This new procedure of choledochojejunostomy by the way behind antrue pyloricum was easy and safe to perform with no mortality and low complication rates.     

Effects of phosphodiesterase 4 inhibitor on cough response in guinea pigs sensitized and challenged with ovalbumin

Background There is currently considerable interest in the potential value of selective inhibitors of cyclic nucleotide phosphodiesterase 4 in the treatment of asthma. However, whether they influence eosinophilic airway inflammation-associated cough remains unclear. The objective of this study was to investigate the effects of selective phosphodiesterase 4 inhibitor SB207499 on cough response and airway inflammation in guinea pigs sensitized and challenged with ovalbumin. Methods Forty sensitized guinea pigs were randomly divided into four groups: control (n=10), challenge (n=10), SB207499 (n=10) and aminophylline (n=10), then challenged with aerosol of 1% ovalbumin or saline. Two hours later, animals were intraperitoneally injected with either saline, 25 mg/kg of SB207499 or aminophylline. At the 24th hour, the injection was repeated with 2.5 mg/kg and 25 mg/kg SB207499 or aminophylline, then cough response to inhaled capsaicin and airway responsiveness to methacholine inducing a 150% of the peak airway pressure to the baseline (PC150) was measured. Finally, total cell number and differentials in bronchoalveolar lavage fluid were analysed. Results The cough frequency per 3 minutes and PC150 in the challenge group were (22±4) times/3 minutes and (198±54) μg/ml, which were significantly different from (6±2) times/3 minutes and (691±81) μg/ml in the control group (P&lt;0.05, respectively). The injection of 25 mg/kg SB207499 significantly inhibited the increased cough response and airway hyperresponsiveness, the cough frequency and PC150 in guinea pigs were (13±2) times/3 minutes and (680±81) μg/ml (P&lt;0.05), which differed significantly from (18±2) times/3 minutes and (400±86) μg/ml after the administration of the same dose of aminophylline (P&lt;0.05). The inhibition of SB207499 on cough response was dose-dependent. Similarly, SB207499 decreased the total cell number and percentage of eosinophils in bronchoalveolar lavage fluid to (2.1±0.5)×10（6）/ml and (20±5)% respectively, which were significantly different from (3.2±0.5)×10（6）/ml and (29±5)% in the aminophylline group (P&lt;0.05, respectively) or (4.2±0.7)×10（6）/ml and (35±4)% in the challenge group (P&lt;0.05, respectively). Conclusion Phosphodiesterase 4 inhibitor may be more useful than aminophylline for cough associated with eosinophilic airway inflammation via inhibiting airway inflammation and airway hyperresponsiveness.     

侧脑室微量注射ghrelin对大鼠小肠转运和肌电活动的影响及作用机制

目的 探讨侧脑室微量注射ghrelin对大鼠小肠转运和小肠消化间期复合肌电活动(interdigestive myoelectric complex,IMC)的影响及作用机制. 方法 ①大鼠分别接受十二指肠导管置入及侧脑室套管置入,在禁食状态下经侧脑室给予ghrelin(0.4,1.6或6.4μg/kg),经十二指肠导管注入伊文氏蓝溶液,观察不同剂量ghrelin对禁食大鼠小肠转运的影响.另两组大鼠分别经侧脑室给予ghrelin受体拮抗剂(D-Lys3) GHRP-6(3.7μg/kg),(D-Lys3) GHRP-6(3.7 μg/kg)+ ghrelin(6.4μg/kg),探讨ghrelin的作用机制.②大鼠分别接受肠道电极置入及侧脑室套管置入,在禁食状态下采用多道生理记录仪监测消化间期MC,观察侧脑室微量注射ghrelin(6.4μg/kg)对大鼠小肠IMC的影响.经侧脑室给予(D-Lys3) GHRP-6(3.7 μg/kg)和NPY抗体(4μg/kg),再经侧脑室给予ghrelin(6.4μg/kg);经静脉给予阿托品(1 mg/kg)、酚妥拉明(1 mg/kg)或普萘洛尔(1 mg/kg),再经侧脑室给予ghrelin(6.4μg/kg),观察侧脑室注射ghrelin对IMC影响的作用机制. 结果 ①侧脑室微量注射ghrelin 0.4μg/kg对大鼠小肠转运无显著影响,ghrelin 1.6 μg/kg和6.4 μg/kg促进小肠转运,此促进作用可被(D-Lys3) GHRP-6阻断.②侧脑室微量注射ghrelin促进十二指肠和空肠IMC,使IMC周期缩短,Ⅲ相频率和振幅增加,Ⅲ相时程缩短,对Ⅲ相占IMC周期百分比无显著性改变.阿托品和(D-Lys3) GHRP-6不同程度地抑制ghrelin的促动力效应;酚妥拉明和普萘洛尔对ghrelin的促动力作用无显著影响. 结论 侧脑室微量注射ghrelin可促进小肠运动,这可能是通过外周胆碱能通路起作用,ghrelin受体GHS-R参与其促动力作用.     

A Study on the Traditional Chinese Medicine Jinyebaidu for Prevention and Treatment of Intrauterine Infection with Guinea Pigs Cytomegalovirus

The purpose is to study the prophylactic and therapeutic effect of the traditional Chinese Medicine （TCM）-Jinyebaidu （JYBD） to guinea pig cytomegalovirus （GPCMV） intrauterine infection. The virus-free female and male guinea pigs were screened with nest-polymerase chain reaction （N-PCR）. After inbred, pregnant guinea pigs were selected and divided into 3 groups randomly： 5 guniea pigs of the blank control group were not given either GPCMV or JYBD. 31 guniea pigs of the positive control group were inoculated 1 mL （107 TCID50 ） suspension of GPCMV intraperitoneal. 10 gunlea pigs of the experimental group were inoculated GPCMV firstly and then perfused stomach with JYBD for 14 days （Dosage in accordance with the modulus of the weight ratio of human to guniea pig）. The effects of JYBD on the intrauterine infection of GPCMV were observed. The results showed that JYBD could decrease the maternal infection rate from 100 % （31/31） to 50 % （5/10） （P〈0. 001）, the intrauterine infection rate from 100% （72/72） to 75 % （21/28） （P〈 0. 001）, and the rate of abnormal outcome of pregnancy from 64.4 % （29/45） to 25.0 % （7/28） （P〈0. 001）, the infective symptoms being relieved. It can be concluded that traditional Chinese medicine- JYBD can prevent and treat GPCMV intrauterine infection, and can be expected a prophylactic drug for HCMV intrauterine infection.     

Antiviral Effect of Chinonin against Herpes Simplex Virus

In order to investigate the antiviral effect of chinonin against Herpes simplex virus (HSV), the encephalitis model in mice and skin infection model in guinea pigs were established by HSV-Ⅰ and HSV-Ⅱ infection respectively. Acyclovir was used as the positive reference drug to evaluate the antiviral capacity of chinonin. Chinonin showed an obvious therapeutic effect on encephalitis in mice at doses of 25 and 50 mg/kg. At both dosages, chinonin demonstrated stronger protection than acyclovir (1 and 5 mg/kg) to the infected mice from death. It was also found that chinonin could treat the skin infection in guinea pigs effectively. The therapeutic effect of chinonin was similar to that of acyclovir (5 mg/kg) at 25 mg/kg but obviously better than that at 50 and 75 mg/kg. In conclusion, chinonin is a potential candidate for the treatment against HSV.     

The Relationship of the Expression of Estrogen Receptor in Cartilage Cell and Osteoarthritis Induced by Bilateral Ovariectomy in Guinea Pig

To investigate the estrogen receptor（ER） expression in cartilage cell in the development of oste0arthritis induced by bilateral ovariectomy in guinea pig and to find their relationship. 30 two-month-old female guinea pigs were randomly divided into two groups （n=15 each） ： sham operation （control）group and ovariectomized group （OVX）; Scanning electorne microscope （SEM） and transmission electron microscope （TEM） were obtained to analysis the cartilage degeneration of the hind limb knee joint after 6 and 12 weeks of ovariectomy. Dextran-Coated-Charcoal （DCC） was taken to quantitively detect the expression of ER. The serum levels of estrogen and gestone were detected by immune contest assay. The results showed that ER do exist in the cartilages of the guinea pigs, with higher expression in the control group than in OVX group at the same time point （P〈0. 05）. It was increased also at 12 th week after operation than that of preoperation. The blood serum levels of estrogen and gestone showed a similar tendency to the expression of ER. Joint cartilage degeneration detected by SEM and TEM could be found at 6 th week, but severe degenerative lesions at 12 th week in the OVX group compared with the control group （P〈0.01）. The data suggested that bilateral ovariectomy in guinea pig lead to severe os.teoarthritis which mighgt be related to the lower serum level of estrogen and the downregulation of the expression of ER in the cartilage also.     

Ghrelin对豚鼠胃平滑肌收缩的影响及机制

目的:探讨并比较Ghrelin及其拟似剂生长激素释放肽6(GHRP6)对豚鼠胃平滑肌舒缩活动的影响及机制。方法:采用电场刺激豚鼠胃底和胃窦部肌间神经方法,观察Ghrelin和GHRP6对胃平滑肌舒缩活动的影响,并通过观察一氧化氮合酶(NOS)抑制剂Nω硝基L精氨酸(LNNA)、一氧化氮前体L精氨酸(LAA)对Ghrelin和GHRP6调控胃平滑肌运动的影响以阐明其机制。结果:不同频率(1~16 Hz)电刺激胃底部肌间神经,平滑肌条呈现开电刺激(onresponse)的舒张波和随后出现的断电刺激(offresponse)收缩波,其中产生的开电刺激舒张效应可被LNNA消除,而断电刺激诱导的收缩效应可被阿托品和胍乙啶(NANC)阻断。在胃底部,Ghrelin和GHRP6可使开电刺激诱导的平滑肌舒张活动减弱,断电刺激诱导的肌条收缩活动增强,且Ghrelin作用明显强于GHRP6。LNNA可显著增强Ghrelin和GHRP6的促平滑肌收缩效应,但LAA可显著减弱该作用。在胃窦部,电场刺激肌间神经,舒张波消失,仅出现断电刺激的收缩波。Ghrelin和GHRP6均可使该收缩作用增强。结论:Ghrelin和GHRP6均可通过肌间神经丛促进胃底部、胃窦部平滑肌收缩,该效应可能与一氧化氮通路有关。     

Ghrelin对大鼠小肠转运及消化间期移行性复合肌电活动的作用及机制

目的 探讨ghrelin对大鼠小肠转运和消化间期移行性复合肌电活动(MMC)的影响及作用机制.方法 大鼠禁食24 h,观察静脉给予不同剂量ghrelin对小肠转运的影响,及静脉给予ghrelin受体拮抗剂(D-Lys3)GHRP-6对ghrelin作用的影响.采用多道生理记录仪在大鼠清醒、禁食状态下监测消化间期MMC,观察静脉给予ghrelin对胃肠MMC的影响.分别给予阿托品、酚妥拉明、普萘洛尔、L-精氨酸及(D-Lys3)GHRP-6,探讨ghrelin对MMC的作用机制.结果 静脉给予ghrelin剂量依赖性地促进小肠转运,此作用可被(D-Lys3)GHRP-6阻断.静脉给予ghrelin促进胃肠MMC.阿托,品、L-精氨酸和(D-Lys3)GHRP-6不同程度地抑制ghrelin的促动力效应;酚妥拉明和普萘洛尔对ghrelin的促动力作用无显著影响.结论 Ghrelin可促进胃肠运动,这可能是通过胆碱能通路起作用,与NO通路关系密切,ghrelin受体GHS-R参与其促动力作用.     

Ghrelin 对大鼠十二指肠肌电活动的影响及作用机制

目的:探讨ghrelin对大鼠进食期和消化间期十二指肠肌电活动的影响及作用机制.方法:大鼠十二指肠埋置银丝电极,采用多道生理记录仪监测十二指肠肌电活性,观察进食期和消化间期静脉.给予ghrelin对大鼠十二指肠肌电活动的影响.分别给予阿托品、酚妥拉明、普萘洛尔、L-精氨酸及ghrelin受体拮抗剂(D-Lys3)GHRP-6拮抗ghrelin,探讨ghrelin对肌电活动的作用机制.结果:进食期给予ghrelin提前诱发十二指肠移行性复合肌电活动(migrating myoelectrical complex,MMC);消化间期给予曲ghrelin可使十二指肠MMC周期和Ⅲ相时程缩短,Ⅲ相频率和振幅增加,但Ⅲ相占MMC周期百分比无显著性改变.阿托品、L-精氨酸和(D-Lys3)GHRP-6可抑制这种效应;酚妥拉明和普纂洛尔对此效应无影响.结论:Ghrelin可促进大鼠十二指肠MMC,这可能是通过胆碱能通路起作用,与NO关系密切.Ghrelin受体GHS-R参与其促动力作用.     

Ghrelin对血管紧张素Ⅱ诱导的脐静脉内皮细胞 氧化应激和内皮功能损伤的影响

目的：探讨促生长激素释放肽（ghrelin）对血管紧张素Ⅱ（angiotensinⅡ, Ang Ⅱ）诱导的离体培养的脐静脉内皮细胞（human umbilicus vein endothelial cell-12,HUVEC-12）损伤的保护作用。方法：(1)在培养的人脐静脉内皮细胞中加入10-9~10-6mol/L AngⅡ共培养24 h,或用10-9~10-6mol/L ghrelin预处理2 h后与10-6mol/L AngⅡ共培养24 h。用MTT法测量内皮细胞活力和用AnnexinV-FITC凋亡试剂盒在流式细胞仪下测量内皮细胞凋亡率。(2)HUVEC用10-9,10-8,10-7,10-6mol/L AngⅡ分别培养3,6,12或24 h,10-9,10-8,10-7或10-6 mol/L ghrelin预处理2 h后与10-6mol/L AngⅡ共培养24 h。生长激素促分泌剂受体1a(growth hormone secretagogue receptor 1a,GHSR1a)受体阻断剂 ［D-Lys3］GHRP-6加入 10-6 mol/L ghrelin预处理2 h后与10-6mol/L AngⅡ共培养24 h组,DCF荧光探针法测细胞内活性氧（reactive oxygen species, ROS）。(3)HUVEC分别与 10-9,10-8,10-7或10-6mol/L AngⅡ和 ghrelin共培养24 h,与10-6mol/L AngⅡ孵育3,6,12或24 h,或10-9,10-8,10-7或10-6mol/L ghrelin预处理30 min,1 h或2 h后与10-6mol/LAngⅡ培养24 h,加入丝裂原活化蛋白激酶/细胞外信号调节激酶信号通路（mitogen-activated protein kinase /extracullar signal regulated kinase 1/2,MAPK/ERK1/2）信号通路抑制剂PD98058、磷脂酰肌醇3-激酶/丝-苏氨酸激酶（phosphoinositide-3-kinase/serine threonine kinase,PI3K/Akt ）阻断剂 wortmannin和［D-Lys3］GHRP-6 共培养24 h,与用AngⅡ和ghrelin孵育的HUVEC 比较上清液中NO产量,HUVEC用ghrelin,PD98059, wortmannin, ［D-Lys3］GHRP-6预处理2 h后与10-6mol/L AngⅡ共培养24 h,或用ghrelin加上PD98059,wortmannin及［D-Lys3］GHRP-6预处理2 h后与10-6mol/L AngⅡ共培养24 h。内皮细胞上清中的一氧化氮（nitric oxide, NO）用Griess法测量。(4)HUVEC用空白对照或AngⅡ在有或没有用ghrelin或ghrelin和wortmannin 一起预处理的情况下孵育,用免疫印迹法（Western blot ）测量内皮型一氧化氮合酶（endothelial nitric oxide synthase, eNOS）的蛋白表达及丝苏氨酸激酶（serine threonine kinase,Akt）磷酸化蛋白表达。结果：AngⅡ引起内皮细胞损伤,增加HUVEC细胞凋亡率,减少培养的HUVEC细胞上清中NO含量,而ghrelin保护HUVEC免受AngⅡ损伤;Ghrelin减少与AngⅡ共同孵育的HUVEC ROS的产生。这种作用被［D-Lys3］GHRP-6消除。PD98059能阻止AngⅡ导致的HUVEC分泌NO减少,Wortmannin和［D-Lys3］GHRP-6消除Ghrelin保护HUVEC释放NO的作用;AngⅡ减少eNOS 的表达,但ghrelin能增加eNOS表达,Wortmannin消除Ghrelin的这种作用;Ghrelin 能刺激p-Akt的表达并在10~20 min达到高峰。结论：Ghrelin在AngⅡ导致的HUVEC损伤中起保护作用,其机制与通过GHSR1a受体减少氧化应激、增加eNOS蛋白表达和激活PI3K/Akt信号通路有关。     

Ghrelin对血管紧张素Ⅱ诱导的脐静脉内皮细胞氧化应激和内皮功能损伤的影响（英文）

目的：探讨促生长激素释放肽（ghrelin）对血管紧张素Ⅱ（angiotensinⅡ,AngⅡ）诱导的离体培养的脐静脉内皮细胞（human umbilicus vein endothelial cell-12,HUVEC-12）损伤的保护作用。方法：（1）在培养的人脐静脉内皮细胞中加入10-9～10-6mol/L AngⅡ共培养24h,或用10-9～10-6mol/Lghrelin预处理2h后与10-6mol/L AngⅡ共培养24h。用MTT法测量内皮细胞活力和用AnnexinV-FITC凋亡试剂盒在流式细胞仪下测量内皮细胞凋亡率。（2）HUVEC用10-9,10-8,10-7,10-6mol/L AngⅡ分别培养3,6,12或24h,10-9,10-8,10-7或10-6mol/L ghrelin预处理2h后与10-6mol/L AngⅡ共培养24h。生长激素促分泌剂受体1a（growth hormone secretagogue receptor1a,GHSR1a）受体阻断剂[D-Lys3]GHRP-6加入10-6mol/L ghrelin预处理2h后与10-6mol/L AngⅡ共培养24h组,DCF荧光探针法测细胞内活性氧（reactive oxygen species,ROS）。（3）HUVEC分别与10-9,10-8,10-7或10-6mol/L AngⅡ和ghrelin共培养24h,与10-6mol/L AngⅡ孵育3,6,12或24h,或10-9,10-8,10-7或10-6mol/L ghrelin预处理30min,1h或2h后与10-6mol/LAngⅡ培养24h,加入丝裂原活化蛋白激酶/细胞外信号调节激酶信号通路（mitogen-activated protein kinase/extracullar signal regulated kinase1/2,MAPK/ERK1/2）信号通路抑制剂PD98058、磷脂酰肌醇3-激酶/丝-苏氨酸激酶（phosphoinositide-3-kinase/serine threonine kinase,PI3K/Akt）阻断剂wortmannin和[D-Lys3]GHRP-6共培养24h,与用AngⅡ和ghrelin孵育的HUVEC比较上清液中NO产量,HUVEC用ghrelin,PD98059,wortmannin,[D-Lys3]GHRP-6预处理2h后与10-6mol/L AngⅡ共培养24h,或用ghrelin加上PD98059,wortmannin及[D-Lys3]GHRP-6预处理2h后与10-6mol/L AngⅡ共培养24h。内皮细胞上清中的一氧化氮（nitric oxide,NO）用Griess法测量。（4）HUVEC用空白对照或AngⅡ在有或没有用ghrelin或ghrelin和wortmannin一起预处理的情况下孵育,用免疫印迹法（Western blot）测量内皮型一氧化氮合酶（endothelial nitric oxide synthase,eNOS）的蛋白表达及丝苏氨酸激酶（serine threonine kinase,Akt）磷酸化蛋白表达。结果：AngⅡ引起内皮细胞损伤,增加HUVEC细胞凋亡率,减少培养的HUVEC细胞上清中NO含量,而ghrelin保护HUVEC免受AngⅡ损伤;Ghrelin减少与AngⅡ共同孵育的HUVEC ROS的产生。这种作用被[D-Lys3]GHRP-6消除。PD98059能阻止AngⅡ导致的HUVEC分泌NO减少,Wortmannin和[D-Lys3]GHRP-6消除Ghrelin保护HUVEC释放NO的作用;AngⅡ减少eNOS的表达,但ghrelin能增加eNOS表达,Wortmannin消除Ghrelin的这种作用;Ghrelin能刺激p-Akt的表达并在10～20min达到高峰。结论：Ghrelin在AngⅡ导致的HUVEC损伤中起保护作用,其机制与通过GHSR1a受体减少氧化应激、增加eNOS蛋白表达和激活PI3K/Akt信号通路有关。     

Ghrelin对肾功能不全大鼠胃肠移行性复合运动的影响

目的探讨ghrelin对肾功能不全(CRF)大鼠胃肠移行性复合运动(MMC)的影响。方法健康雄性SD大鼠30只,随机取出6只做假手术组,其余24只根据是否预先应用ghrelin受体GHS-R的抑制剂D-Lys3-GHRP-6以及ghrelin剂量不同而分为ghrelin 1～4组。采用5/6肾切除法建立CRF大鼠模型。采用多道生理记录仪在大鼠清醒、禁食状态下监测消化间期MMC,观察腹腔注射不同剂量ghrelin对MMC周期的影响,以及给予D-Lys3-GHRP-6对ghrelin作用的影响。结果慢以g性h抑肾rel制功in可能gh以不rel明全in显大的增作鼠加用存。在MM结胃C论肠出动g现h力r的e障li频n碍可率,以表、增明现加显为Ⅲ改胃相善肠出C道现R形F的大态时鼠异间M常、M频的C率M周和M期振C紊增幅乱加,且,,进M呈而M剂C改量周善依期其赖缩胃性短肠(,P动频<力0率.0障5增)碍;加D。-,LⅢys相3-G振H幅RP减-6低可;     

Ghrelin对糖尿病大鼠海马DKK-1表达和学习记忆功能的影响

目的：观察Ghrelin对糖尿病大鼠DKK-1和WNT信号通路的表达变化,探讨其参与学习记忆功能的机制。方法60只SD大鼠随机均分为对照组（NC组）、糖尿病组（DM组）、糖尿病+Ghrelin组（DM1组）、糖尿病+Ghrelin+D-lys3-GHRP-6组（DM2组）。腹腔注射STZ（60 mg/kg）建立糖尿病大鼠模型,Morris水迷宫实验检测大鼠空间学习和记忆能力;电镜观察大鼠海马CA1区超微结构,普通显微镜下HE染色观察大鼠海马CA1区细胞形态;ELISA检测大鼠血清DKK-1的表达;荧光定量PCR及Western blotting分别检测大鼠海马DKK-1及β-catenin mRNA和蛋白水平。结果与NC组相比,DM组大鼠逃避潜伏期延长,穿越平台次数减少（P<0.05）;神经元细胞肿胀、线粒体空泡变性等（P<0.05）;神经元细胞排列紊乱,细胞核固缩等;血清、海马组织中DKK-1的表达明显升高（P<0.05）,海马中β-catenin表达下降（P<0.05）。与DM组相比,DM1组大鼠逃避潜伏期缩短,穿越平台次数增多（P<0.05）;神经元细胞形态完整,线粒体发达、密度增加等（P<0.05）;神经元细胞排列整齐、细胞层数清晰;血清、海马组织中DKK-1表达明显降低（P<0.05）,海马中β-catenin的表达升高（P<0.05）。联合应用Ghrelin和GHSR-1a受体拮抗剂Ghrelin+D-lys3-GHRP-6后,Ghrelin上述作用被阻断（P<0.05）。结论 WNT信号通路可能参与糖尿病脑病的发生发展过程, Ghrelin改善糖尿病大鼠学习记忆功能的机制至少部分与下调海马DKK-1的表达、调控WNT信号通路有关。     

Ghrelin及其受体在大鼠中枢神经系统的分布特征及比较

目的观察ghrelin及其受体生长激素促分泌素受体(GHS-R)在大鼠中枢的分布特征及表达水平。方法采用免疫组织化学和图像分析方法观察ghrelin和GHS-R在大鼠中枢的分布特征及在与消化功能相关脑区的表达水平。结果 Ghrelin与GHS-R免疫阳性表现为细胞膜染色,部分有细胞质着色,在中枢的海马(HIP)、弓状核(ARC)、室旁核(PVN)、杏仁核、延髓、小脑等部位均有表达。在与消化功能密切相关的脑区,二者表达量较多。Ghrelin在下丘核的PVN和下丘脑外侧区(LH)表达最多,免疫反应阳性细胞数分别为116.31±7.76和107.02±8.80,明显高于ARC(75.12±5.59)、下丘脑背内侧核(DMH)(56.84±7.07)和下丘脑腹内侧核(VMH)(62.54±6.93),差异有统计学意义(P<0.05);在HIP的CA3区表达最多,免疫反应阳性细胞数为123.20±11.17,明显高于CA1区(67.46±6.93)、CA2区(37.91±5.21)和CA4区(59.96±6.73),差异有统计学意义(P<0.05)。GHS-R在下丘脑的PVN和ARC表达最多,免疫反应阳性细胞数分别为111.06±11.80和97.06±10.37,明显高于LH(74.70±6.04)、DMH(58.65±7.28)和VMH(48.24±6.58),差异有统计学意义(P<0.05);在HIP的CA3和CA4区表达最多,免疫反应阳性细胞数分别为103.79±9.23和86.48±7.25,明显高于CA1区(51.16±6.56)和CA2区(43.39±5.11),差异有统计学意义(P<0.05)。Ghrelin和GHS-R在杏仁内侧核均有强阳性表达,免疫反应阳性细胞数分别为140.22±9.07和115.66±7.35,明显高于其他核区,差异有统计学意义(P<0.05)。结论 Ghrelin可能作为神经调节递质广泛作用于中枢各脑区,尤其是HIP、杏仁核、下丘脑等与消化功能相关的脑区,通过其受体GHS-R发挥各种生理调节作用。     

四氧嘧啶联合饮食失节法诱导家兔糖尿病胃轻瘫模型探讨

目的研究使用四氧嘧啶联合饮食失节法诱导家兔糖尿病胃轻瘫模型的制作方法。方法根据给药剂量不同对家兔分组，使用四氧嘧啶腹腔注射，配合高糖高脂不规律进食制造糖尿病胃轻瘫模型。观察糖尿病家兔成模率、体重、摄食、胃排空率的变化。结果与其他组比较，实验组以300μg／kg剂量连续两天注射，隔日注射相同剂量，糖尿病家兔成模率高。与对照组比较，糖尿病家兔高糖高脂不规律进食2周，其体重、摄食、胃排空率均有明显变化。结论四氧嘧啶3次腹腔注射，第1日、第2日、第4日300μg/kg剂量，配合高糖高脂不规律进食可成功制造家兔糖尿病胃轻瘫模型。     

超声观察Ⅱ型糖尿病胃动力及西沙比利对其改善作用

目的 探讨Ⅱ型糖尿病患者胃液排空功能,同时观察西沙比利对糖尿病胃轻瘫的疗效。方法 应用超声检查对22例正常组及20例糖尿病患者的胃液半排空时间进行比较,并用西沙比利（10 mg tid）治疗其中11名有胃轻瘫的病人,4周后复查对照。结果 正常组胃液半排空间为（t1/2）（14.80±4.35）min,Ⅱ型糖尿病组的胃半排空时间为（28.00±8.94）min,二者之间有显著的差异。口服西沙比利后11名患者的胃半排空时间由（33.64±5.05）min减至（20.91±5.39）min。结论 糖尿病患者胃排空有明显的障碍,西沙比利可改善糖尿病胃轻瘫,本实验为糖尿病人临床应用西沙比利治疗提供了理论依据。