Study on Mechanism of Jinxin Oral Liquid Regulating Cardiolipin Metabolism against Respiratory Syncytial Virus Infection
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摘要:
目的 研究金欣口服液及其主要成分黄芩苷对呼吸道合胞病毒(Respiratory syncytial virus, RSV)诱导心磷脂代谢异常的影响及可能的作用机制。 方法 将BALB/c小鼠随机分为空白组、模型组和金欣口服液给药组。前3 d以RSV混悬液滴鼻建立RSV感染模型, 从第3天下午开始, 金欣口服液组予金欣口服液(27.6 g·kg-1·d-1)连续灌胃给药3 d。体外实验采用RSV感染Raw264.7细胞的方式建立病毒感染模型，黄芩苷组给予黄芩苷(100 mg·kg-1·d-1)干预。采用色谱质谱联用的方式分别检测小鼠肺组织及Raw264.7细胞心磷脂代谢谱的变化。qPCR法检测RSV表面蛋白 RSV-F、RSV-G, 炎症因子IL-6、IL-1β、Tnf-α, 心磷脂代谢酶Tafazzin(TAZ)、心磷脂合成酶1( Crls1)、心磷脂转运酶(PLSCR3)以及自噬受体蛋白SQSTM1/P62(p62) 的转录水平。Western blot法检测p62蛋白表达水平，分子对接检测p62与CL14∶0-16∶1-16∶1-18∶2的结合能力。 结果 RSV感染小鼠及细胞模型中 IL-6、IL-1β、Tnf-α mRNA表达增加(P < 0.05，P < 0.01，P < 0.001), 金欣口服液及黄芩苷可以起到一定的回调作用; RSV感染后心磷脂代谢谱发生了变化，金欣口服液及黄芩苷可起到一定的调节作用；模型组小鼠肺组织中的Crls1、TAZ mRNA表达显著上调(P < 0.01), 给予金欣口服液之后Crls1、TAZ mRNA表达明显降低下调(P < 0.01)，细胞模型组中Crls1 mRNA表达明显增加(P < 0.001), 黄芩苷干预后Crls1 mRNA表达显著降低(P < 0.000 1)；RSV感染小鼠肺组织p62蛋白表达明显下降(P < 0.001), 给予金欣口服液之后p62蛋白表达显著增加(P < 0.01)。Raw264.7细胞模型组p62蛋白表达明显增加(P < 0.05), 给予黄芩苷后p62蛋白水平进一步显著升高(P < 0.000 1)；分子对接结果表明心磷脂CL14∶0-16∶1-16∶1-18∶2可与p62的UBA结构发生阈结合。 结论 金欣口服液以及黄芩苷可改善RSV诱导的心磷脂代谢紊乱, 调节线粒体功能，进而发挥了抗RSV感染作用。 Abstract:OBJECTIVE To study the possible effects and mechanism of Jinxin oral liquid and its main ingredient baicalin on Respiratory syncytial virus (RSV)-induced abnormal cardiolipin metabolism. METHODS BALB/c mice were randomly divided into control group, RSV group and Jinxin oral liquid treatment group. Mice were nasally administrated of RSV suspension to establish RSV infection model at the first 3 days. Then Jinxin oral liquid 27. 6 g·kg-1·d-1 was administrated intragastrically for the next 3 days from the afternoon of the third day. Raw264.7 cells were infected by RSV to establish an in vitro infection model and baicalin (100 mg·kg-1·d-1) was given for intervention. The changes of cardiolipin metabolism profiles in mice lung tissue and Raw264. 7 cells were detected by chromatography-mass spectrometry. Transcriptional mRNA levels of RSV surface proteins RSV-F and RSV-G, inflammatory cytokines IL-6, IL-1β and Tnf-α, cardiolipin metabolic enzymes Tafazzin (TAZ), Cardiolipin Synthase 1 (Crls1), Phospholipid Scramblase 3 (PLSCR3) and autophagy-related protein p62 were detected by qPCR. The protein level of p62 was detected by Western blot. Molecular docking was used to detect the binding ability of p62 to CL14∶0-16∶1-16∶1-18∶2. RESULTS The expression of IL-6, IL-1β, and Tnf-α mRNA increased in RSV-infected mice and cell models (P < 0.05, P < 0.01, P < 0.001). Jinxin oral liquid and baicalin plays a certain restoration effect. The cardiolipin metabolism profile changes after RSV infection, and Jinxin oral liquid and baicalin can play a certain regulatory role. The expression of Crls1 and TAZ mRNA in the lung tissue of mice in the model group was significantly up-regulated (P < 0. 01), after administration of Jinxin oral liquid, the expression of Crls1 and TAZ mRNA was significantly reduced (P < 0. 01); the expression of Crls1 mRNA in the cell model group was significantly increased (P < 0. 001), and after baicalin intervention, the expression of Crls1 mRNA was significantly reduced (P < 0.000 1). The expression of p62 protein in the lung tissue of RSV-infected mice was significantly reduced (P < 0.001), and the expression of p62 protein was significantly increased after administration of Jinxin oral liquid (P < 0.01). The p62 protein expression in the Raw264.7 cell model group increased significantly (P < 0.05), and the p62 protein level further increased significantly after administration of baicalin (P < 0.000 1). Molecular docking results showed that cardiolipin CL14∶0-16∶1-16∶1-18∶2 can threshold-bind with the UBA structure of p62. CONCLUSION Jinxin oral liquid and baicalin can improve RSV-induced cardiolipin metabolism disorder, regulate mitochondrial function, and thus exert anti-RSV infection effects. -
Key words:
- cardiolipin metabolism /
- respiratory syncytial virus /
- Jinxin oral liquid /
- baicalin
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图 1 金欣口服液化学成分鉴定
注: a.正离子模式下金欣口服液定性信息; b.负离子模式下金欣口服液定性信息; c.正离子模式下标准品检测信息; d.负离子模式下标准品检测信息
Figure 1. Identification of chemical components of Jinxin oral liquid
图 2 金欣口服液及黄芩苷对RSV复制的抑制作用
注: a~b.BALB/c小鼠模型; c~d.Raw264.7细胞模型
Figure 2. Inhibitory effect of Jinxin oral liquid and baicalin on RSV replication
图 3 金欣口服液及黄芩苷对炎症因子的抑制作用
注: a.BALB/c小鼠肺组织HE染色; b.Raw264.7细胞形态图; c~e.BALB/c小鼠模型; f~h: Raw264.7细胞模型
Figure 3. Inhibitory effect of Jinxin oral liquid and baicalin on inflammatory factors
图 4 金欣口服液及黄芩苷对心磷脂代谢谱的影响
注: a~b.BALB/c小鼠模型PCA图及热图; c~d.Raw264.7细胞模型PCA图及热图; e.BALB/c小鼠模型CL 14:0_16:1_16:1_18:2的归一化峰强度图；f. Raw264. 7细胞模型CL 14:0_16:1_16:1_18:2的归一化峰强度图
Figure 4. Effects of Jinxin oral liquid and baicalin on cardiolipin metabolism profiles
图 5 金欣口服液及黄芩苷对Crls1、TAZ mRNA的影响
注: a~b.BALB/c小鼠; c~d.Raw264.7细胞
Figure 5. Effects of Jinxin oral liquid and baicalin on Crls1 and TAZ mRNA
图 6 金欣口服液及黄芩苷对心磷脂外翻及自噬的影响
注: a~b及e~f.BALB/c小鼠模型; c~d及g~h.Raw264.7细胞模型
Figure 6. Effects of Jinxin oral liquid and baicalin on cardiolipin eversion and autophagy
图 7 蛋白2MGW(a)、2JY7(b)与CL14 ∶ 0_16 ∶ 1_16 ∶ 1_18 ∶ 2分子对接相互作用图
Figure 7. Molecular docking interaction diagram of proteins 2MGW(a), 2JY7(b) and CL14 ∶ 0_16 ∶ 1_16 ∶ 1_18 ∶ 2
图 8 分子模拟过程中自噬受体蛋白的RMSD、SASA、Rg和RMSF随时间的变化曲线
Figure 8. The curve of changes of RMSD, SASA, Rg and RMSF of autophagy receptor proteins over time during molecular simulation
表 1 标准品的信息
Table 1. Information of standards
名称 批号 厂家 纯度 黄芩苷 16092401 成都普菲德生物技术有限公司 93% 白花前胡甲素 73609-25-7 成都普菲德生物技术有限公司 92% 盐酸麻黄碱 S-004-120927 中国药品生物制品检定所 99% 芥子碱 D-020-120331 中国药品生物制品检定所 93% 大黄素 0756-200110 中国药品生物制品检定所 98% 苦杏仁苷 H-027-120912 四川省维克奇生物 98% 野黄芩苷 H-029-121002 四川省维克奇生物 98% 汉黄芩素 D-017-120830 成都瑞芬思生物 98% 虎杖苷 120317 成都瑞芬思生物 98% 白藜芦醇 110753-201314 成都瑞芬思生物 98% 黄芩素 Y-174-120823 成都瑞芬思生物 98% 
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表 2 动物及细胞相关基因的引物序列
Table 2. Primer sequences of mice and cell related genes
基因 上游引物(5’→3’) 下游引物(5’→3’) RSV-F AACAGATGTAAGCAGCTCCGTTATC GATTTTTATTGGATGCTGTACATTT RSV-G CGGCAAACCACAAAGTCACA TTCTTGATCTGGCTTGTTGCA Mouse GAPDH GGAGAGTGTTTCCTCGTCCC ATGAAGGGGTCGTTGATGGC Mouse IL-6 TAGTCCTTCCTACCCCAATTTCC TTGGTCCTTAGCCACTCCTTC Mouse IL-1β GCAACTGTTCCTGAACTCAACT ATCTTTTGGGGTCCGTCAACT Mouse Tnf-α CCCTCACACTCAGATCATCTTCT GCTACGACGTGGGCTACAG Mouse TAZ TATGAGCTCATTGAGAACCGAG ATCAACTTCAGGTTCCAGATGT Mouse PLSCR3 CAAGACTCCCCTCCTGATTCC TGAAGGGGCATAGCCTTTGG Mouse P62 TGTGGAACATGGAGGGAAGAG TGTGCCTGTGCTGGAACTTTC Mouse Bax AAGTAGAAGAGGGCAACCAC GATGGCAACTTCAACTGGG Mouse Bid CCTGGAAATAGGGAGACG GATACGGCAAGAATTGTGAA
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表 3 金欣口服液指标性成分定性分析
Table 3. Qualitative analysis of the index components of Jinxin oral liquid
检测模式 序号 物质名称 分子式 tR/min 离子形式 m/z ppm 正离子模式检测 1 麻黄碱 C10H15NO 4.03 [M+H]+ 166.122 6 5.4 2 苦杏仁苷 C20H27NO11 5.72 [M+Na]+ 480.147 6 1.6 3 芥子碱 C16H24NO5 6.63 [M]+ 310.164 9 4.0 4 虎杖苷 C20H22O8 9.46 [M+H]+ 391.138 7 -5.9 5 野黄芩苷 C21H18O12 9.74 [M+H]+ 463.087 1 -5.0 6 黄芩苷 C21H18O11 12.51 [M+H]+ 447.092 2 -4.6 7 黄芩素 C15H10O5 17.61 [M+H]+ 271.060 1 -4.5 8 汉黄芩素 C16H12O5 20.70 [M+H]+ 285.075 8 0.5 9 白花前胡甲素 C21H22O7 24.62 [M+Na]+ 387.143 8 0.6 负离子模式检测 2 苦杏仁苷 C20H27NO11 5.72 [M-H]- 456.150 0 5.7 4 虎杖苷 C20H22O8 9.47 [M-H]- 389.123 1 4.7 5 野黄芩苷 C21H18O12 9.72 [M-H]- 461.071 5 2.1 6 黄芩苷 C21H18O11 12.49 [M-H]- 445.076 5 4.9 7 黄芩素 C15H10O5 17.68 [M-H]- 269.044 5 1.6 8 汉黄芩素 C16H12O5 20.66 [M-H]- 283.060 1 1.1 10 大黄素 C15H10O5 23.29 [M-H]- 269.044 5 3.7
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表 4 蛋白2MGW、2JY7与CL 14 ∶ 0_16 ∶ 1_16 ∶ 1_18 ∶ 2的MMPBSA分析
Table 4. MMPBSA analysis of proteins 2MGW, 2JY7 and CL 14 ∶ 0_16 ∶ 1_16 ∶ 1_18 ∶ 2
相互作用能 2MGW 2JY7 范德华相互作用/(kJ·mol-1) -212.756 -308.340 静电相互作用/(kJ·mol-1) -52.065 -13.446 极性溶剂化相互作用/(kJ·mol-1) 121.740 88.484 非极性溶剂化相互作用/(kJ·mol-1) -29.938 -37.851 总结合能/(kJ·mol-1) -173.019 -271.153 TΔS/(kJ·mol-1) 68.634 42.997 结合自由能/(kJ·mol-1) -104.386 -228.156
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