酶解前后鹿筋体外抗炎活性与其氨基酸组成的相关性

Correlation between in Vitro Anti-inflammatory Activity and Amino Acid Composition of Deer Sinew before and after Enzymolysis

目的:分析酶解前后鹿筋对TNF-α诱导的MH7A细胞抗炎作用的影响。方法:采用MTT法检测鹿筋天然蛋白(DSNP)和鹿筋酶解蛋白(DSEP)不同分子量的超滤组分的增殖抑制活性,筛选增殖抑制活性最佳的组分,测定活性组分对MH7A细胞分泌炎症因子(NO、IL-6)的影响,分析鹿筋酶解前后活性组分中氨基酸组成及含量。采用多元统计分析找出酶解前后鹿筋的差异性氨基酸与抑制MH7A细胞分泌炎症因子之间的关系。结果:酶解前后鹿筋不同的超滤组分中,DSNP-5、DSEP-5对MH7A细胞增殖抑制的作用最强(P<0.05),且能够抑制细胞炎症因子NO、IL-6的分泌。质量浓度为25~200μg/mL时,DSNP-5给药组炎症因子(NO、IL-6)的释放量均低于DSEP-5给药组,具有显著差异(P<0.05),说明DSNP-5抑制能力更强。酶解前后鹿筋的氨基酸组成基本一致,但含量存在明显差异,其中甘氨酸既是特征性差异成分,又与抑制MH7A细胞分泌炎症因子NO、IL-6密切相关,且DSNP-5的甘氨酸含量(31.01%)高于DSEP-5的甘氨酸含量(28.91%)。结论:甘氨酸为鹿筋酶解前后与抗炎活性相关的差异氨基酸,初步探究酶解对鹿筋抗炎活性的影响,为鹿筋的开发应用提供可靠依据。

Objective:To analyze the anti-inflammatory effect on TNF-αinduced MH7A cells before and after deer sinew enzymolysis.Methods:Proliferative inhibitory activity of each peptide segment from deer sinew natural protein(DSNP)and deer sinew enzymolysis protein(DSEP)in ultrafiltration fraction was determined by MTT assay,and the components with best activity were screened.The effects of active component on the secretion of inflammatory factors(NO and IL-6)by MH7A cells were determined by ELLSA,and analysised the amino acid composition and content before and after deer sinew enzymolysis.Multivariate statistical analysis was used to find out the relationship between the differential amino acids before and after deer sinew enzymolysis and the inhibition of MH7A cells secreting inflammatory factors.Results:DSNP-5 and DSEP-5 had the best inhibitory effect on the proliferation of MH7A cells(P<0.05),and inhibited the secretion of NO and IL-6.When the concentration was 25~200μg/mL,the release of inflammatory factors(NO and IL-6)in DSNP-5 was lower than that in DSEP-5,with significant difference(P<0.05),indicating that DSNP-5 had better inhibition ability.The amino acid composition before and after deer sinew enzymolysis was basically the same,but the content was significantly different.Gly was not only a characteristic difference component,but also closely related to the inhibition of MH7A cells secreting inflammatory factors NO and IL-6.The Gly content of DSNP-5(31.01%)was higher than that of DSEP-5(28.91%).Conclusion:Gly is the difference amino acid related to anti-inflammatory activity before and after deer sinew enzymolysis.The effect of enzymolysis on anti-inflammatory activity of deer sinew is explored,which provide reliable basis for the development and application.