甲泼尼龙对呼吸机相关性肺损伤大鼠肺组织TRPV4/MMP-2/MMP-9信号通路的影响

Effect of methylprednisolone on TRPV4/MMP-2/MMP-9 signaling pathway in lung tissue of the rats with ventilator-induced lung injury

目的评价甲泼尼龙对呼吸机相关性肺损伤(VILI)大鼠肺组织瞬时受体电位香草酸4 (TRPV4)/基质金属蛋白酶2/9(MMP-2/MMP-9)信号通路的影响。方法清洁级雄性SD大鼠100只,采用随机数字表法分为5组(n=20):对照组(C组)、机械通气组(V组)、甲泼尼龙组(Mp组)、甲泼尼龙+GSK1016790A组(MpG组)、HC-067047组(H组)。C组不行机械通气,自主呼吸空气4 h;V组机械通气(RR 40次/min,VT 40 m L/kg,I∶E 1∶1,PEEP 0,Fi O221%) 4 h;Mp组在机械通气前20 min静脉输注甲泼尼龙10.0 mg/kg;MpG组在给予甲泼尼龙前20 min静脉输注GSK1016790A 0.025 mg/kg;H组机械通气前30 min静脉输注HC-067047 10.0 mg/kg。机械通气4 h时,检测支气管肺泡灌洗液(BALF)中白细胞介素-1(IL-1)、肿瘤坏死因子-α(TNF-α)、总蛋白浓度,测定肺通透指数(LPI)、肺湿/干质量比(W/D),观察肺组织病理学结果。Western blot法检测肺组织TRPV4、MMP-2、MMP-9的表达水平。结果与C组比较,V组和MpG组BALF中IL-1(ng/m L:84.56±5.35 vs. 144.85±9.39、121.56±7.69)、TNF-α(ng/m L:179.65±45.73 vs. 486.18±94.79、316.93±69.71)、总蛋白(mg/m L:321.29±28.76 vs. 687.78±65.78、476.39±46.67)升高,肺组织LPI [(2.47±0.17)×10^(-3)vs.(6.19±0.29)×10^(-3)、(4.24±0.25)×10^(-3)]、W/D比值(4.42±0.19 vs. 8.83±0.61、6.32±0.41)升高,TRPV4(1.85±0.25 vs.5.81±0.92、3.87±0.65)、MMP-2 (0.44±0.06 vs. 1.16±0.23、0.85±0.11)、MMP-9(0.19±0.03 vs. 0.46±0.09、0.34±0.07)表达上调(P<0.05);与V组比较,Mp组、MpG组和H组BALF中IL-1(ng/m L:144.85±9.39 vs. 89.78±5.91、121.56±7.69、94.23±6.78)、TNF-α(ng/m L:486.18±94.79 vs. 186.42±49.37、316.93±69.71、193.71±51.41)、总蛋白(mg/m L:687.78±65.78 vs. 348.78±31.52、476.39±46.67、359.68±36.12)降低,肺组织LPI [(6.19±0.29)×10^(-3)vs.(2.85±0.14)×10^(-3)、(4.24±0.25)×10^(-3)、(2.97±0.21)×10^(-3)]、W/D比值(8.83±0.61 vs. 4.75±0.22、6.32±0.41、4.82±0.25)降低,TRPV4(5.81±0.92 vs. 2.13±0.29、3.87±0.65、2.35±0.37)、MMP-2 (1.16±0.23 vs. 0.48±0.08、0.85±0.11、0.52±0.08)、MMP-9(0.46±0.09 vs. 0.22±0.04、0.34±0.07、0.25±0.05)表达下调(P<0.05),肺组织病理损伤减轻;与Mp组比较,MpG组BALF中IL-1 (ng/m L:89.78±5.91 vs. 121.56±7.69)、TNF-α(ng/m L:186.42±49.37 vs. 316.93±69.71)、总蛋白(mg/m L:348.78±31.52 vs. 476.39±46.67)升高,肺组织LPI [(2.85±0.14)×10^(-3)vs.(4.24±0.25)×10^(-3)]、W/D比值(4.75±0.22 vs. 6.32±0.41)升高,TRPV4(2.13±0.29 vs. 3.87±0.65)、MMP-2(0.48±0.08 vs. 0.85±0.11)、MMP-9(0.22±0.04 vs. 0.34±0.07)表达上调(P<0.05)。结论甲泼尼龙可减轻大鼠VILI,与其抑制TRPV4/MMP-2/MMP-9信号通路有关。

Objective To evaluate the effect of methylprednisolone on transient receptor potential vanilloid 4( TRPV4)/matrix metalloproteinase 2/9( MMP-2/MMP-9) signal pathway in lung tissue of ventilator-induced lung injury rats. Methods 100 male SD rats of clean grade were randomly divided into 5 groups( n = 20) : control group( group C),mechanical ventilation group( group V),methylprednisolone group( group Mp), methylprednisolone + GSK1016790 A group( group MpG),HC-067047 group( group H). In group C,mechanical ventilation was not available,and spontaneous breathing air was conducted for 4 hours;The rats in group V were mechanically ventilated( RR 40 times/min,VT 40 m L/kg,I ∶ E 1 ∶ 1,PEEP 0,Fi O221%) for 4 h;In group Mp,10. 0 mg/kg methylprednisolone was given intravenously 20 minutes before mechanical ventilation;0. 025 mg/kg GSK1016790 A were infused intravenously in group MpG 20 minutes before methylprednisolone administration;In group H,10 mg/kg HC-067047 was intravenously infused 30 min before mechanical ventilation. At 4 h after mechanical ventilation,the levels of IL-1,TNF-α,total protein in bronchoalveolar lavage fluid( BALF) were detected,lung permeability index( LPI),lung wet/dry weight( W/D) ratio were measured,and lung histopathological results were observed. Western blot was used to detect the expression levels of TRPV4,MMP-2,MMP-9 in lung tissue. Results Compared with group C,the levels of IL-1( ng/m L: 84. 56 ± 5. 35 vs. 144. 85 ± 9. 39,121. 56 ± 7. 69),TNF-α( ng/m L: 179. 65 ± 45. 73 vs. 486. 18 ± 94. 79,316. 93 ± 69. 71) and total protein( mg/m L: 321. 29 ±28. 76 vs. 687. 78 ± 65. 78,476. 39 ± 46. 67) in BALF of group V and MpG were increased,the LPI[( 2. 47 ± 0. 17)×10^(-3) vs.( 6. 19 ± 0. 29)×10^(-3),( 4. 24 ± 0. 25)×10^(-3)] and W/D ratio( 4. 42 ±0. 19 vs. 8. 83 ± 0. 61,6. 32 ± 0. 41) in lung tissue were increased,and the expressions of TRPV4( 1. 85 ±0. 25 vs. 5. 81 ± 0. 92,3. 87 ± 0. 65),MMP-2( 0. 44 ± 0. 06 vs. 1. 16 ± 0. 23,0. 85 ± 0. 11) and MMP-9( 0. 19 ± 0. 03 vs. 0. 46 ± 0. 09,0. 34 ± 0. 07) were up-regulated( P<0. 05);Compared with group V,the levels of IL-1( ng/m L: 144. 85 ± 9. 39 vs. 89. 78 ± 5. 91,121. 56 ± 7. 69,94. 23 ±6. 78),TNF-α( ng/m L: 486. 18 ± 94. 79 vs. 186. 42 ± 49. 37,316. 93 ± 69. 71,193. 71 ± 51. 41),total protein( mg/m L: 687. 78 ± 65. 78 vs. 348. 78 ± 31. 52,476. 39 ± 46. 67,359. 68 ± 36. 12) in BALF of group Mp,group MpG and group H were decreased,LPI [( 6. 19 ± 0. 29)×10^(-3) vs.( 2. 85 ±0. 14)×10^(-3),( 4. 24 ±0. 25)×10^(-3),( 2. 97 ± 0. 21)×10^(-3)]and W/D ratio( 8. 83 ± 0. 61 vs. 4. 75 ±0. 22,6. 32 ± 0. 41,4. 82 ± 0. 25) of lung tissue were decreased,the expression of TRPV4( 5. 81 ± 0. 92 vs. 2. 13 ± 0. 29,3. 87 ± 0. 65,2. 35 ± 0. 37),MMP-2( 1. 16 ± 0. 23 vs. 0. 48 ± 0. 08,0. 85 ± 0. 11,0. 52 ± 0. 08) and MMP-9( 0. 46 ± 0. 09 vs. 0. 22 ± 0. 04,0. 34 ± 0. 07,0. 25 ± 0. 05) were down-regulated( P<0. 05),and the pathological injury of lung tissue was alleviated;Compared with group Mp,the levels of IL-1( ng/m L: 89. 78 ± 5. 91 vs. 121. 56 ± 7. 69),TNF-α( ng/m L: 186. 42 ± 49. 37 vs. 316. 93 ± 69. 71) and total protein( mg/m L: 348. 78 ± 31. 52 vs. 476. 39 ± 46. 67) in BALF of group MpG were increased,LPI [( 2. 85 ± 0. 14)×10^(-3) vs.( 4. 24 ± 0. 25)×10^(-3)] and W/D ratio( 4. 75 ± 0. 22 vs. 6. 32 ± 0. 41) of lung tissue were increased,and the expressions of TRPV4( 2. 13 ±0. 29 vs. 3. 87 ± 0. 65),MMP-2( 0. 48 ± 0. 08 vs. 0. 85 ± 0. 11) and MMP-9( 0. 22 ± 0. 04 vs. 0. 34 ±0. 07) were up-regulated( P<0. 05). Conclusions Methylprednisolone can reduce VILI in rats,which is related to the inhibition of TRPV4/MMP-2/MMP-9 signaling pathway.