牡丹皮对脂多糖诱导大鼠系膜细胞的保护作用

目的 观察牡丹皮对脂多糖（LPS）诱导的大鼠系膜细胞（GMC）转化生长因子-β1（TGF-β1）、血小板衍生性生长因子（PDGF）和血管内皮生长因子(VEGF)表达的影响。方法 用脂多糖 (20 mg·L^-1)刺激大鼠系膜细胞,经牡丹皮(50、100、200 mg·L^-1)培养48 h后,采用ELISA法检测转化生长因子-β1的分泌水平;实时定量PCR方法检测血小板衍生性生长因子和血管内皮生长因子 mRNA的表达。结果 牡丹皮可以抑制脂多糖诱导的大鼠系膜细胞转化生长因子-β1的分泌,下调血小板衍生性生长因子和血管内皮生长因子的表达。结论 牡丹皮可能通过减少转化生长因子-β1的分泌,抑制血小板衍生性生长因子和血管内皮生长因子的表达,对受损的大鼠系膜细胞起保护作用。     

Characterization of a B2-bradykinin receptor in rat renal mesangial cells

The specific binding of bradykinin (BK) was investigated using membrane fractions from mesangial cells in primary culture, a cloned cell line, and in intact adherent cells with three different radiolabelled BK analogues: ¹²⁵I-[Tyr⁰]BK, ¹²⁵I-[Tyr⁵]BK and ¹²⁵I-[Tyr⁸]BK. The best radioligand was ¹²⁵I-[Tyr⁰]BK, and assay conditions were determined to ensure maximal stable binding. Binding appeared to be reversible and not to be inhibited by a wide variety of protease inhibitors including converting enzyme inhibitor and phosphoramidon. The maximum density of binding sites (B_max) was about 88 ± 18 fmol/mg protein, which is equivalent to about 6000 sites/cell, and the dissociation constant averaged 2 nM. No significant difference in B_max was observed between membranes from cells in primary culture and those from cloned cells. Of the BK analogues tested, unmodified BK exhibited the highest inhibition constant (close to 10⁻¹⁰ M). No displacement of ¹²⁵I-[Tyr⁰]BK was observed in the presence of the B₁ agonist des-Arg⁹-BK or several unrelated peptides, including atrial natriuretic factor and angiotensin I and II, whereas 50% inhibition of binding was achieved with the B₂ antagonist [D-Arg,Hyp³,D-Phe⁷]BK (10⁻⁹ M). Addition of BK for 3 min to the incubation medium of cloned mesangial cells induced a dose- and time-dependent increase in PGE₂ unlike des-Arg⁹-BK, which showed no such effect. The secretion was strongly inhibited by prior incubation with the B₂ antagonist [D-Arg,Hyp³,D-Phe⁷]BK. The pharmacological profile of the binding site determined with various BK agonists and antagonists, and the stimulating effect of binding site activation on prostaglandin release strongly suggest that B₂-kinin-like receptors are present in rat mesangial cells.     

Platelet-derived growth factor triggers PKA-mediated signalling by a redox-dependent mechanism in rat renal mesangial cells

Inflammatory glomerular kidney diseases are often accompanied with a massive production of reactive oxygen species (ROS) that affect the function of the glomerular filtration barrier and contribute to mesangiolysis via the induction of cell death in mesangial cells. Intriguingly, ROS also trigger fine-tuned signalling processes that affect gene expression and cell proliferation or migration. To define such redox-driven signalling devices, a proteomics approach was performed to identify the formation of protein complexes induced by ROS. To this end, protein lysates of human podocytes were treated with or without hydrogen peroxide (250 μM). Thereafter cell lysates were subjected to diagonal 2D gel electrophoresis and putative redox-affected proteins were analysed by MS/MS analysis. Among others, the regulatory subunit of protein kinase A (PKA) could be identified that forms homodimers under oxidative conditions. To evaluate whether ROS dependent dimerization of PKA also occurs in a more physiological setting, rat mesangial cells were treated with platelet-derived growth factor-BB (PDGF-BB) to induce ROS formation. This regimen resulted in a redox dependent dimerization of the R-subunits of PKA. To demonstrate whether PDGF-BB induced ROS formation affects PKA dependent pathways, the effects of PDGF-BB on phosphorylation of serine 157 of vasodilator stimulated protein (VASP) a classical target of PKA were analysed. Interestingly PDGF-BB induced VASP phosphorylation in a ROS dependent manner but independent of changes in cAMP levels. Taken together, we demonstrate a redox-mediated activation of PKA by PDGF-BB thus highlighting a physiological role of ROS as regulator of PKA activity in rat mesangial cells.     

薏苡仁油对体外大鼠系膜细胞端粒酶表达的影响

目的观察以薏苡仁油为主要成分的康莱特注射液(KLT)对大鼠端粒酶活性的影响,为该药用于肾炎治疗提供实验基础。方法应用端粒重复序列扩增(TRAP)TRAP-ELISA及聚丙烯酰胺凝胶电泳银染技术,检测肾小球系膜细胞株(MC)在加入KLT6 h后再加白介素(IL)-1或者加入IL-1 6 h后再加KLT作用下端粒酶活性的表达。结果IL-1 10μg.L-1刺激下MC端粒酶活性(ΔA=A450 nm-A690 nm)升高(1.49±0.51),KLT 100 mL.L-1抑制MC端粒酶活性(0.29±0.17)。两者序贯加入,无论先后都抑制端粒酶表达〔(0.47±0.19),(0.39±0.07),P<0.05,n=3〕。结论KLT注射液对MC的端粒酶表达有抑制作用,并可干预IL-1的刺激效应。     

Regulation of glomerular mesangial cell proliferation in culture by adrenomedullin.

Adrenomedullin is a recently discovered vasodilatory peptide that has been shown to be a potent activator of adenylate cyclase in a variety of cell systems, including rat mesangial cells. The major aim of the present study was to determine the regulation of rat mesangial cell proliferation (using [3H]thymidine incorporation as an index), apoptosis (using nucleosome-associated cytoplasmic DNA fragmentation as an index) and mitogen-activated protein kinase (MAPK) cascade, specifically extracellular signal-regulated kinase (ERK), jun-amino terminal kinase (JNK) and P38 mitogen-activated protein kinase (P38 MAPK) activities, by adrenomedullin-stimulated cyclic AMP-protein kinase-A pathway. Adrenomedullin increased cAMP levels significantly above basal and the response was inhibited by the adrenomedullin receptor antagonist, adrenomedullin-(22-52). Adrenomedullin also decreased [3H]thymidine incorporation and increased nucleosome-associated cytoplasmic DNA fragmentation, in a concentration-dependent fashion. Both these responses were receptor mediated as, adrenomedullin-(22-52) inhibited these effects. The decrease in proliferation and increase in apoptosis were both mimicked by forskolin, a direct adenylate cyclase activator. Adrenomedullin-mediated decrease in proliferation and increase in apoptosis were inhibited by H89 [[N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, hydrochloride]], a potent protein kinase-A inhibitor. Associated with the changes in proliferation and apoptosis, adrenomedullin decreased ERK2 activity, and increased JNK1 and P38 MAPK activities. All these kinase activities, except the increase in JNK1 activity could be simulated using forskolin. In addition, only adrenomedullin-mediated changes in ERK2 and P38 MAPK activities were inhibited by H89 while, adrenomedullin-stimulated JNK1 was not consistently inhibited by the protein kinase-A inhibitor. These results suggest that adrenomedullin might play an important role in mesangial cell turnover and that although adrenomedullin-mediated responses are primarily cAMP-dependent, it does not preclude the involvement of cAMP-independent pathways.     

二苯乙烯苷对高糖诱导系膜细胞衰老相关信号的调节作用

高糖引起的细胞衰老与氧化应激、糖基化终产物的产生增加、信号转导通路的活化密切相关~([1]).二苯乙烯苷(TSG)是何首乌中提取的一种具有明显药理活性水溶性有效成分~([2]),具有抗氧化作用、延缓衰老、改善脂质代谢等功能.     

Positive inotropic effects of adrenomedullin on rat papillary muscle

Adrenomedullin is a peptide recently isolated from pheochromocytoma that has vasorelaxant and long-lasting hypotensive activities. Plasma levels of adrenomedullin are elevated in patients with congestive heart failure, but the effects of adrenomedullin on the cardiac function are unclear. We, thus, investigated the effects of adrenomedullin on the contraction of rat papillary muscles. We measured the isometric tension and cAMP contents of isolated rat papillary muscles. Adrenomedullin exhibited concentration-dependent inotropic effects. Adrenomedullin also significantly increased intracellular contents of cAMP. Addition of the calcitonin gene-related peptide (CGRP) receptor antagonist inhibited both contractile force and cAMP generation of papillary muscles stimulated by adrenomedullin. The adrenomedullin-induced inotropic effect was further increased in the presence of the phosphodiesterase inhibitor, 3-isobutyl-1-methyl-xanthine (IBMX), while the effect was significantly suppressed by KT5720 and Rp-8-bromoadenosine-3',5'-cyclic monophosphorothioate (Rp-8-Br-cAMPS), protein kinase A inhibitors. These results indicate that adrenomedullin has positive inotropic effects on the heart, at least partially through a cAMP-dependent pathway.     

Characterization of a B2-bradykinin receptor in rat renal mesangial cells

The specific binding of bradykinin (BK) was investigated using membrane fractions from mesangial cells in primary culture, a cloned cell line, and in intact adherent cells with three different radiolabelled BK analogues: ¹²⁵I-[Tyr⁰]BK, ¹²⁵I-[Tyr⁵]BK and ¹²⁵I-[Tyr⁸]BK. The best radioligand was ¹²⁵I-[Tyr⁰]BK, and assay conditions were determined to ensure maximal stable binding. Binding appeared to be reversible and not to be inhibited by a wide variety of protease inhibitors including converting enzyme inhibitor and phosphoramidon. The maximum density of binding sites (B_max) was about 88 ± 18 fmol/mg protein, which is equivalent to about 6000 sites/cell, and the dissociation constant averaged 2 nM. No significant difference in B_max was observed between membranes from cells in primary culture and those from cloned cells. Of the BK analogues tested, unmodified BK exhibited the highest inhibition constant (close to 10⁻¹⁰ M). No displacement of ¹²⁵I-[Tyr⁰]BK was observed in the presence of the B₁ agonist des-Arg⁹-BK or several unrelated peptides, including atrial natriuretic factor and angiotensin I and II, whereas 50% inhibition of binding was achieved with the B₂ antagonist [D-Arg,Hyp³,D-Phe⁷]BK (10⁻⁹ M). Addition of BK for 3 min to the incubation medium of cloned mesangial cells induced a dose- and time-dependent increase in PGE₂ unlike des-Arg⁹-BK, which showed no such effect. The secretion was strongly inhibited by prior incubation with the B₂ antagonist [D-Arg,Hyp³,D-Phe⁷]BK. The pharmacological profile of the binding site determined with various BK agonists and antagonists, and the stimulating effect of binding site activation on prostaglandin release strongly suggest that B₂-kinin-like receptors are present in rat mesangial cells.     

Cyclosporin A augments vasoconstrictor-induced rise in intracellular free calcium in rat renal mesangial cells

Pretreatment of rat renal mesangial cells with the immunosuppressive drug cyclosporin A caused a dose-dependent increase in the angiotensin II, [Arg8]vasopressin and noradrenalin-stimulated rise in intracellular free calcium as measured with quin 2. Cyclosporin A had no significant effect on basal cytosolic free calcium. However, cyclosporin A increased the basal 45Ca2+ influx. This stimulated 45Ca2+ influx was not blocked by nifedipine (10(-6) M). Cyclosporin A also augmented the angiotensin II, [Arg8]vasopressin and noradrenalin-stimulated efflux of 45Ca2+ from mesangial cells. These results suggest that cyclosporin A stimulates transmembrane Ca2+ influx in mesangial cells and also augments the vasoconstrictor-induced increases in cytosolic free calcium.     

Inhibitory effects of honokiol on lipopolysaccharide-induced cellular responses and signaling events in human renal mesangial cells

Honokiol has been shown to possess a lot of pharmacologic benefits, including antioxidative, antiangiogenic and antineoplastic effects. In the present study, we investigated the anti-inflammatory effects of honokiol and the signaling mechanisms involved in lipopolysaccharide (LPS)-induced conditions in human renal mesangial cells (HRMCs). Honokiol did not significantly change HRMC viability when used at a concentration of < 20 μmol/l but markedly altered cell viability at concentrations of > 40 μmol/l. In this study, LPS treatment led to a marked upregulation of the levels of IL-1β, IL-18, TNF-α, TGF-β1, CCL2, CCL3, and CCL5 in HRMCs. The expression of COX-2, iNOS, and their products PGE₂ and NO also increased. The upregulation of these molecules was significantly abolished by honokiol in a dose-dependent manner. Moreover, honokiol almost completely reversed IL-1β, CCL3, and NO expression at 10 μmol/l, and IL-18, TNF-α, TGF-β1, and COX-2 expression at 20 μmol/l. In addition, phospho-NF-κB p65 at Ser536, phospho-Akt, and phospho-p42/44 were dramatically suppressed by honokiol in LPS-treated HRMCs. These results indicate that honokiol can inhibit the LPS-induced expression of inflammatory cytokines and mediators in HRMCs. The anti-inflammatory mechanisms of honokiol are partly due to the suppression of the phospho-NF-κB p65, phospho-Akt and phospho-p42/44 pathways.     

The stimulation of somatostatin release by hpGRF44 from rat hypothalamic cells and fragments in vitro

Growth hormone releasing factor (GHRF) was examined to determine whether it affects somatostatin (SRIF) release from cultured rat hypothalamic cells and fragments in vitro. The hypothalami of rat fetuses were collected on the 17th day of pregnancy under a dissection microscope. Thirty hypothalami were placed in phosphate buffered saline, and the cells were dispersed with 0.1% collagenase. The dispersed cells were cultured in Dulbecco's modified Eagle medium (DMEM) containing 10% horse serum and 2.5% fetal calf serum at 37 degrees C under 5% CO2 in air. On the 12th day of culture, the cells were washed with Krebs Ringer bicarbonate buffer containing glucose (KRBG), and then incubated with KRBG for 1 hour. The medium was replaced with KRBG alone (control) or KRBG containing test substances, and incubated for another hour. SRIF released into the medium was quantitated by RIA. The mean basal release of SRIF was 14.7 +/- 0.9 pg/dish/hour. One-tenth, 1, and 10nM hpGRF44 stimulated SRIF release by 1.4, 1.5, and 1.8 fold respectively in a dose-related manner. Ten nM ovine corticotropin releasing factor (o-CRF) also stimulated SRIF release by 2.3 fold. One, 10, and 100 nM hpGRF44, 10nM o-CRF, 10nM thyrotropin releasing hormone (TRH) and 60 mM K+ also stimulated SRIF release from rat hypothalamic fragments. Removal of Ca++ from the medium resulted in a decrease of basal release of SRIF. In Ca++ free medium, 10nM hpGRF44 failed to release SRIF. One-tenth nM hpGRF44, 10nM GnRH, and 10nM VIP have no effect on SRIF release statistically. The results of this study demonstrate that a high concentration of GHRF stimulates SRIF release from the hypothalamus in vitro, suggesting a possibility that GHRF may increase the release of SRIF from the median eminence and the hypothalamus in vivo under certain conditions.     

糖基化终产物对人肾小球系膜细胞细胞外基质金属蛋白酶促进因子基因表达的影响

目的 探讨糖基化终产物(AGEs)对人肾小球系膜细胞(HMRC)细胞外基质金属蛋白酶诱导因子(EMMPRIN)基因表达的影响.方法 在培养的人肾系膜细胞(HRMC)中分别加入不同浓度AGEs干预24 h和同一浓度AGEs干预HRMC不同的时间点,以无血清培养基(DMEM)和相应浓度的牛血清白蛋白(BSA)为对照.RT-PCR法检测EMMPRIN mRNA的表达水平.结果 与对照组相比,经各浓度及各时间点AGEs干预后, HMRC的EMMPRIN mRNA水平与对照组相比有显著差异(P＜0.01),且随AGEs浓度以及干预时间增加,EMMPRIN mRNA水平也明显逐渐降低(P＜0.05).结论 EMMPRIN可能是AGEs所致糖尿病肾病的因素之一.     

Pleiotropic effects of cadmium in mesangial cells

The mesangial cell of the renal glomerulus is exposed to circulating toxic substances and is a target involved in the glomerular component of chronic occupational and environmental exposure to cadmium. We review evidence for the involvement of cadmium in mesangial cell pathology, including effects on cell signaling, oncogene expression, and cell death. Previously we have shown that cadmium can inhibit apoptosis initiated through both the extrinsic (death ligand receptor) and intrinsic (mitochondrial) pathways, whereas exposure of mesangial cells to 10 μM CdCl₂ for 6 h initiates caspase-independent cell death through both apoptotic and apoptotic-like (annexin V positive, propidium iodide staining) mechanisms. Apoptotic death is dependent upon activation of Ca²⁺/calmodulin-dependent protein kinase II (CaMK-II). In the present study we show that low level exposure of mesangial cells to Cd²⁺ (0.5 μM) initiates cell survival signals including PI3 kinase/Akt signaling, also dependent on CaMK-II, that are eventually overcome resulting in caspase-dependent cell death. These studies underscore the roles of cell signaling in various modes of cell death, and in particular the central role of CaMK-II in cadmium toxicology of the mesangial cell.     

Proinflammatory cytokines enhance COX-1 gene expression in cultured rat glomerular mesangial cells

Glomerular mesangial cells (GMC) exert an essential maintaining effect on hemodynamic integrity and immune competence of the kidney through arachidonate metabolism. To clarify this, cultured rat GMC were measured for the expression and production of cyclooxygenase (COX) and excretion of prostaglandin (PG). The rat GMC spontaneously expressed type 1 cyclooxygenase (COX-1), but not COX-2. The PGE2 and thromboxane B2 (TXB2) were spontaneously produced by the cells. Interleukin (IL)-1beta (25 ng/ml), IL-8 (25 ng/ml), growth-related oncogene-alpha (GRO, 50 ng/ml) and tumor necrosis factor-alpha (TNF-alpha, 25 ng/ml) stimulated the COX-1 protein production as demonstrated by Western blot and enhanced PGE2 synthesis in GMC, beginning on 2 h of incubation, and steadily enhanced TXB2 synthesis over a 24-h period. Lipopolysaccharide (LPS, 100 ng/ml) enhanced both PGE2 and TXB2 syntheses from 2 h to at least 24 h of incubation. Collectively, the proinflammatory cytokines could enhance COX-1 but not COX-2 expression in GMC leading to increased PGE2 and TXB2 production. These biochemical events may be implicated in normal renal physiology as well as in pathogenesis of glomerular diseases.     

大鼠FasL基因重组慢病毒载体介导大鼠肾细胞体外转染

目的 探讨慢病毒载体对大鼠肾细胞体外转染的可行性.方法 三质粒系统共转染包装细胞293-T,合成重组慢病毒载体,P24gag EIISA对其定量测定.流式细胞术测定SD大鼠肾细胞Fas、FasL的表达.Western blot、RT-PCR检测转染后目的 基因的表达.结果 定量测定目的 基因载体、空白载体浓度分别为11.6 ng/ml、13.8 ng/ml,Western blot、RT-PCR分别检测到目的 基因的表达.结论 慢病毒载体体外成功转染SD大鼠肾细胞,并表达目的 基因.     

大鼠糖皮质激素受体基因腺病毒载体的构建及在肾系膜细胞中的表达

目的 利用AdEasy系统构建大鼠糖皮质激素受体(GR)基因重组腺病毒载体,转染大鼠肾小球系膜细胞(GMC),并通过Western Blot法检测GR蛋白的表达.方法 将质粒pcDNA1 Neo-Rat GR扩增、凝胶回收获得的Rat GR cDNA双酶切片段,插入腺病毒穿梭载体质粒pShuttle-CMV的巨细胞病毒(CMV)启动子下游,构建重组穿梭载体pShuttle-CMV-Rat GR,线性化后与骨架载体AdEasy-1在细菌BJ5183内经同源重组得到腺病毒重组质粒pAd-Rat GR,经人胚肾293A细胞包装后得到复制缺陷型重组腺病毒Ad-Rat GR;用包装后的病毒上清转染大鼠GMC,提取细胞中总蛋白,通过Western Blot方法 检测GR蛋白的表达.结果 连接、重组后通过酶切和测序法筛选出pAd-Rat GR重组质粒,经人胚肾293细胞包装,48 h后观察到增强型绿色荧光蛋白(EGFP)明显表达,氯化铯梯度离心纯化最终获得1×109 PFU /ml 滴度的重组病毒;用该滴度的Ad-Rat GR,转染大鼠GMC 48 h后提取细胞总蛋白,Western Blot检测GR蛋白有明显表达.结论 构建的大鼠GR基因腺病毒载体能够在大鼠GMC 中表达GR蛋白,并有上调作用.