CCR5第2胞外环特异性拮抗短肽对哮喘小鼠肺组织自噬相关基因的表达和自噬泡形成的影响
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摘要
目的研究CC趋化因子受体5(CCR5)第2胞外环特异性拮抗短肽对哮喘小鼠肺组织自噬相关基因Beclin1、ATG5、LC3的表达和自噬泡形成的影响。方法将40只BALB/c小鼠随机分为5组,每组各8只,空白对照组小鼠予生理盐水腹腔注射致敏、生理盐水滴鼻激发,致敏组小鼠予鸡卵白蛋白(OVA)致敏、生理盐水激发,哮喘模型组小鼠予OVA致敏、OVA激发,地塞米松磷酸钠(Dex)组小鼠予OVA致敏、激发后予Dex尾静脉注射,拮抗短肽组予OVA致敏、激发后予拮抗短肽尾静脉注射。分别采用实时荧光定量PCR、蛋白免疫印迹法和透射电镜评估哮喘小鼠肺组织中自噬水平的变化。结果哮喘模型组的Beclin1、ATG5和LC3 mRNA表达水平及LC3蛋白表达水平均低于空白对照组(P均<0.05),拮抗短肽组的Beclin1、ATG5、LC3mRNA和蛋白表达水平均高于哮喘模型组(P均<0.05)。透射电镜显示,哮喘模型组小鼠肺组织中自噬泡数量较空白对照组稍减少,自噬泡主要位于具有免疫功能的2型肺泡上皮细胞。拮抗短肽组、Dex组中自噬泡数量较模型组增多,但多为未成熟的自噬泡,且自噬泡同样主要位于2型肺泡上皮细胞中。结论哮喘小鼠存在自噬功能障碍,CCR5第2胞外环的特异性拮抗短肽能升高哮喘小鼠肺组织中自噬相关蛋白Beclin1、ATG5和LC3的表达水平。
Objective To investigate the effects of antagonistic peptide specifically binding to the second extracellular membrane loop of CC chemokine receptor 5(CCR5) on the expression levels of autophagy-related genes, such as Beclin1, ATG5, LC3 and the formation of autophagosomes in the lung tissues of asthmatic mice. Methods Forty BALB/c mice were randomly assigned into five groups(n = 8). In the blank control group, the mice were sensitized by intraperitoneal injection of normal saline and induced by nasal drops of normal saline. In the sensitization group, the animals were sensitized by ovalbumin(OVA) and induced by normal saline. In the asthmatic model group, the mice were sensitized and induced by OVA. In the dexamethasone sodium phosphate(Dex) group, the mice were treated with caudal injection of Dex after they were sensitized and induced by OVA. In the antagonistic peptide group, the mice were treated with caudal injection of antagonistic peptide after they were sensitized and induced by OVA. The levels of autophagy in the lung tissues were assessed by real-time PCR, Western blot and transmission electron microscope(TEM). Results In the asthmatic model group, the expression levels of Beclin1, ATG5, LC3 mRNA and LC3 protein were significantly down-regulated than those in the blank control group(all P<0.05). In the antagonistic peptide group, the expression levels of Beclin1, ATG5, LC3 mRNA and protein levels were remarkably upregulated compared with those in the asthmatic model group(all P<0.05). TEM demonstrated that the quantity of autophagosomes in the lung tissues of the asthmatic mice was less than that in the blank control group. The autophagosomes were primarily distributed in the typeⅡ alveolar epithelial cells with immune function. The quantity of autophagosomes in the antagonistic peptide and Dex groups was larger than that in the asthmatic model group. However, a majority of the autophagosomes were immature and mainly distributed in the type Ⅱ alveolar epithelial cells. Conclusions Autophagy dysfunction exists in the asthmatic mice. The specific binding of antagonistic peptide to the second extracellular membrane loop of CCR5 effectively up-regulates the expression levels of autophagy-related genes, such as Beclin1, ATG5 and LC3 in the lung tissues of asthmatic mice.
Objective To investigate the effects of antagonistic peptide specifically binding to the second extracellular membrane loop of CC chemokine receptor 5(CCR5) on the expression levels of autophagy-related genes, such as Beclin1, ATG5, LC3 and the formation of autophagosomes in the lung tissues of asthmatic mice. Methods Forty BALB/c mice were randomly assigned into five groups(n = 8). In the blank control group, the mice were sensitized by intraperitoneal injection of normal saline and induced by nasal drops of normal saline. In the sensitization group, the animals were sensitized by ovalbumin(OVA) and induced by normal saline. In the asthmatic model group, the mice were sensitized and induced by OVA. In the dexamethasone sodium phosphate(Dex) group, the mice were treated with caudal injection of Dex after they were sensitized and induced by OVA. In the antagonistic peptide group, the mice were treated with caudal injection of antagonistic peptide after they were sensitized and induced by OVA. The levels of autophagy in the lung tissues were assessed by real-time PCR, Western blot and transmission electron microscope(TEM). Results In the asthmatic model group, the expression levels of Beclin1, ATG5, LC3 mRNA and LC3 protein were significantly down-regulated than those in the blank control group(all P<0.05). In the antagonistic peptide group, the expression levels of Beclin1, ATG5, LC3 mRNA and protein levels were remarkably upregulated compared with those in the asthmatic model group(all P<0.05). TEM demonstrated that the quantity of autophagosomes in the lung tissues of the asthmatic mice was less than that in the blank control group. The autophagosomes were primarily distributed in the typeⅡ alveolar epithelial cells with immune function. The quantity of autophagosomes in the antagonistic peptide and Dex groups was larger than that in the asthmatic model group. However, a majority of the autophagosomes were immature and mainly distributed in the type Ⅱ alveolar epithelial cells. Conclusions Autophagy dysfunction exists in the asthmatic mice. The specific binding of antagonistic peptide to the second extracellular membrane loop of CCR5 effectively up-regulates the expression levels of autophagy-related genes, such as Beclin1, ATG5 and LC3 in the lung tissues of asthmatic mice.
引文
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[2]Zeki AA,Yeganeh B,Kenyon NJ,Post M,Ghavami S.Autophagy in airway diseases:a new frontier in human asthma?Allergy,2016,71(1):5-14.
[3]Scurci I,Martins E,Hartley O.CCR5:established paradigms and new frontiers for a‘celebrity’chemokine receptor.Cytokine,2018,109:81-93.
[4]梁蓉蓉,李雯静,刘娟,沈溪明,黄花荣.CCR5第二胞外环的拮抗短肽对哮喘小鼠炎症细胞浸润和TNF-α表达的影响.中国病理生理杂志,2017,33(4):596-602.
[5]Puleston DJ,Simon AK.Autophagy in the immune system.Immunology,2014,141(1):1-8.
[6]Parzych KR,Klionsky DJ.An overview of autophagy:morphology,mechanism,and regulation.Antioxid Redox Signal,2014,20(3):460-473.
[7]Farooq MB,Walsh GM.Autophagy and asthma.Clin Exp Allergy,2016,46(1):7-9.
[8]Gu W,Cui R,Ding T,Li X,Peng J,Xu W,Han F,Guo X.Simvastatin alleviates airway inflammation and remodelling through up-regulation of autophagy in mouse models of asthma.Respirology,2017,22(3):533-541.
[9]Jiao X,Velasco-Velázquez MA,Wang M,Li Z,Rui H,Peck AR,Korkola JE,Chen X,Xu S,DuHadaway JB,GuerreroRodriguez S,Addya S,Sicoli D,Mu Z,Zhang G,Zhang X,Cristofanilli M,Fatatis A,Gray JW,Zhong JF,Prendergast GC,Pestell RG.CCR5 governs DNA damage repair and breast cancer stem cell expansion.Cancer Res,2018,78(7):1657-1671.
[10]Ye X,Liu S,Hu M,Song Y,Huang H,Zhong Y.CCR5expression in inflammatory bowel disease and its correlation with inflammatory cells andβ-arrestin2 expression.Scand JGastroenterol,2017,52(5):551-557.
[11]Venuti A,Pastori C,Pennisi R,Riva A,Sciortino MT,Lopalco L.Class Bβ-arrestin2-dependent CCR5 signalosome retention with natural antibodies to CCR5.Sci Rep,2016,6:39382.
[12]宋杨达,刘思雪,宋铱航,沈溪明,黄花荣,钟英强.CCR5第一和第二胞外环特异性拮抗短肽对大鼠结肠炎的炎症细胞浸润及NF-κB/TNF-α信号通路的影响.新医学,2018,49(5):309-314.
[13]Cadwell K.Crosstalk between autophagy and inflammatory signalling pathways:balancing defence and homeostasis.Nat Rev Immunol,2016,16(11):661-675.
[14]Pan H,Zhang Y,Luo Z,Li P,Liu L,Wang C,Wang H,Li H,Ma Y.Autophagy mediates avian influenza H5N1 pseudotyped particle-induced lung inflammation through NF-κB and p38MAPK signaling pathways.Am J Physiol Lung Cell Mol Physiol,2014,306(2):L183-L195.