联合间充质干细胞培养对大鼠胰岛的保护作用

目的 探讨间充质干细胞与胰岛联合培养在延长胰岛体外存活时间、保护胰岛功能方面的作用.方法 以4周龄Wistar大鼠作为供体,分离纯化骨髓间充质干细胞并培养鉴定;Histopaque-1077一步法分离纯化Wistar大鼠胰岛;将胰岛培养分为单纯胰岛基础培养、单纯胰岛高糖培养、胰岛与间充质干细胞联合基础培养、胰岛与间充质于细胞联合高精培养4组,每组又分3个时间段(3、7、14 d)观察胰岛形态变化及胰岛存活率,酶联免疫吸附法检测胰岛素分泌量及刺激指数.结果 骨髓间充质干细胞传代培养3代后,流式细胞术检测CD45及CD90,二者荧光强度差异有统计学意义(P＜0.05);联合培养组3、7、14 d的胰岛存活率均高于单纯培养组(P＜0.01);高糖刺激下联合培养组7 d的胰岛素分泌量及刺激指数均高于单纯培养组(P＜0.01).结论 间充质干细胞与胰岛联合培养可以明显延长胰岛体外存活时间并保持其活性,对胰岛具有良好的保护作用.

Abstract：

Objective To observe the effect of mesenchymal stem cells (MSCs) on enhancing rat islets viability and function in vitro by a pretransplant co-culture. Methods 4-week-old Wistar rats were used as donors, bone mesenchymal stem cells were isolated and subcultured. Islets of Wistar rats were isolated and purified by one-step single-layer Histopaque-1077. Then islets were divided into four groups randomly, 2 groups co-cultured with mesenchymal stem cells (MSCs) (one using low-glucose medium; the other using high-glucose medium ); 2 groups were cultured alone (low-glucose medium; high-glucose medium), each group was further stratified into 3 subgroups(3, 7, 14 d); the survival and functionality of these islets were observed and evaluated. The amount of glucose stimulated secreted insulin were measured wth a rat/mouse insulin enzyme-linked immunosorbent assay (ELISA) kit and stimulation index was also calculated. Results Compared with those not co-cultured, islets co-cultured with MSCs demonstrated significantly higher survival rates and viability both in 3th, 7th and 14th day ( P ＜ 0. 01 ); furthermore, cocultured islets revealed higher levels of glucose stimulated insulin secretion and secretion indexes in 7th day (P＜0.01). Conclusion Rat islet cells co-cultured with MSCs have longer in vitro survival and better functions.     

骨髓基质干细胞及其来源的产胰岛素细胞移植对受体胰岛和新生血管的影响

Objective To investigate the proliferative impact of transplantation of bone marrow mesenchymal stem cells (MSCs) and insulin producing ceils (IPCs) from them on recipients' remnant islets and neovascularization around. Methods MSCs were induced to IPCs in vitro. The rats with type Ⅰ diabetic meilitus were randomly divided into control group, MSCs transplant group, and IPCs transplant group. When the blood glucose level of recipient rats was decreased to 10 mmol/L, the right kidney and pancreas were removed from each group simultaneously. All the thin tissue slices were estimated by immu-nohistochemistry staining of PCNA antibody,insulin antibody and CD31 anbibody. Results The passaged MSCs differentiated into IPCs. In the grafts, insulin-positive and CD31 -positive cells were observed in the right kidney. In control group, few proliferative cells located in the atrophic islets. In the transplant groups, a lot of proliferative islet cells were obsereved, around which few CD31 -positive ceils existed;Pancreatic proliferative islet cell rate in MSCs and IPCs transplant group was ( 20.84±3.48 ) % and ( 18.43± 2.84% ) ( P > 0.05 ) respectively. Conclusion Obviously, MSCs and IPCs promoted recipient neovascu-larization surrounding the islets, which enhanced the proliferation of remnant islet cells. MSCs differentiated to IPCs and vascular endothelial cells in the recipient transplant location.     

骨髓基质干细胞及其来源的产胰岛素细胞移植对受体胰岛和新生血管的影响

Objective To investigate the proliferative impact of transplantation of bone marrow mesenchymal stem cells (MSCs) and insulin producing ceils (IPCs) from them on recipients' remnant islets and neovascularization around. Methods MSCs were induced to IPCs in vitro. The rats with type Ⅰ diabetic meilitus were randomly divided into control group, MSCs transplant group, and IPCs transplant group. When the blood glucose level of recipient rats was decreased to 10 mmol/L, the right kidney and pancreas were removed from each group simultaneously. All the thin tissue slices were estimated by immu-nohistochemistry staining of PCNA antibody,insulin antibody and CD31 anbibody. Results The passaged MSCs differentiated into IPCs. In the grafts, insulin-positive and CD31 -positive cells were observed in the right kidney. In control group, few proliferative cells located in the atrophic islets. In the transplant groups, a lot of proliferative islet cells were obsereved, around which few CD31 -positive ceils existed;Pancreatic proliferative islet cell rate in MSCs and IPCs transplant group was ( 20.84±3.48 ) % and ( 18.43± 2.84% ) ( P > 0.05 ) respectively. Conclusion Obviously, MSCs and IPCs promoted recipient neovascu-larization surrounding the islets, which enhanced the proliferation of remnant islet cells. MSCs differentiated to IPCs and vascular endothelial cells in the recipient transplant location.     

骨髓基质干细胞及其来源的产胰岛素细胞移植对受体胰岛和新生血管的影响

Objective To investigate the proliferative impact of transplantation of bone marrow mesenchymal stem cells (MSCs) and insulin producing ceils (IPCs) from them on recipients' remnant islets and neovascularization around. Methods MSCs were induced to IPCs in vitro. The rats with type Ⅰ diabetic meilitus were randomly divided into control group, MSCs transplant group, and IPCs transplant group. When the blood glucose level of recipient rats was decreased to 10 mmol/L, the right kidney and pancreas were removed from each group simultaneously. All the thin tissue slices were estimated by immu-nohistochemistry staining of PCNA antibody,insulin antibody and CD31 anbibody. Results The passaged MSCs differentiated into IPCs. In the grafts, insulin-positive and CD31 -positive cells were observed in the right kidney. In control group, few proliferative cells located in the atrophic islets. In the transplant groups, a lot of proliferative islet cells were obsereved, around which few CD31 -positive ceils existed;Pancreatic proliferative islet cell rate in MSCs and IPCs transplant group was ( 20.84±3.48 ) % and ( 18.43± 2.84% ) ( P > 0.05 ) respectively. Conclusion Obviously, MSCs and IPCs promoted recipient neovascu-larization surrounding the islets, which enhanced the proliferation of remnant islet cells. MSCs differentiated to IPCs and vascular endothelial cells in the recipient transplant location.     

骨髓基质干细胞及其来源的产胰岛素细胞移植对受体胰岛和新生血管的影响

Objective To investigate the proliferative impact of transplantation of bone marrow mesenchymal stem cells (MSCs) and insulin producing ceils (IPCs) from them on recipients' remnant islets and neovascularization around. Methods MSCs were induced to IPCs in vitro. The rats with type Ⅰ diabetic meilitus were randomly divided into control group, MSCs transplant group, and IPCs transplant group. When the blood glucose level of recipient rats was decreased to 10 mmol/L, the right kidney and pancreas were removed from each group simultaneously. All the thin tissue slices were estimated by immu-nohistochemistry staining of PCNA antibody,insulin antibody and CD31 anbibody. Results The passaged MSCs differentiated into IPCs. In the grafts, insulin-positive and CD31 -positive cells were observed in the right kidney. In control group, few proliferative cells located in the atrophic islets. In the transplant groups, a lot of proliferative islet cells were obsereved, around which few CD31 -positive ceils existed;Pancreatic proliferative islet cell rate in MSCs and IPCs transplant group was ( 20.84±3.48 ) % and ( 18.43± 2.84% ) ( P > 0.05 ) respectively. Conclusion Obviously, MSCs and IPCs promoted recipient neovascu-larization surrounding the islets, which enhanced the proliferation of remnant islet cells. MSCs differentiated to IPCs and vascular endothelial cells in the recipient transplant location.     

SDF-1/CXCR4轴在骨髓间充质干细胞移植促进胰岛再生中的作用

目的 观察同种异体骨髓间充质干细胞(MSCs)移植入受体后,基质细胞衍生因子(SDF)-1/CXCR4轴在促进残存胰岛及其周围新生血管增殖中的作用.方法 对大鼠MSCs进行体外培养、鉴定.链脲佐菌素(STZ)诱导的糖尿病大鼠随机分为A组(MSCs移植组)、B组(MSCs移植+SDF-I/CXCR4轴阻断剂AMD组)和C组(糖尿病对照组),另设D组(正常大鼠对照组).移植MSCs后第30天取出各组大鼠胰腺和血清,胰腺组织采用苏木素-伊红(HE)染色和免疫组织化学法观察CD31、增殖细胞核抗原(PCNA)、胰腺干细胞标志物(PDX)-1在胰腺组织的表达水平.血糖仪检测血糖水平、放免法检测胰岛素水平、酶联免疫吸附试验(ELISA)检测SDF-1水平.结果 (1)A组残存胰岛周围可见新生血管,CD31、PCNA、PDX-1染色阳性率分别为(71.2±5.3)%、(76.5±4.5)%、(69.8±6.7)%;B组残存胰岛周围基本未见新生血管,CD31、PCNA、PDX-1染色阳性率分别为(7.4±2.1)%、(5.5±3.7)%、(8.8±2.9)%,两组比较差异有统计学意义(P＜0.05).(2)移植后第25天,A组血糖浓度基本正常,低于B组和C组,而胰岛素水平明显高于B组和C组(P＜0.05).(3)A组与B组血清SDF-1水平差异无统计学意义(P＞0.05),但都明显高于C组(P＜0.05).结论 MSCs促进胰岛再生和新生血管形成,AMD3100能抑制MSCs的作用,进而提示SDF-1/CXCR4轴在胰岛再生和血管形成中具有重要作用.

Abstract：

Objective To investigate the role of stromal cell derived factor-1 (SDF-1)/CXCR4axis in recipients' remnant islets regeneration and neovascularization after the transplantation of allogeneic bone marrow mesenchymal stem cells (MSCs). Methods MSCs were isolated from SD rats, cultured in vitro and identified by testing the phenotypes with flow cytometry ( FCM ). The diabetic rats induced by streptozotozin were randomly divided into group A ( MSCs transplant group), group B ( MSCs transplant +AMD group) and group C ( DM control group). Group D serve as the normal control. The pancreata were removed and blood serum was retrieved from each group simultaneously at the 13th day after MSCs transplant. The expression of CD31, proliferating cell nuclear antigen (PCNA) and PDX-1 in each group of pancreas tissue was detected by using immunohistochemistry, and the morphological changes in the isletswere observed by Hematoxylin and Eosin (HE) staining. Serum glucose and insulin levels were determined by blood glucose monitor, radioimmunoscintigraphy, and SDF-1 in serum was by enzyme linked immunosorbent assay (ELISA). Results Neovascularization was observed in the remnant islets of the recipient pancreatic tissue and CD31 -positive cells (71.2 ± 5.3 ) %, PCNA-positive cells ( 76. 5 ± 4. 5 ) %, PDX-1-positive cells (69. 8 ±6. 7)% were highly expressed in group A. As compared with group A, seldom-positive cells[CD31 (7.4±2. 1)%, PCNA (5.5 ±3.7)% and PDX-1 (8.8 ±2.9)%]and rarely neovascularization were observed in group B (P ＜0. 05 ). Serum glucose level in group A was lower than that in group B and group C, but serum insulin level in group A was significantly higher than that in group B and group C (P ＜ 0. 05 ). There was no significant difference between group A and group B in serum SDF-1level ( P ＞ 0. 05 ), but that was higher in groups A and B than in group C ( P ＜ 0. 05 ). Conclusion Obviously, MSCs promote recipient neovascularization surrounding the islets, which enhances the proliferation and regeneration of remnant islets. AMD 3100 has the function of intervening SDF-1/CXCR4 axis,which inhibits the effect of MSCs on promoting islets regeneration. It is suggested that SDF-1/CXCR4 axis may play an important role in vascularization and islets regeneration.     

与大鼠胰岛细胞联合移植的骨髓间充质干细胞的免疫调节作用

目的 研究同种异体或自体的大鼠骨髓间充质干细胞(MSC)与胰岛肝内联合移植对胰岛移植物的免疫调节作用及其机制.方法 以链佐星制备Lewis大鼠的糖尿病模型,作为胰岛移植受者,分为3组:单纯移植组大鼠经门静脉单独移植SD大鼠胰岛6000 IEQ/kg;同系MSC组大鼠经门静脉共同移植1×109/L的Lewis大鼠MSC 1 ml与SD大鼠胰岛6000 IEQ/kg;同种MSC组大鼠经门静脉共同移植1×109/L的SD大鼠MSC 1 ml与SD大鼠胰岛6000 IEQ/kg.检测受鼠的血糖变化,术后1、3 d大鼠外周血γ干扰素(IFN-γ)、白细胞介素(IL)-2、IL-4和IL-10的含量.结果 3组大鼠术后第1天血糖均下降到13.9 mmol/L以下.同系MSC组移植物存活时间为(11.38±4.03)d,同种MSC组为(5.50±2.07)d,单纯移植组为(2.88±1.25)d(P＜0.01).术后1、3 d,单纯移植组IFN-γ和IL-2的含量显著高于同系MSC组和同种MSC组(P＜0.01),同种MSC组IFN-γ和IL-2的含量高于同系MSC组(P＜0.05);单纯移植组IL-10的含量低于同系MSC组和同种MSC组(P＜0.01),同系MSC组IL-10的含量与司种MSC组相比较,差异无统计学意义(P＞0.05);各组IL-4含量的差异无统计学意义(P＞0.05).结论 MSC与同种胰岛共移植可以延长胰岛移植物存活时间,应用同系MSC的效果优于同种异体MSC.共移植的MSC主要通过减少TH1类细胞因子(IFN-y和IL-2)的表达使受者TH1/TH2平衡向TH2方向偏移.

Abstract：

Objective To compare the immune regulation of syngenic and allogenic mesenchymal stem cells (MSCs) in the transplantation combined with islets. Methods After induction of diabetes in 30 Lewis rats with streptozotocin (STZ), the recipient Lewis rats received islets from SD rats combined with syngenic (group B) or allogenic (group C) MSCs injection via the portal vein. The group of islets transplanted alone served as control (group A). The survival time of grafts in all groups was assessed by the level of blood glucose. ELISA was used to detect the levels of interferon-γ (IFN-γ), interleukin 2 (IL-2), IL-4 and IL-10 in the peripheral blood on the 1st and 3rd day after transplantation. Results The blood glucose levels in all three groups were decreased in a normal range (13. 9 mmol/L) and the survival time of grafts in group B (11.38 ± 4. 03 days) was significantly longer than in group C (5. 50± 2. 07 days) as well as group A (2. 88 ± 1.25 days). On the 1 st and 3rd day after transplantation, the levels of TH 1 cytokines IFN-γ and IL-2 in group A were significantly higher than in groups C and B (P＜0.05). Meanwhile the levels of TH 2 cytokine IL-10were increased in group B, but there was no significant difference between groups A and C (P＞0.05). The levels of IL-4 had no significant difference among these three groups (P ＞ 0.05).Conclusion Islet transplantation combined with MSCs could prolong the survival time of grafts.Syngenic MSCs, superior to allogenic ones, were more effective in changing the balance of TH1/TH2to TH2. Decreased expression of TH1 cytokine (IFN-γ, IL-2), which was closely related to the induction of immune tolerance, was beneficial to the long-term survival of grafts.     

局部应用缓释免疫抑制剂对胰岛细胞移植的影响

Objective To explore the local application of immunosuppressant in improving the survival rate of the transplanted islet cells and systemic side effects.Methods The streptozocin of 200 ms/kg was injected into the abdominal cavity of the Wistar rats,the blood sugar was tested after 48,and 72 hours,and the rats with two consecutive measurements ≥20 mol/L were taken as the experimental animal model.The dose of pancreatic islet cells transplanted into the abdominal cavity was 8 000 IE,/kg,and that of cyclosporine dosage was 1.5 mg/(100 g·d).The pancreatic islet cells were divided into three groups：(1)systemic immunosuppressive agents through stomach lavage with the intraperitoneal injection of microencapsulated islet cells;(2)pure intraperitoneal injection of microencapsulated islet cells;(3)intraperitoneal injection microencapsulated activated carbon particles loaded with immunosuppressants,and mieroencapsulated islet cells.Changes of blood glucose and pathological in rats after transplantation were detected.Results The blood glucose of group 3 and group 1 showed no significant difference(P＞0.05),as well as compared with group 2(P＞0.05).But the local application of immune agents could prolong the effective time of the islet cells and attenuate the fibrotic extent of the surrounding islets when compared with the control group,the C peptide level in applicating immunosuppressive agents group was significantly hisher in the immunosuppressive group than the pure transplantation group.Conclusion Compared with the systemic immune suppression via stomach lavage,local application of slow-release immunosuppressive agents showed the same effects of activated carbon particles,with a prolonged the effective time of islet cell and reduced topical side effects in the latter.     

肝脏缺血/再灌注后Toll样受体4表达情况及丙泊酚对其表达的影响

Objective To observe the changes in TLR4 expression during hepatic ischemia/reperfusion (I/R) and the effects of propofol on the expression of TLR4 induced by I/R injury in rats.Methods Fifty six male SD rats were randomly divided into min before isehemia in P groups,and the same volume of sodium lactate Ringer's solution was infused in sham group and I/R groups.Plasma ALT,AST,TNF-α levels were measured,and the expression of Tlr4 was detected 2,6,24 h after reperfusion.Results The levels of plasma ALT,AST and TNF-α were lower,and Tlr4 expression was weaker in P groups than those in I/R groups (P＜0.05).Conclusion Propofol decreases hepatic ischemia/reperfusion (I/R)-induced Tlr4 expression and exerts property of liver protection.     

Ghrelin uses Galphai2 and activates voltage-dependent K+ channels to attenuate glucose-induced Ca2+ signaling and insulin release in islet beta-cells: novel signal transduction of ghrelin

Ghrelin reportedly serves as a physiological regulator of insulin release. This study aimed to explore signaling mechanisms for insulinostatic ghrelin action in islet beta-cells, with special attention to heterotrimeric GTP-binding proteins and K(+) channels. Plasma insulin and growth hormone (GH) concentrations in rats were measured by enzyme-linked immunosorbent assay (ELISA). Islets were isolated from rats, ghrelin-knockout (Ghr-KO) mice, and wild-type mice by collagenase digestion, and insulin release was determined by ELISA. In rat single beta-cells, cytosolic Ca(2+) concentration ([Ca(2+)](i)) was measured by fura-2 microfluorometry, and membrane potentials and whole cell currents by patch-clamp technique. In rats, systemic ghrelin administration decreased plasma insulin concentrations, and this effect was blocked by treatment with pertussis toxin (PTX), whereas stimulation of GH release remained unaffected. In rat islets, ghrelin receptor antagonist increased and exogenous ghrelin suppressed glucose-induced insulin release in a PTX-sensitive manner. Glucose-induced insulin release from islets was greater in Ghr-KO than wild-type mice, and this enhanced secretion was blunted with PTX. Ghrelin PTX sensitively increased voltage-dependent K(+) (Kv) currents without affecting ATP-sensitive K(+) channels in rat beta-cells. In the presence of Kv channel blockers, ghrelin failed to suppress insulin release. Ghrelin attenuated glucose-induced action potentials and [Ca(2+)](i) increases in beta-cells. Suppressions of [Ca(2+)](i) increase and insulin release by ghrelin were blunted in beta-cells treated with PTX and with antisense oligonucleotide specific for G-protein Galpha(i2)-subunit. Ghrelin attenuates glucose-induced insulin release via PTX-sensitive Galpha(i2)-mediated activation of Kv channels and suppression of [Ca(2+)](i) in beta-cells, representing the unique signaling of ghrelin distinct from that for GH release.     

Blockade of pancreatic islet-derived ghrelin enhances insulin secretion to prevent high-fat diet-induced glucose intolerance

The gastric hormone ghrelin and its receptor, growth hormone secretagogue receptor (GHSR), are expressed in pancreas. Here, we report that ghrelin is released from pancreatic islets to regulate glucose-induced insulin release. Plasma concentrations of ghrelin, as well as insulin, were higher in pancreatic veins than in arteries. GHSR antagonist and immunoneutralization of endogenous ghrelin enhanced glucose-induced insulin release from perfused pancreas, whereas exogenous ghrelin suppressed it. GHSR antagonist increased plasma insulin levels in gastrectomized and normal rats to a similar extent. Ghrelin knockout mice displayed enhanced glucose-induced insulin release from isolated islets, whereas islet density, size, insulin content, and insulin mRNA levels were unaltered. Glucose tolerance tests (GTTs) in ghrelin knockout mice showed increased insulin and decreased glucose responses. Treatment with high-fat diet produced glucose intolerance in GTTs in wild-type mice. In ghrelin knockout mice, the high-fat diet-induced glucose intolerance was largely prevented, whereas insulin responses to GTTs were markedly enhanced. These findings demonstrate that ghrelin originating from pancreatic islets is a physiological regulator of glucose-induced insulin release. Antagonism of the ghrelin function can enhance insulin release to meet increased demand for insulin in high-fat diet-induced obesity and thereby normalize glycemic control, which may provide a potential therapeutic application to counteract the progression of type 2 diabetes.     

Ghrelin is present in pancreatic alpha-cells of humans and rats and stimulates insulin secretion.

Ghrelin, a novel growth hormone-releasing peptide isolated from human and rat stomach, is a 28-amino acid peptide with a posttranslational acylation modification that is indispensable for stimulating growth hormone secretion by increasing intracellular Ca(2+) concentration. It also functions in the regulation of feeding behavior, energy metabolism, and gastric acid secretion and motility. Using two different antibodies against the NH(2)- and COOH-terminal regions of ghrelin, we studied its localization in human and rat pancreas by immunohistochemistry. Ghrelin-immunoreactive cells were identified at the periphery of pancreatic islets in both species. Ghrelin co-localized exclusively with glucagon in rat islets, indicating that it is produced in alpha-cells. We identified ghrelin and des-acyl ghrelin in the rat pancreas using reverse-phase high-performance liquid chromatography combined with two radioimmunoassays. We also detected mRNA encoding ghrelin and its receptor in the rat pancreatic islets. Ghrelin increased the cytosolic free Ca(2+) concentration in beta-cells and stimulated insulin secretion when it was added to isolated rat pancreatic islets. These findings indicate that ghrelin may regulate islet function in an endocrine and/or paracrine manner.     

Endogenous ghrelin in pancreatic islets restricts insulin release by attenuating Ca2+ signaling in beta-cells: implication in the glycemic control in rodents

Ghrelin, isolated from the human and rat stomach, is the endogenous ligand for the growth hormone (GH) secretagogue receptor, which is expressed in a variety of tissues, including the pancreatic islets. It has been shown that low plasma ghrelin levels correlates with elevated fasting insulin levels and type 2 diabetes. Here we show a physiological role of endogenous ghrelin in the regulation of insulin release and blood glucose in rodents. Acylated ghrelin, the active form of the peptide, was detected in the pancreatic islets. Counteraction of endogenous ghrelin by intraperitoneal injection of specific GH secretagogue receptor antagonists markedly lowered fasting glucose concentrations, attenuated plasma glucose elevation, and enhanced insulin responses during the glucose tolerance test (GTT). Conversely, intraperitoneal exogenous ghrelin GH-independently elevated fasting glucose concentrations, enhanced plasma glucose elevation, and attenuated insulin responses during GTT. Neither GH secretagogue receptor antagonist nor ghrelin affected the profiles of the insulin tolerance test. In isolated islets, GH secretagogue receptor blockade and antiserum against acylated ghrelin markedly enhanced glucose-induced increases in insulin release and intracellular Ca2+ concentration ([Ca2+]i), whereas ghrelin at a relatively high concentration (10 nmol/l) suppressed insulin release. In single beta-cells, ghrelin attenuated glucose-induced first-phase and oscillatory [Ca2+]i increases via the GH secretagogue receptor and in a pertussis toxin-sensitive manner. Ghrelin also increased tetraethylammonium-sensitive delayed outward K+ currents in single beta-cells. These findings reveal that endogenous ghrelin in islets acts on beta-cells to restrict glucose-induced insulin release at least partly via attenuation of Ca2+ signaling, and that this insulinostatic action may be implicated in the upward control of blood glucose. This function of ghrelin, together with inducing GH release and feeding, suggests that ghrelin underlies the integrative regulation of energy homeostasis.     

一氧化氮抑制ghrelin诱导的生长激素腺瘤GH3细胞生长激素分泌和细胞增殖及其机制

目的 检测一氧化氮(NO)对ghrelin诱导的大鼠GH3细胞的生长激素(GH)分泌和细胞增殖的影响,探讨NO的作用机制.方法 首先应用ghrelin在不同浓度分别作用2 h;应用ghrelin在工作浓度1×10-7mol/L,分别作用不同时间,检测对GH3细胞GH分泌的影响;然后检测NO的供体(SNAP,1×10-5 mol/L)和NO合成酶的抑制剂(NAME,1×10-5mol/L)对ghrelin诱导的GH分泌和细胞增殖的影响;用酶联免疫吸附试验(ELISA)方法检测GH水平,噻唑蓝(MTT)比色法检测细胞增殖,Western免疫印迹法检测细胞内信号通路蛋白的活性变化.结果 ghrelin刺激GH3细胞分泌GH呈时间和浓度依赖性(P＜0.01),ghrelin明显刺激GH3细胞的增殖(P＜0.05);SNAP可抑制基础的和ghrelin刺激的GH分泌(P＜0.01),对细胞增殖也有明显影响(P＜0.01),而NAME对GH的分泌和细胞增殖无影响;ghrelin可诱导细胞外信号调节激酶(ERK)的活化,SNAP可以抑制这种效应.结论 NO抑制ghrelin诱导GH3细胞GH分泌和细胞增殖,其机制可能阻断了ghrelin激活的ERK信号通路.     

Ghrelin attenuates cAMP-PKA signaling to evoke insulinostatic cascade in islet β-cells

OBJECTIVE

Ghrelin reportedly restricts insulin release in islet β-cells via the Gα_i2 subtype of G-proteins and thereby regulates glucose homeostasis. This study explored whether ghrelin regulates cAMP signaling and whether this regulation induces insulinostatic cascade in islet β-cells.

RESEARCH DESIGN AND METHODS

Insulin release was measured in rat perfused pancreas and isolated islets and cAMP production in isolated islets. Cytosolic cAMP concentrations ([cAMP]_i) were monitored in mouse MIN6 cells using evanescent-wave fluorescence imaging. In rat single β-cells, cytosolic protein kinase-A activity ([PKA]_i) and Ca²⁺ concentration ([Ca²⁺]_i) were measured by DR-II and fura-2 microfluorometry, respectively, and whole cell currents by patch-clamp technique.

RESULTS

Ghrelin suppressed glucose (8.3 mmol/L)-induced insulin release in rat perfused pancreas and isolated islets, and these effects of ghrelin were blunted in the presence of cAMP analogs or adenylate cyclase inhibitor. Glucose-induced cAMP production in isolated islets was attenuated by ghrelin and enhanced by ghrelin receptor antagonist and anti-ghrelin antiserum, which counteract endogenous islet-derived ghrelin. Ghrelin inhibited the glucose-induced [cAMP]_i elevation and [PKA]_i activation in MIN6 and rat β-cells, respectively. Furthermore, ghrelin potentiated voltage-dependent K⁺ (Kv) channel currents without altering Ca²⁺ channel currents and attenuated glucose-induced [Ca²⁺]_i increases in rat β-cells in a PKA-dependent manner.

CONCLUSIONS

Ghrelin directly interacts with islet β-cells to attenuate glucose-induced cAMP production and PKA activation, which lead to activation of Kv channels and suppression of glucose-induced [Ca²⁺]_i increase and insulin release.Ghrelin, an acylated 28-amino acid peptide, is the endogenous ligand for the growth hormone secretagogue receptor (GHS-R) (1,2). Ghrelin is produced predominantly in the stomach and stimulates growth hormone release and feeding and exhibits positive cardiovascular effects, suggesting its possible clinical application (3). Ghrelin and GHS-R are located in the pancreatic islets (4–6). Furthermore, ghrelin O-acyltransferase (GOAT), which has been identified as the enzyme that promotes the acylation of the third serine residue of ghrelin, is highly expressed in the pancreatic islets (7–9). Administration of ghrelin at 10⁻⁹–10⁻⁸ mol/L, the concentrations higher than the circulating levels of 10⁻¹⁰–10⁻⁹ mol/L, attenuates insulin release and deteriorates glucose tolerance in rodents and humans, whereas desacyl ghrelin has no effects (5,10,11). This effective concentration of ghrelin that is approximately one log order higher than the circulating level is considered physiological in the pancreatic islets for the following reasons: 1) ghrelin is produced in the pancreatic islets (5), and the ghrelin level in the pancreatic vein is one log order higher than that in the pancreatic artery (12), indicative of release of ghrelin from the pancreas; 2) ghrelin immunoneutralization and GHS-R antagonists augment glucose-induced insulin release from perfused pancreas and isolated islets (5,12); and 3) ghrelin knockout mice display enhanced glucose-induced insulin release from isolated islets without altering islet density and size, insulin content, or insulin mRNA levels, indicating increased secretory activity in the knockout mouse islets (12). Furthermore, glucose intolerance in high-fat diet–induced obese (DIO) mice is prevented in the ghrelin knockout mice as a result of enhanced insulin secretory response to glucose (12). These findings suggest that the islet-derived ghrelin regulates insulin release in a paracrine and/or autocrine manner and that manipulation of the ghrelin action could provide a novel tool to optimize insulin release (13,14).It is currently thought that GHS-R is coupled primarily to G₁₁-phospholipase C signaling (2). Intriguingly, our previous data indicated that the insulinostatic ghrelin signaling is produced via pertussis toxin (PTX)-sensitive G-protein Gα_i2 in β-cells; ghrelin PTX-sensitively activates voltage-dependent K⁺ (Kv) channels, attenuates membrane excitability, and suppresses glucose-induced Ca²⁺ concentration ([Ca²⁺]_i) increases in β-cells to attenuate insulin release (15). However, the mechanism that links GHS-R and Gα_i2 to these activities in β-cells remains to be clarified. It is known that PTX-sensitive G_i-proteins inhibit adenylate cyclase, which produces cyclic AMP in the cells. In pancreatic β-cells, intracellular cAMP signals are generated by nutrient secretagogues and play a critical role in regulating insulin secretion (16–18). However, implication of cAMP signaling in the insulinostatic function of ghrelin has been unclear. In this study, we aimed to clarify whether ghrelin regulates cAMP pathway in islet β-cells and whether this regulation leads to insulinostatic cascade in islet β-cells. We here show a novel signaling mechanism for ghrelin that operates in islet β-cells; GHS-R–mediated attenuation of cAMP and protein kinase-A (PKA) signaling leads to activation of Kv channels and suppression of glucose-induced [Ca²⁺]_i increase and insulin release.     

脑肠肽对脓毒症相关性肾损伤的保护作用研究

目的 探讨脑肠肽对内毒素所致大鼠脓毒症相关性肾损伤的影响.方法 健康雄性SD大鼠36只,随机分为3组：正常对照组,急性肾损伤（acute kidney injury,AKI）组,脑肠肽治疗组.采用内毒素静注制备脓毒症AKI模型,脑肠肽治疗组于造模前后30 min给予皮下注射脑肠肽（1.0mg/kg）,选择不同时间点（6 h、12 h、24 h）处死动物后留取血标本和肾组织,检测血清肌酐（SCr）、尿素氮（BUN）、血清肿瘤坏死因子-α（tumor necrosis factor-α,TNF-α）、观察肾组织的病理变化并检测肾脏组织中核因子κB的活化.结果 与同时间点比较,AKI组6h和12 h大鼠血清中TNF-α表达水平明显升高（P＜0.01）,24 h降至对照组水平（P＞0.05）,AKI组6h、12 h和24 h大鼠血清中BUN水平逐渐升高（P＜0.05）,24 h血清中SCr水平明显升高（P＜0.01）;AKI组24 h大鼠肾脏组织核因子κB p65核阳性率明显升高（P＜0.01）.脑肠肽治疗组对应时间点血清BUN和SCr水平较AKI组明显降低（P＜0.01）.TNF-α和核因子κB p65表达明显低于AKI组（P＜0.01）.病理显示脑肠肽治疗组大鼠肾损伤减轻,SCr、BUN、TNF-α、光镜检查均未见明显差异.结论 脑肠肽可通过抑制肾组织核因子κB的表达,下调TNF水平,对脓毒症相关性肾损伤发挥保护作用.     

Ghrelin improves renal function in mice with ischemic acute renal failure

Growth hormone and IGF-1 have been suggested to have tissue-protective effects. Ghrelin is a stomach-derived growth hormone secretagogue. The effects of ghrelin on ischemia/reperfusion-induced renal failure in mice were examined. Ischemic acute renal failure was induced by bilateral renal artery clamping for 45 min and reperfusion for 24 h. Ghrelin (100 microg/kg mouse) or vehicle was injected subcutaneously six times before surgery and three times after surgery every 8 h. Twenty-four hours after reperfusion, the right kidney was isolated and perfused. Acetylcholine (ACh)- and adrenomedullin-induced endothelium-dependent vasorelaxation of renal vessels significantly improved in ghrelin-pretreated mice (%Delta renal perfusion pressure by 10(-7) M ACh -63.5 +/- 3.7 versus -41.2 +/- 5.5%; P < 0.05). This change was associated with significant increases of nitric oxide release in the kidneys of ghrelin-treated mice (10(-7) M ACh 35.5 +/- 5.8 versus 16.9 +/- 3.5 fmol/g kidney per min; P < 0.05). Serum concentration of urea nitrogen (53 +/- 7 versus 87 +/- 15 mg/dl; P < 0.05) and renal injury score were significantly lower in the ghrelin group (2.5 +/- 0.8 versus 5.3 +/- 1.5; P < 0.01). Tubular apoptotic index was significantly lower in the ghrelin group (5 +/- 5 versus 28 +/- 4; P < 0.05). Furthermore, the survival rate after the 60-min ischemic period was higher in the ghrelin group (80 versus 20%; P < 0.05). Ghrelin treatment significantly increased the serum level of IGF-1. However, such renal protective effects of ghrelin on ischemia/reperfusion injury were not observed in insulin receptor substrate-2 knockout mice. These results suggest that ghrelin may protect the kidneys from ischemia/reperfusion injury and that this effect is related to an improvement of endothelial function through an IGF-1-mediated pathway.     

Exogenous Ghrelin Enhances Endocrine and Exocrine Regeneration in Pancreatectomized Rats

Aim  Ghrelin, the most important modulator of endocrine and exocrine pancreatic functions, has a role in the development of islets of Langerhans during embryogenesis. The aim of this study was to evaluate the effects of ghrelin on pancreatic regeneration in rats with 90% pancreatectomy. Materials and Methods  Two- to 3-week-old Wistar rats were used in the study. After anesthesia, 90% pancreatectomy was performed. In the ghrelin group, 90% pancreatectomy was performed. Ten nanomoles per kilogram per day of ghrelin was administered intraperitoneally from the first postoperative day. In the antagonist group, 90% pancreatectomy was performed. From the first postoperative day, rats received the ghrelin receptor antagonists and substance P intraperitoneally at 1 μmol/kg. In the control group, 90% pancreatectomy was performed, and intraperitoneal saline was administered. The sham group did not receive pancreatectomy. Eight rats from each group were randomly selected and sacrificed on the second, third, and 30th days. Results  Blood glucose levels in pacreatectomized rats were significantly higher than in rats in the sham group. The number of beta islet cells, serum insulin levels, and PDX-1 and cytokeratin staining scores decreased in rats with pancreatectomy when compared to the sham-group rats. In the ghrelin-receiving rats, blood glucose levels tended to decrease from the 15th postoperative day. Ghrelin treatment increased insulin levels, insulin-positive islet cell number, and 5-bromo-2-deoxyuridine and PDX-1 staining, whereas ghrelin antagonist administration resulted in significant decreases in these parameters. Ghrelin treatment significantly improved glucose tolerance test results. Conclusion  Exogenous ghrelin administration decreased blood glucose levels after 90% pancreatectomy by increasing islet cell numbers and enhancing endocrine and exocrine regeneration. Kerem M and Salman B contributed equally to this work; Kerem M, Salman B, and Bedirli A designed experiments; Kerem M, Salman B, Pasaoglu H, Ozsoy S, Haziroglu R, and Yilmaz Tu performed experiments; Kerem M, Salman B, and Bedirli A analyzed data; Kerem M, Salman B, and Bedirli A wrote the paper.