1,25-二羟维生素D_3对糖尿病大鼠肺脏的保护作用及机制

Protective effect of 1,25-dihydroxyvitamin D_3 on lung tissues of diabetic rats

目的观察1,25-二羟维生素D3[1,25-(OH)2D3]对糖尿病大鼠炎性水平及肺组织Toll样受体4(TLR4)表达的影响,探讨其肺保护作用及机制。方法将90只SD大鼠随机分为对照组(C组)10只及模型组80只,模型组经尾静脉注射STZ制备糖尿病模型,C组注射等体积柠檬酸缓冲液;将模型组造模成功的75只大鼠随机分为糖尿病组(D组),1,25-(OH)2D3大、中、小剂量干预组(H、M、L组)及精蛋白锌胰岛素干预组(Y组)各15只,其中H、M、L组分别予0.3、0.15、0.025μg/(kg·d)的1,25-(OH)2D3灌胃,Y组予胰岛素颈后皮下注射,C组和D组予蒸馏水2 mL灌胃,均为1次/d、连续16周,16周后所有动物均采用股动脉放血处死,立即取肺脏组织置于10%甲醛及液氮中保存。测定各组血糖及血清CRP水平;采用HE、Masson染色法观察肺组织形态学变化,免疫组织化学法检测TLR4、NF-κB p65的蛋白表达;采用PCR法定量检测TLR4、MyD88、NF-κB p65的mRNA表达。结果①D组血糖及血清CRP水平均显著高于C组(P<0.01),各干预组均不同程度低于D组,尤以H组为著(P<0.01)。②D组肺泡壁重度增厚、肺间质明显增生、大量炎症细胞浸润,H、Y、M及L组肺泡壁轻到中度增厚、炎细胞浸润明显;与C组比较,余5组TLR4、NF-κB p65蛋白表达有所升高,其中H、Y组显著低于D组。③D组TLR4、MyD88和NF-κB p65的mRNA表达显著高于C组(P<0.01),各干预组表达水均低于D组。肺组织TLR4 mRNA表达与MyD88 mRNA(r=0.610)、NF-κB p65 mRNA表达(r=0.744)均呈正相关,P均<0.001;MyD88 mRNA与NF-κB p65mRNA表达亦呈正相关(r=0.609),P<0.001。结论 1,25-(OH)2D3可能通过TLR4介导的炎症途径对STZ诱导的糖尿病大鼠肺脏病变发挥保护作用。

Objective % observe the effect of I, 25-dihydroxyvitamin D3（1, 25-（OH）2D3） on inflammatory level and Toll-like receptor 4 （TLR4） expression of lung tissues in diabetic rats, and to investigate the mechanism of the lung protective effect. Methods Ninety SD rats were randomly divided into the control group （group C, n = 10） and diabetic model group （n =80）. Streptozotocin （STZ） was injected into caudal veins to generate diabetic models, with 75 diabetic rats generated. Cit rate buffer was injected into group C. Seventy-five diabetic rats were randomly divided into five groups （n =15）, including dia betic group （group D）, high-dose （group H）, medium-dose （group M） and low-dose 1,25-（OH）2D3 intervention groups （group L） and pretamine zinc insulin group （group Y）. Rats in group H, M and L were administered 0.3 μg/（kg . d）, 0. 15μG/ （kg- d）, 0.025 pg/（kg . d）1,25-（OH）2D3 intragastrieally respectively. Group Y was given 16 U/（kg^-1 . d） insulin hypoder mic injection in posterior of the neck. Group D and C were given distilled water intragastrically. All rats were sacrificed after 16 weeks by femoral artery hemorrhage. Lung samples were placed in 10% neutral formalin and liquid nitrogen. Blood glucose and serum CRP levels were determined. PAthological alterations in lung tissues were observed through HE and Masson stain. The pro tein expression of TLR4 and NF-r,B p65 was detected by immunohistochemistry. The expression of TLR4, MyD88 and NF-KB 1365 mRNA was quantitatively measured by PCR. Results 1. Blood glucose and serum CRP levels were significantly higher in groupD than those in group C （P 〈 0. 01 ）, and lower in all intervention groups, especially in group H （P 〈 0. 01 ）. 2. Group D showed severe alveolar wall thickening, significant interstitial proliferation and massive inflammatory cell infiltration. Group H, Y, M and L showed mild to moderate alveolar wall thickening and inflammatory cell infiltration. TLR4 and NF-KB p65 protein expression was significantly higher in all 5 diabetic model groups than that in group C, and significantly lower in group H and Y as compared with that of group D. 3. The expression levels of TLR4, MyD88, and NF-KB 1365 mRNA were significantly higher in group D than those in group C （P 〈 0. O1 ）, and lower in all intervention groups as compared with that in group D. TLR4 mRNA expression was positively correlated with both MyD88 （ r = 0. 610, P 〈 0. 001 ） and NF-KB p65 （ r = 0. 744, P 〈 0.001 ） mRNA ex- pression. MyD88 and NF-KB p65 mRNA expression was also positively correlated （r =0. 609, all P 〈0. 001 ）. Cotwdusion 1, 25-（OH）2D3 may exert lung protective effect in STZ-induced diabetic rats by down-regulating TLR4-mediated inflammatory path- way.