选择性激动黑皮质素4型受体对脓毒症大鼠急性肾损伤的保护作用和机制研究

发布时间：2018-06-18 08:46

本文选题：盲肠结扎穿孔 + 急性肾损伤　；参考：《安徽医科大学》2013年硕士论文

【摘要】：目的：急性肾损伤(acute kidney injury, AKI)是危重患者最常见的临床问题之一,脓毒症一直是AKI的首要原因,脓毒症合并AKI患者的死亡率高达70%。因此,探讨脓毒症时肾脏的保护治疗有着重要的临床意义。黑皮质素4型受体(melanocortin4receptor, MC4R)在摄食、能量代谢、性功能、心血管功能等方面起着重要调节作用。有文献报道选择性激动MC4R可以激活胆碱能抗炎通路,显著抑制重症急性胰腺炎大鼠全身炎症进展,保护胰腺组织。当脓毒症合并AKI时,能否通过选择性激动MC4R,激活胆碱能抗炎通路对抗炎症反应,实现对脓毒症患者肾脏的保护作用尚未见报道。本课题采用盲肠结扎穿孔术(cecal ligation and puncture, CLP),建立脓毒症大鼠模型,观察脓毒症对肾脏的影响,探讨脓毒症AKI动物模型的制备。在脓毒症AKI大鼠模型的基础上,通过选择性激动MC4R进行干预,观察对肾脏的影响,阐述选择性激动MC4R对脓毒症大鼠AKI的保护作用和机制,为临床预防和治疗脓毒症合并AKI提供实验基础和理论依据。方法：采用CLP法制备脓毒症大鼠模型(1)实验大鼠随机分为假手术组(Sham组)和盲肠结扎穿孔组(CLP组),每组各18只。二组均在造模后设置6h、12h和24h三个时间点取材,检测大鼠心率(heart rate, HR)和平均动脉压(mean arterial blood perssuer, MABP);测定血清中肿瘤坏死因子-a(tumor necrosis factor-a, TNF-a)和白细胞介素-6(interleukin-6, IL-6)的表达水平及血清肌酐(creatine,CRE)和尿素氮(blood urea nitrogen, BUN)水平；观察大鼠肾脏组织结构的改变；检测肾脏组织中高迁移率族蛋白B1(high mobility group box1protein, HMGB1) mRNA的表达水平和核因子-κB(nuclear factor-KB, NF-κB)的活化。(2)实验大鼠随机分假手术组(Sham组)、假手术+Ro27-3225治疗组(Sham+Ro27组)、盲肠结扎穿孔组(CLP组)和盲肠结扎穿孔+Ro27-3225治疗组(CLP+Ro27组)。每组各6只。四组均在造模后24h取材,检测大鼠HR和MABP;测定血清CRE和BUN水平；观察大鼠肾脏组织结构的改变；检测肾脏组织中HMGB1mRNA的表达水平和NF-κ3的活化。结果：(1)与同时间点Sham组比较,CLP组6h和12h大鼠HR和MABP明显升高(p0.01),24h MABP降低(p0.01)；CLP组6h和12h大鼠血清中TNF-a和IL-6表达水平明显升高(p0.01),24h降至Sham组水平(p0.05)；CLP组6h、12h和24h大鼠血清中BUN水平升高(p0.05和p0.01),24h血清中CRE水平明显升高(p0.01)；CLP组12h大鼠肾脏组织中HMGB1mRNA表达水平出现升高,24h明显升高(p0.01)；CLP组6h大鼠肾脏组织NF-kB p65核阳性率升高,且随病程延长,NF-kB p65核阳性率明显升高(p0.01)。Sham组各组大鼠肾小体形态正常,肾小管上皮细胞未见肿胀；CLP组各组大鼠肾小体中血管球淤血、皱缩,肾小囊腔扩大,且随病程延长,淤血、皱缩的血管球增多,并伴有肾小管上皮细胞肿胀。(2)Sham+Ro27组大鼠HR和MABP较Sham组明显降低(p0.01),CLP组大鼠MABP低于Sham组(p0.01),CLP+Ro27组大鼠MABP较CLP组升高(p0.01)；CLP组大鼠血清BUN和CRE水平较Sham组明显升高(p0.01),CLP+Ro27组大鼠血清BUN和CRE水平较CLP组明湿降低(p0.01)；CLP组大鼠肾脏组织中HMGB1mRNA的表达水平较Sham组明显升高(p0.01)；CLP+Ro27组大鼠肾脏组织中HMGB1mRNA的表达水平较CLP组降低(p0.01)；CLP组大鼠肾脏组织NF-kB p65核阳性率较Sham组升高；CLP+Ro27组大鼠肾脏组织中NF-kB p65核阳性率较CLP组降低(p0.01)；Sham组和Sham+Ro27组大鼠肾小体形态正常,肾小管上皮细胞未见肿胀,CLP组大鼠肾小体中血管球淤血、皱缩,肾小囊腔扩大,伴有肾小管上皮细胞肿胀,CLP+Ro27组大鼠也可见肾小体中血管球淤血、皱缩和肾小囊腔扩大,但数量明显少于CLP组,间质充血、水肿及炎性细胞浸润也有所改善。结论：采用CLP方法能成功的复制出脓毒症AKI大鼠模型,引起大鼠肾脏结构和功能的改变,引起肾脏组织中HMGB1mRNA的表达水平升高和NF-kB p65核阳性率增加；选择性激动MC4R对脓毒症AKI大鼠肾脏的结构和功能都有保护作用,并且抑制肾脏组织中HMGB1mRNA的表达水平升高和NF-κB p65核阳性率增加。
[Abstract]:Objective: acute kidney injury (AKI) is one of the most common clinical problems in critically ill patients. Sepsis is the primary cause of AKI. The mortality of patients with sepsis with AKI is as high as 70%., so the protective treatment of kidney in sepsis has an important clinical significance. The 4 type of melanin receptor (melanocortin4receptor,) MC4R) plays an important regulatory role in feeding, energy metabolism, sexual function and cardiovascular function. It is reported that selective excitation of MC4R can activate the cholinergic anti-inflammatory pathway, significantly inhibit the progression of severe acute pancreatitis in rats and protect the pancreatic tissue. When AKI is combined with sepsis, it can be activated by selectively activating MC4R and activating the bile. The protective effect of alkaline energy anti-inflammatory pathway against inflammatory reaction has not yet been reported. This subject uses cecal ligation and puncture (CLP) to establish a rat model of sepsis, to observe the effect of sepsis on the kidney, and to explore the preparation of AKI animal model in sepsis. In the AKI rat model of sepsis, the model of sepsis AKI rat model is made. On the basis of selective stimulation of MC4R, the effect of MC4R on the kidney was observed, and the protective effect and mechanism of selective excitant MC4R on septic rat AKI were described, and the experimental basis and theoretical basis were provided for the clinical prevention and treatment of sepsis with AKI.
Methods: the rat model of sepsis (1) was prepared by CLP method. The experimental rats were randomly divided into sham operation group (group Sham) and cecum ligation group (group CLP), each group was 18. The two groups were set up 6h, 12h and 24h at three time points, and the heart rate (heart rate, HR) and mean arterial pressure (mean arterial blood) were measured. The level of the expression of tumor necrosis factor -a (tumor necrosis factor-A, TNF-a) and interleukin -6 (interleukin-6, IL-6) in serum and the level of serum creatinine (creatine, CRE) and urea nitrogen (blood urea), and the changes of renal tissue structure in rats were observed and the high mobility group protein in renal tissue was detected. OUP box1protein, HMGB1) mRNA expression level and the activation of nuclear factor kappa B (nuclear factor-KB, NF- kappa B). (2) experimental rats were randomly divided into sham operation group (Sham group), sham operation +Ro27-3225 treatment group (Sham+Ro27 group), caecum ligation and perforation group and cecum ligation perforation treatment group. Each group was made up of four groups. After 24h, HR and MABP were measured and the levels of serum CRE and BUN were measured. The changes of renal tissue structure of rats were observed. The expression of HMGB1mRNA in renal tissue and the activation of NF- kappa 3 were detected.
Results: (1) compared with group Sham at the same time point, HR and MABP in CLP group 6h and 12h rats increased significantly (P0.01) and 24h MABP decreased (P0.01). The level of HMGB1mRNA expression in renal tissue of 12h rats in group CLP was increased and 24h increased significantly (P0.01), and the positive rate of NF-kB p65 in renal tissue of group 6h rats increased, and the positive rate of NF-kB p65 nucleus increased obviously with the course of disease, and the renal corpuscle of renal tubules was not normal in all groups of rats. Swelling, vascular bulging, shrinkage and enlargement of renal capsule cavity in the renal corpuscle of rats in group CLP, with the extension of the course, the increase of blood vessel and crumpled blood vessel, and the swelling of renal tubular epithelial cells. (2) the HR and MABP in the group Sham+Ro27 rats were significantly lower than those in the Sham group (P0.01), and the MABP in the group CLP was lower than that in the Sham group (P0.01), and the CLP+Ro27 group MABP was higher than that of the group. The level of serum BUN and CRE in the group CLP rats was significantly higher than that in the Sham group (P0.01). The level of serum BUN and CRE in the CLP+Ro27 group was lower than that in the CLP group (P0.01), and the expression level of the kidneys in the rats of the CLP group was significantly higher than that in the Sham group; the expression level of the kidney in the rats was lower than that in the group of rats. (P0.01); the positive rate of NF-kB p65 nucleus in kidney tissue of rats in group CLP was higher than that in group Sham, and the positive rate of NF-kB p65 in kidney tissue of group CLP+Ro27 was lower than that in group CLP (P0.01), and the renal corpuscle cells in Sham group and Sham+Ro27 group were normal, no swelling of renal tubular epithelial cells, vascular congestion, shrinkage and enlargement of renal cysts in renal corpuscles of rats. The epithelial cells of renal tubule were swollen, and the rats in group CLP+Ro27 also found vascular congestion, contraction and enlargement of the renal capsule cavity in the renal corpuscle, but the number was significantly less than that in the CLP group. The interstitial congestion, edema and inflammatory cell infiltration were also improved.
Conclusion: CLP can successfully replicate the AKI rat model of sepsis, cause changes in the structure and function of kidney in rats, increase the level of HMGB1mRNA expression in renal tissue and increase the positive rate of NF-kB p65 nucleus; selective excitant MC4R has protective effect on the structure and function of kidney in septic AKI rats, and inhibits the kidney. The expression level of HMGB1mRNA and NF- kappa B p65 were increased in tissues.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R459.7

【共引文献】