丁酸钠对50%总体表面积烫伤大鼠肺血管内皮糖萼层保护作用和机制研究

目的研究丁酸钠对严重烫伤大鼠肺血管内皮糖萼层的保护作用及可能机制。 方法将144只成年雄性SD大鼠按照随机数字表法分成假伤＋盐水组(n＝24)，假伤＋丁酸钠组(n＝24)，烫伤＋盐水组(n＝48)，烫伤＋丁酸钠组(n＝48)。制作50%总体表面积Ⅲ度烫伤模型：烫伤＋盐水组和烫伤＋丁酸钠组大鼠麻醉、备皮后置于80 ℃水浴锅中(背部15 s，腹部8 s，双下肢15 s)，假伤＋盐水组和假伤＋丁酸钠组大鼠浸入37 ℃水相同时间。假伤＋丁酸钠组和烫伤＋丁酸钠组立即皮下注射丁酸钠(300 mg/kg)；假伤＋盐水组和烫伤＋盐水组立即皮下注射0.5 mL 0.9%氯化钠溶液。于伤后3、6 h分别处死动物并取材，测定肺泡灌洗液蛋白(BALF)浓度，肺湿干重比；采用蛋白质印迹法和酶联免疫法检测肺组织多配体蛋白聚糖-1(SDC-1)、基质金属蛋白酶9(MMP-9)和血管内皮生长因子(VEGF)的蛋白表达和含量率的变化。数据比较采用单因素方差分析和Newman-Kueuls法。 结果(1)伤后3、6 h，烫伤＋盐水组BALF蛋白浓度分别为(0.657 5±0.045 5)、(0.684 1±0.076 0) mg/mL，高于假伤＋盐水组[(0.476 6±0.065 8) mg/mL]，差异均有统计学意义(q＝7.188、8.243，P值均小于0.05)；伤后3、6 h，烫伤＋盐水组肺湿干重比分别为4.545 1±0.444 4，4.795 1±0.606 6，均高于假伤＋盐水组(2.636 0±0.157 5)，差异均有统计学意义(q＝10.950、12.380，P值均小于0.05)。伤后3、6 h，烫伤＋丁酸钠组BALF蛋白浓度分别为(0.541 0±0.071 )、(0.538 1±0.063 6) mg/mL，均低于烫伤＋盐水组[(0.657 5±0.045 5)、(0.684 1±0.076 0) mg/mL]，差异均有统计学意义(q＝4.610、5.799，P值均小于0.05)；且伤后3、6 h，烫伤＋丁酸钠组肺湿干重比值3.626 6±0.446 1、3.477 9±0.510 7，均低于烫伤＋盐水组，差异均有统计学意义(q＝5.269、7.555，P值均小于0.05)。(2)蛋白质印迹法检测结果显示，伤后3、6 h，烫伤＋盐水组肺组织SDC-1表达水平分别为0.376 1±0.075 7、0.329 8±0.074 9，假伤＋盐水组肺组织SDC-1表达水平均为1.000 0±0，2组比较差异均有统计学意义(q＝13.280、14.260，P值均小于0.05)。伤后3、6 h，烫伤＋丁酸钠组肺组织SDC-1表达水平分别为0.760 5±0.207 1、0.678 3±0.117 9，均高于烫伤＋盐水组，差异均有统计学意义(q＝8.180、7.417，P值均小于0.05)。伤后3、6 h，烫伤＋盐水组肺组织MMP-9、VEGF表达水平分别为(MMP-9：2.683 4±0.318 8、2.655 6±0.448 9；VEGF：3.806 3±0.703 8、2.850 0±0.396 3)，假伤＋盐水组MMP-9、VEGF表达水平均为1.000 0±0，烫伤＋盐水组均低于假伤＋盐水组，差异均有统计学意义[(MMP-9: q＝9.356、9.202)，(VEGF: q＝12.700、8.375); P值均小于0.05 ]。伤后3、6 h，烫伤＋丁酸钠组肺组织MMP-9、VEGF表达水平分别为(MMP-9：0.995 2±0.378 5、1.527 1±0.342 8；VEGF：1.574 6±0.216 0、1.721 5±0.341 8)，均低于烫伤＋盐水组，差异均有统计学意义[(MMP-9: q＝9.383、10.100)，(VEGF: q＝6.272、5.108); P值均小于0.05]。(3)酶联免疫法检测结果显示，伤后3、6 h，烫伤＋盐水组与假伤＋盐水组比较，肺组织MMP-9、VEGF蛋白含量均显著增高，差异均有统计学意义[(MMP-9: q＝8.850、8.436), (VEGF：q＝9.090、9.186); P值均小于0.05]；SDC-1蛋白含量显著降低，差异均有统计学意义(q＝8.296、8.331，P值均小于0.05)。伤后3、6 h，烫伤＋丁酸钠组肺组织MMP-9、VEGF蛋白含量均低于烫伤＋盐水组，差异均有统计学意义[(MMP-9: q＝6.579、6.939)，(VEGF：q＝7.853、7.768)；P值均小于0.05]；SDC-1蛋白含量高于烫伤＋盐水组(q＝6.265、4.456，P值均小于0.05)。 结论丁酸钠能减轻严重烫伤大鼠肺组织水肿，保护肺血管内皮糖萼层，其作用机制可能与抑制MMP-9和VEGF通路相关。

Study on the protective effect and mechanism of sodium butyrate on endothelial glucocalyx layer in pulmonary vessels of rats with 50% total body surface area scald

Objective To study the protective effect of sodium butyrate on glycocalyx layer of pulmonary vascular endothelium in severely scalded rats and its possible mechanism.Methods One hundred and fourty four adult male SD rats were randomly divided into sham injury+saline group(n=24),sham injury+sodium butyrate group(n=24),scald+saline group(n=48),scald+sodium butyrate group(n=48)according to the random number table method.The full thickness degree scald models with 50%total body surface area:Rats in the scald+saline group and scald+sodium butyrate group were anesthetized and prepared in a water bath at 80℃(back 15 s,abdomen 8 s,lower limbs 15 s).The rats in the sham injury+saline group and the sham injury+sodium butyrate group were immersed in water at 37℃for the same time.The sham injury+sodium butyrate group and the scald+sodium butyrate group were injected subcutaneously with sodium butyrate(300 mg/kg);the sham injury+saline group and the scald+saline group were immediately injected subcutaneously with 0.5 mL of 0.9%sodium chloride solution.Rats were sacrificed at 3 and 6 h after scald,and samples were collected.The broncho-alveolar lavage fluid(BALF)concentration,lung wet-dry weight ratio were measured;lung polyligand proteoglycan-1(SDC-1),matrix metalloproteinase 9(MMP-9)and vascular endothelial growth factor(VEGF)were measured using Western blotting and enzyme-linked immunosorbent assay.Data were compared by One-way analysis of variance and Newman-Kueuls method.Results(1)At 3 and 6 h after scald,the BALF protein concentration in the scald+saline group was(0.6575±0.0455)and(0.6841±0.0760)mg/mL,which were higher than those in the sham injury+saline group(0.4766±0.0658)mg/mL,the differences were statistically significant(q=7.188,8.243;with P values below 0.05);the lung wet-dry weight ratio of the scald+saline group were 4.5451±0.4444,4.7951±0.6066,which were higher than that of the sham injury+saline group(2.6360±0.1575),the differences were statistically significant(q=10.950,12.380;with P values below 0.05).At 3 and 6 h after scald,the BALF protein concentration in the scald+sodium butyrate group was(0.5415±0.0714)and(0.5381±0.0636)mg/mL,which were lower than those in the scald+saline group[(0.6575±0.0455)and(0.6841±0.0760)].the differences were statistically significant(q=4.610,5.799;with P values below 0.05);and at 3 and 6 h after scald,the lung wet-dry weight ratio of the scald+sodium butyrate group were 3.6266±0.4461,3.4779±0.5107,which were lower than those in the scald+saline group,and the differences were statistically significant(q=5.269,7.555;with P values below 0.05).(2)The results of Western blotting analysis showed that at 3 and 6 h after scald,the expression levels of SDC-1 in the lung tissue of the scald+saline group was 0.3761±0.0757 and 0.3298±0.0749,respectively,and that of the sham injury+saline group were all 1.0000±0,and the differences were statistically significant(q=13.280,14.260;with P values below 0.05).At 3 and 6 h after scald,the expression level of SDC-1 in the lung tissues of the scald+sodium butyrate group were 0.7605±0.2071 and 0.6783±0.1179,respectively.The scald+sodium butyrate group was significantly higher than those of the scald+saline group,the differences were statistically significant(q=8.180,7.417;with P values below 0.05).At 3 and 6 h after scald,the expression levels of MMP-9 and VEGF in the lung tissues of the scald+saline group were(MMP-9:2.6834±0.3188,2.6556±0.4489;VEGF:3.8063±0.7038,2.8500±0.3963).The expression levels of MMP-9 and VEGF in the sham injury+saline group were both 1.0000±0,and the scald+saline group was lower than those in the sham injury+saline group,and the differences were statistically significant[(MMP-9:q=9.356,9.202)(VEGF:q=12.7,8.375);with P values below 0.05].At 3 and 6 h after scald,the expression levels of MMP-9 and VEGF in the lung tissue of the scald+sodium butyrate group were(MMP-9:0.9952±0.3785,1.5271±0.3428;VEGF:1.5746±0.2160,1.7215±0.3418),which were lower than the scald+saline group,and the differences were statistically significant[(MMP-9:q=9.383,10.1),(VEGF:q=6.272,5.108);with P values below 0.05].(3)The results of enzyme-linked immunosorbent assay showed that at 3 and 6 h after scald,the levels of MMP-9 and VEGF protein in lung tissue in the scald+saline group were higher than those in the sham injury+saline group,the differences were statistically significant[(MMP-9:q=8.850,8.436);(VEGF:q=9.090,9.186);with P values below 0.05];the content of SDC-1 protein was significantly reduced,and the differences were statistically significant(q=8.296,8.331;with P values below 0.05).The contents of MMP-9 and VEGF in the lung tissues of the scald+sodium butyrate group were statistically significantly lower than those of the scald+saline group at 3 and 6 hours after the scald,the differences were statistically significant[(MMP-9:q=6.579,6.939),(VEGF:q=7.853,7.768);with P values below 0.05],SDC-1 protein content was significantly higher than those in the scald+saline group,the differences were statistically significant(q=6.265,4.456;with P values below 0.05).Conclusion Sodium butyrate can restore the damage of pulmonary vascular glycocalyx layer in scald rats,the mechanism may be related to MMP-9 and VEGF pathways.