【作者】 刘书娟；

【导师】 倪鑫； 朱晓燕；

【作者基本信息】 第二军医大学 ， 生理学， 2010， 博士

【摘要】 感染应激时,炎症反应可通过激活HPA轴促进肾上腺皮质分泌大量糖皮质激素(glucocorticoids, GCs)。GCs的抗炎作用已为人们所熟知,也是它成为临床上治疗多种急、慢性炎症性疾病的一线药物的主要原因。然而近年来的研究表明GCs在某些情况下还具有促进炎症反应的作用,学者们认为这是GCs限制其自身作用的一种方式,从而避免其发挥过度的抗炎和免疫抑制效应、减少副作用。然而有关GCs调节抗炎-促炎平衡的具体机制仍未完全阐明。虽然GCs在机体面临炎症反应时对于维持内环境的稳定起着重要作用,但是大量临床资料表明在包括脓毒症在内的重症炎症性疾病患者常出现肾上腺功能不全的表现,患者GCs基础分泌水平降低或对促肾上腺皮质激素(adrenocorticotrophic hormone, ACTH)刺激反应低下,而且GCs分泌不足通常还伴随患者死亡率显著增高。因此,明确炎症性疾病时肾上腺功能不全的机制对重症炎症性疾病的治疗具有非常重要的意义。在本论文的第一部分,我们首先发现巨噬细胞不仅可以产生已知具有显著促炎作用的气体分子一氧化氮(nitric oxide, NO),同时还可生成新型气体分子硫化氢(hydrogen sulfide, H2S),而且后者可以通过抑制NO的生成而具有一定抗炎作用。在进一步的工作中,我们还发现GCs可同时抑制巨噬细胞生成NO和H2S,并重点探讨了GCs抑制H2S生成的作用机制。这一部分的工作为阐明GCs在调节抗炎一促炎平衡中的作用机制提供了重要的实验依据。已有研究表明,微量内毒素(lipopolysaccharide, LPS)预处理单核细胞/巨噬细胞等天然免疫细胞后可以导致该细胞对后续的内毒素刺激反应低下,这种现象被称之为“内毒素耐受”。目前内毒素耐受导致脓毒症时免疫受抑的观点已被广泛接受,,在本论文的第二部分,我们提出内毒素耐受可能也发生在肾上腺水平,从而导致了脓毒症时肾上腺的低反应性。为验证这种假说,我们首先观察了LPS对大鼠肾上腺束状带-网状带(zona fasciculata-reticularis, F/R)细胞皮质酮分泌和皮质酮相关调节蛋白和代谢酶表达的影响,继而探讨了LPS预处理对LPS和ACTH诱导的F/R细胞皮质酮生成的影响及其作用机制。主要实验结果如下：一、巨噬细胞内H2S的生成及功能1.RT-PCR和western blot结果显示巨噬细胞上有内源性硫化氢合成酶胱硫醚-γ-裂解酶CSE mRNA和蛋白的表达,而无CBS mRNA和蛋白的表达。H2S的实时定量检测结果也显示巨噬细胞能够产生内源性的H2S。2.荧光实时定量PCR和western blot结果显示内毒素LPS能够剂量依赖性地诱导巨噬细胞内CSE的mRNA和蛋白表达。H2S的实时定量检测结果也显示LPS能够显著增加内源性H2S的生成。提示在革兰氏阴性菌导致的炎症反应中,巨噬细胞是内源性H2S生成的来源之一3.一氧化氮测定和western blot结果显示H2S的前体L-半胱氨酸(L-cysteine)和外源性的H2S供体NaHS均显著抑制了LPS诱导的NO的生成和诱导性一氧化氮合酶(inducible nitric oxide synthase, iNOS)表达,而CSE的抑制剂DL-propargylglycine (PAG)则显著阻断了L-半胱氨酸的作用。用siRNA阻断CSE基因表达能显著促进LPS诱导的NO生成和iNOS表达,而CSE过表达则能显著抑制LPS诱导的NO生成和iNOS表达。提示H2S可能可以通过抑制NO生成而发挥抗炎作用。二、糖皮质激素对H2S生成的调节及机制1.荧光实时定量PCR、western blot、H2S检测、NO检测结果显示,地塞米松能够剂量依赖性地抑制LPS诱导的巨噬细胞内CSE mRNA和蛋白表达及H2S生成量,同时也能显著抑制iNOS蛋白表达及NO生成量。提示这一作用可能是糖皮质激素在炎症过程中调节促炎、抗炎平衡的重要机制之一。2.荧光实时定量PCR、western blot、H2S检测结果显示NO前体L-精氨酸(L-arginine, L-arg)剂量依赖性地促进LPS诱导的CSE表达和H2S生成,而NOS抑制剂NG-nitro-L arginine methyl ester (L-NAME)则剂量依赖性地抑制LPS诱导的CSE表达和H2S生成。提示地塞米松可能通过抑制NO生成而间接抑制CSE表达和H2S生成。3.荧光实时定量PCR、western blot、H2S检测结果显示,在L-NAME存在时地塞米松仍然能够显著抑制LPS诱导的CSE表达和H2S生成。提示地塞米松还可以通过不依赖于NO的方式直接抑制CSE表达和H2S生成。4.荧光实时定量PCR结果显示地塞米松使原代腹腔巨噬细胞CSE mRNA稳定性显著降低。我们以往荧光素酶报告基因检测的结果显示地塞米松显著抑制RAW264.7巨噬细胞中LPS诱导的CSE启动子转录激活。提示糖皮质激素可能通过不同的机制抑制原代巨噬细胞和巨噬细胞系的CSE表达,GCs对CSE表达的调节机制可能具有细胞特异性。三、LPS对大鼠原代F/R细胞皮质酮生成的影响及机制1.免疫荧光组织化学和细胞化学结果显示LPS的受体toll样受体4(toll-like receptor 4, TLR4)可表达于肾上腺组织和原代培养的F/R细胞,提示LPS可能可以通过与TLR4结合而直接作用于肾上腺。2.皮质酮检测、荧光实时定量PCR和western blot结果显示LPS能够刺激原代F/R细胞分泌皮质酮,同时可诱导类固醇合成急性调节蛋白(steroidogenic acute regulatory protein, StAR)、细胞色素P450 11β-羟化酶(cytochrome P450 11β-hydroxylase, CYP11B1)表达,但LPS并不影响F/R细胞中胆固醇侧链裂解酶(p450 side chain cleavage enzyme, CYP11A1)、3β-羟化类固醇脱氢酶(3β-hydroxysteroid dehydrogenase, HSD3B)和细胞色素P450 21β-羟化酶(cytochrome P450 21β-hydroxylase, CYP21)的表达。ACTH则可以促进StAR和所有皮质酮合成相关酶(CYP11A1,HSD3B,CYP21和CYP11B1)的表达。上述结果表明ACTH和LPS在诱导皮质酮生成的过程中所涉及到的调节蛋白或代谢酶并不相同,因此两者作用于肾上腺皮质细胞后所介导的细胞内信号转导机制可能并不一致。四、LPS预处理对LPS/ACTH诱导的F/R细胞皮质酮生成的影响及机制1.皮质酮检测结果显示LPS预处理原代培养的F/R细胞后,该细胞接受后续LPS和ACTH二次刺激时皮质酮生成量显著减少。这一结果提示LPS预处理可能导致在肾上腺皮质细胞中产生了内毒素耐受,这可能是脓毒症时肾上腺功能不全的机制之一。2. Western blot结果显示LPS能够抑制TLR4蛋白表达。同时荧光实时定量PCR和western blot结果也显示F/R细胞经LPS预处理后,后续LPS诱导的StAR和代谢酶CYP11B1表达也都被完全抑制了。结合上述结果,我们认为LPS预处理后TLR4表达被抑制可能是F/R细胞对二次LPS刺激反应性低下的主要原因。3. Western blot结果显示LPS抑制了F/R细胞中ACTH受体的表达。然而荧光实时定量PCR和western blot结果显示LPS预处理仅抑制了后续ACTH诱导的CYP11A1,HSD3B和CYP21的表达,但对ACTH诱导的StAR和CYP11B1表达并无显著影响。上述结果表明肾上腺ACTH-R表达降低可能仅仅是LPS预处理导致肾上腺皮质对ACTH反应性降低的部分原因,其他可能参与的机制仍有待后续研究。4.LPS预处理抑制了后续LPS刺激诱导的StAR和CYP11B1的表达,此外还抑制了后续ACTH刺激诱导的CYP11A1,HSD3B和CYP21的表达。由于正常肾上腺皮质中糖皮质激素的储备量非常少,因此类固醇合成酶的表达水平对于维持肾上腺皮质对ACTH的正常反应性非常关键。由此可见,诱导或激活上述被LPS预处理所抑制的类固醇代谢酶可能可以成为临床治疗脓毒症诱导的肾上腺皮质功能不全的有效治疗手段。结论：本研究表明在炎症过程中巨噬细胞是H2S生成的来源之一,H2S可能通过抑制NO生成发挥抗炎作用,抑制H2S和NO的生成可能是GC调节炎症中促炎抗炎因子平衡的一种机制。脓毒症时机体产生内毒素耐受可能是肾上腺功能不全的机制之一；诱导或激活相关合成酶表达可能可以成为临床治疗脓毒症诱导的肾上腺皮质功能不全的有效治疗手段。更多还原

【Abstract】 In severe infectious diseases, inflammatory responses strongly activate the HPA axis and stimulate the release of adrenocorticotropic hormone (ACTH), which in turn stimulates the secretion of glucocorticoids (GCs) from the adrenal cortex. It is well known that GCs have anti-inflammatory effects, which is the main reason for the clinical usage of GCs to treat acute and chronic inflammatory diseases. However, GCs have also been shown to exert pro-inflammatory effects in some cases. Scientists have proposed that GCs could balance between pro- and anti-inflammatory mediators and control their own strength, so as to reduce the side effects of GCs. So far, the mechanisms involved remain to be further elucidated.Although GCs play an important role in maintaining the homeostasis during inflammation, reversible adrenal insufficiency has been frequently diagnosed in critically ill patients with sepsis who have either low basal cortisol levels or low cortisol responses to adrenocorticotrophic hormone (ACTH) stimulation. Moreover, corticosteroid insufficiency is always associated with a high mortality rate. Therefore, it seems to be of great significance to clarify the mechanisms responsible for the occurrence of adrenal insufficiency in severe inflammatory diseases.In the first part of present study, we found that macrophages produced not only the well-known pro-inflammatory gaseous transmitter nitric oxide (NO), but also the newly recognized gas molecule hydrogen sulfide (H2S). In addition, H2S exerts certain anti-inflammatory effects by inhibiting NO production. We also found that GCs decreased the production of both NO and H2S, and we further investigated the mechanisms involved in the inhibitory effects of GCs on H2S production. These findings provide important experimental evidence for the mechanisms by which GCs coordinate the balance between pro- and anti-inflammatory mediators during inflammation.It was found that prior exposure of innate immune cells like monocytes/ macrophages to minute amounts of endotoxin caused them to become refractory to subsequent endotoxin challenge, a phenomenon called "endotoxin tolerance". Now, it is generally accepted that endotoxin tolerance contributes to immunosuppression during sepsis. Therefore, in the second part of this study, we hypothesized that endotoxin tolerance might have occurred in the adrenal gland and led to hyporesponsiveness of adrenal gland during sepsis. To test this, we firstly observed the effect of LPS on corticosterone production in the rat adrenal zona fasciculata-reticularis (F/R) cells and explored the synthetic pathways involved. Then, we studied the effect of LPS pretreatment on LPS- and ACTH-induced corticosterone production in F/R cells.Main results:1.Capacity of H2S biosynthesis in macrophages and the local functions of endogenous H2S1)Macrophages expressed H2S-forming enzyme cystathionine-gamma-lyase (CSE) and produced H2S.2)Lipopolysaccharide (LPS) increased CSE expression and H2S production rate. This suggests that macrophages be one of H2S producing sources during Gram-negative bacteria-induced inflammation.3)L-cysteine reduced LPS-induced nitric oxide (NO) production. CSE inhibitor blocked the inhibitory effect of L-cysteine. CSE knockdown increased, whereas CSE overexpression decreased LPS-induced NO production. Thus, it would suggest that H2S might exert anti-inflammatory effects by inhibiting NO production in macrophages during LPS-induced inflammation.2.Regulation of H2S Production by Glucocorticoids in Macrophages1)Dexamethasone suppressed LPS-induced CSE expression and H2S production rate as well NO production. This may be a mechanism by which glucocorticoids coordinate the balance between pro- and anti-inflammatory mediators during inflammation.2)L-arginine increased whereas NG-nitro-L-arginine methyl ester (L-NAME) decreased LPS-induced CSE expression and H2S production. These suggest that NO is an important endogenous stimulus in the formation of H2S during LPS-induced inflammation.3)Dexamethasone plus L-NAME significantly decreased LPS-induced CSE expression and H2S production compared to L-NAME. This suggests that dexamethasone may directly inhibit CSE expression and H2S production besides NO-dependent way.4)Dexamethasone reduced CSE mRNA stability in primary macrophages, and our previous resultes sugguested that dexamethasone decreased CSE promoter activity in RAW 264.7 macrophages. These suggest that dexamethasone inhibition of CSE expression is through different mechanisms in primary macrophages and macrophage cell line and the mechanism of action of glucocorticoid on CSE expression might be related to cell types. 3. Regulation of corticosterone production by LPS in rat primary F/R cells.1)Toll-like receptor 4 (TLR4) and P450 11β-hydroxylase (CYP11B1) were co-localized in adrenal gland and primary F/R cells.2)LPS stimulates corticosterone production and expression of StAR and CYP11B1 in primary F/R cells, but did not affect the mRNA and protein levels of CYP11A1. ACTH dose-dependently increased both mRNA and protein levels of StAR and all the synthetic enzymes, CYP11A1, HSD3B, CYP21 and CYP11B1. Notably, the synthetic enzymes involved in ACTH stimulation differed from those in LPS stimulation, which indicates that the intracellular signaling mechanisms responsible for ACTH and LPS stimulation of corticosteroid production may be different.4. Regulation of LPS/ACTH-induced corticosterone production by LPS pretreatment.1)LPS pretreatment caused a significant decrease in corticosterone production in response to subsequent LPS or ACTH stimulation in primary F/R cells. These results indicated that endotoxin tolerance might also occur in adrenal gland during sepsis, and endotoxin tolerance of adrenal gland during sepsis is one of mechanisms for adrenocortical insufficiency.2) We found that TLR4 expression was significantly decreased by LPS over a 24 h period of treatment, which was consistent with down-regulation of corticosterone production and the expression of StAR and CYP11B1 in response to second stimulation of LPS. These data indicate that reduced TLR4 expression may account for hypo-responsiveness to second stimulation of LPS in adrenal gland.3)The present study also demonstrated that LPS caused decreased ACTH-R in F/R cells, and LPS pretreatment decreased expression of some(CYP11A1、HSD3B、CYP21), but not all steroidogenic enzymes and proteins, suggesting that reduced ACTH-R in adrenal gland just partly account for hypo-responsiveness to ACTH, and additional mechanisms whereby LPS pretreatment induces hypo-responsiveness to ACTH in adrenal gland might exist.4) The present study indicates that LPS pretreatment suppress both LPS- and ACTH-induced expression of steroidogenic enzymes. The stored GCs can maintain normal rates of secretion for only a few minutes in the absence of continuing biosynthesis. Therefore, any disruption in glucocorticoid biosynthesis will immediately affect the response of adrenal cortex to various stresses. Our findings suggest that induction and activation of these steroid metabolic enzymes can be a promising treatment strategy for adrenocortical insufficiency during sepsis.Conclusions:Our results suggest that macrophages are one of H2S producing sources. H2S might exert anti-inflammatory effects by inhibiting NO production. Dexamethasone may directly inhibit CSE expression and H2S production besides NO-dependent way. Inhibition of H2S and NO production may be a mechanism by which glucocorticoids coordinate the balance between pro- and anti-inflammatory mediators during inflammation.Endotoxin tolerance of adrenal gland during sepsis is one of mechanisms for adrenocortical insufficiency, and activation of synthetic enzymes can be a promising treatment strategy.更多还原