苗药防感香囊活性提取物对聚肌胞苷酸刺激的支气管上皮细胞IFN-α、IL-6水平的影响及机制

doi:10.3877/cma.j.issn.1674-0785.2021.04.007

目的

探讨苗药防感香囊活性提取物甘草酸二铵（DG）与异草苷酸镁（MI）分别对聚肌胞苷酸（PIC）刺激的人正常支气管上皮细胞（NHBE）的作用与影响，明确DG与MI的疗效并探讨可能的作用机制。

方法

使用1 μg/ml的PIC刺激NHBE构建细胞模型，阳性对照组地塞米松（DXMS）的浓度为10 μg/ml，分别使用0.5、1.0、2.0 mg/ml低中高3种浓度的DG与MI进行细胞给药干预。使用CCK8法检测细胞活性，ELISA法检测干扰素α（IFN-α）、白介素-6（IL-6）表达水平，qPCR法检测IFN-α、IL-6 mRNA表达水平，Western blot检测p-TAK1/TAK1 与p-IkB/IkB水平。

结果

CCK8检测结果显示，NHBE细胞活力随药物浓度的增加而下降，DXMS处理的阳性对照组中细胞活力下降更加明显。造模后，相较于空白对照组，IFN-α、IL-6表达量上升，差异有统计学意义（P＜0.05）；相较于模型组，不同剂量的DG与MI处理后，IFN-α、IL-6表达量下降，差异有统计学意义（P＜0.05）。造模后，相较于空白对照组，IFN-α、IL-6 mRNA表达量显著上升（P＜0.05）；干预组相较于模型组，IFN-α、IL-6 mRNA表达量显著下降，差异有统计学意义（P＜0.05）。相较于空白对照组，PIC造模后的通路蛋白p-TAK1/TAK1与p-IkB/IkB比值有明显的升高趋势，差异有统计学意义（P＜0.05）。此外，相较于模型组，经低中高3种浓度的DG与MI处理NHBE细胞后，p-TAK1/TAK1与p-IkB/IkB比值未见明显改变，而在DXMS处理后的阳性对照组细胞中该比值相较模型组显著下降，差异有统计学意义（P＜0.05）。

结论

苗药防感香囊提取物主要活性成分之一为甘草酸铵。DG与MI对PIC诱发NHBE促炎症反应有一定疗效，其可能的作用方式是当细胞处于一种促炎症反应状态时，通过下调IFN-α、IL-6的表达量，减轻PIC诱导的炎症反应，降低炎症对宿主的免疫损伤，从而调节呼吸道免疫功能，抵抗呼吸道感染。对于PIC诱发NHBE促炎症反应机制是否与TAK1与IkB蛋白的磷酸化水平有关，则需要进一步深入研究。

关键词: 苗药防感香囊, 聚肌胞苷酸, 甘草酸二铵, 异草苷酸镁

Objective

To investigate the effects of diammonium glycyrrhizinate (DG) and magnesium isoglycyrrhizinate (MI) on bronchial epithelial cells (NHBE) stimultated with polymyocytic acid (PIC), to determine the therapeutic effects of DG and MI and to explore the possible mechanisms involved.

Methods

NHBE cells were stimulated with 1 μg/ml PIC to construct the cell model. Cells treated with Dexamethasone (DXMS) at 10 μg/ml were used as a positive control. DG and MI at low, medium, and high concentrations (0.5, 1.0, 2.0 mg/ml) were used for cell intervention. Cell activity was detected with CCK8 kit. IFN-α and IL-6 levels were detected by ELISA. IFN-α and IL-6 mRNA levels were detected by qPCR. p-TAK1/TAK1 and p-IkB/IkB expression levels were detected by Western blot.

Results

CCK8 assay showed that the viability of NHBE cells decreased with the increase of drug concentration, and the decrease of cell viability was more obvious in the positive control group. Compared with blank control cells, the expression of IFN-α and IL-6 significantly increased in the model group (cells treated with PIC; P＜0.05). Compared with the model group, the expression levels of IFN-α and IL-6 significantly decreased after treatment with different doses of DG and MI (P＜0.05). The mRNA expression levels of IFN-α and IL-6 in the model group were significantly higher compared with the blank control group (P＜0.05), but these in the intervention groups were significantly decreased (P＜0.05). After modeling, the ratios of p-TAK1/TAK1 and p-IkB/IkB tended to increase significantly (P＜0.05), while they showed no signficant changes in NHBE cells treated with low, medium, and high concentrations of DG and MI. In the positive control group, these ratios decreased compared with those in the model group (P＜0.05).

Conclusion

Ammonium glycyrrhizinate is one of the main active components of Miaoyao Fanggan Sachet. DG and MI have certain curative effects on PIC induced NHBE proinflammatory response possibly by downregulating IFN-α and IL-6 expression and reducing the inflammatory response induced by PIC. Whether the mechamism of PIC induced pro-inflammatory response of NHBE cells is related to the phosphorylation levels of TAK1 and IKB proteins needs to be further studied.

Key words: Miaoyao Fanggan Sachet, Polymyocytic acid, Diammonium glycyrrhizinate, Magnesium isoglycyrrhizinate

表1 NHBE细胞IL-6与IFN-α基因引物序列

引物名称	引物序列	引物长度（bp）	产物长度（bp）	退火温度（℃）
IL-6F	CTTCGGTCCAGTTGCCTTCT	20	228	59.65
IL-6R	GCCTCTTTGCTGCTTTCACA	20	228	59.65
IFN-αF	GAAACTCCCCTGATGAATGC	20	119	57.55
IFN-αR	TCTGCTCTGACAACCTCCC	19	119	57.55
β-actinF	TGGCACCCAGCACAATGAA	19	186	60.8
β-actinR	CTAAGTCATAGTCCGCCTAGAAGCA	25	186	60.8

表1 NHBE细胞IL-6与IFN-α基因引物序列

引物名称	引物序列	引物长度（bp）	产物长度（bp）	退火温度（℃）
IL-6F	CTTCGGTCCAGTTGCCTTCT	20	228	59.65
IL-6R	GCCTCTTTGCTGCTTTCACA	20	228	59.65
IFN-αF	GAAACTCCCCTGATGAATGC	20	119	57.55
IFN-αR	TCTGCTCTGACAACCTCCC	19	119	57.55
β-actinF	TGGCACCCAGCACAATGAA	19	186	60.8
β-actinR	CTAAGTCATAGTCCGCCTAGAAGCA	25	186	60.8

表2 苗药防感香囊多种提取物中主要活性物质的定量结果

组分名称	质量含量（mg/kg）	俗称（CAS No.）
癸酸	1.5×10^-4	334-48-5
甘草酸铵盐	0.14~0.16	1405-86-3
蛋白质	0.05	-
总糖	0.19	-
蒎烯	微量	7785-70-8
β-蒎烯	微量	8172-67-3
P-伞花烃	微量	535-77-3
桉叶油醇	微量	470-82-6
左旋樟脑	微量	464-48-2
L-薄荷醇	微量	2216-51-5
α-松油醇	微量	10492-56-1
丙氨酸	9.69	-
钾元素	46.7	-
铜元素	4.4	-
钙元素	7.7	-

表2 苗药防感香囊多种提取物中主要活性物质的定量结果

组分名称	质量含量（mg/kg）	俗称（CAS No.）
癸酸	1.5×10^-4	334-48-5
甘草酸铵盐	0.14~0.16	1405-86-3
蛋白质	0.05	-
总糖	0.19	-
蒎烯	微量	7785-70-8
β-蒎烯	微量	8172-67-3
P-伞花烃	微量	535-77-3
桉叶油醇	微量	470-82-6
左旋樟脑	微量	464-48-2
L-薄荷醇	微量	2216-51-5
α-松油醇	微量	10492-56-1
丙氨酸	9.69	-
钾元素	46.7	-
铜元素	4.4	-
钙元素	7.7	-

图1 不同浓度DG、MI干预8 h后NHBE细胞的生长曲线（n=3）。图a为DG，IC₅₀=2.190 mg/ml；图b为MI，IC₅₀=3.069 mg/ml

图2 3种药物的不同浓度梯度对NHBE细胞活性的影响（n=3）。图a为DG；图b为MI；图c为DXMS

图3 PIC造模后DG与MI 3种浓度处理对NHBE细胞活性影响（n=3）。图a为不同浓度PIC对细胞活力的影响；图b为PIC造模后，不同浓度DG干预对NHBE细胞活力的影响；图c为PIC造模后，不同浓度MI干预对NHBE细胞活力的影响

图4 ELISA法检测DG与MI分别处理的各组NHBE细胞中IFN-α、IL-6含量（n=3）。图a为不同剂量DG处理NHBE细胞的IFN-α相对表达量；图b为不同剂量MI处理NHBE细胞的IFN-α相对表达量；图c为不同剂量DG处理NHBE细胞的IL-6相对表达量；图d为不同剂量MI处理NHBE细胞的IL-6相对表达量

图5 qPCR检测DG与MI分别处理的各组NHBE细胞中IFN-α、IL-6 mRNA表达情况（n=3）。图a为DG处理NHBE细胞的IFN-α mRNA相对表达量；图b为MI处理NHBE细胞的IFN-α mRNA相对表达量；图c为DG处理NHBE细胞的IL-6 mRNA相对表达量；图d为MI处理NHBE细胞的IL-6 mRNA相对表达量

图6 Western blot检测DG处理的各组NHBE细胞中p-TAK1/TAK1与p-IkB/IkB蛋白表达水平（n=3）。图a为DG处理的各组NHBE细胞蛋白表达条带；图b为p-IkB/IkB相对蛋白表达统计图；图c为p-TAK1/TAK1相对蛋白表达统计图

图7 Western blot检测MI处理的各组NHBE细胞中p-TAK1/TAK1与p-IkB/IkB蛋白表达水平（n=3）。图a为MI处理的各组NHBE细胞蛋白表达条带；图b为p-IkB/IkB相对蛋白表达统计图；图c为p-TAK1/TAK1相对蛋白表达统计图

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Effects of Active Extracts of Miaoyao Fanggan Sachet on bronchial epithelial cells stimulated with polymyocytic acid

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Effects of Active Extracts of Miaoyao Fanggan Sachet on bronchial epithelial cells stimulated with polymyocytic acid