Effect of Jinzhen granule on two coronaviruses: The novel SARS-CoV-2 and the HCoV-229E and the evidences for their mechanisms of action.
Effect of Jinzhen granule on two coronaviruses: The novel SARS-CoV-2 and the HCoV-229E and the evidences for their mechanisms of action.
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DOI：
10.1016/j.phymed.2021.153874
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发表时间：
2022-01
期刊：
影响因子：
--
通讯作者：
Yang Z
中科院分区：
文献类型：
--
作者：
Ma Q;Wang Z;Chen R;Lei B;Liu B;Jiang H;Chen Z;Cai X;Guo X;Zhou M;Huang J;Li X;Dai J;Yang Z
尽管严重急性呼吸综合征冠状病毒2型（SARS-CoV-2）和人冠状病毒229 E（HCoV-229 E）对人类公共卫生构成巨大威胁，但目前尚无特效治疗方法。金珍颗粒是传统的八味中药，对病毒性疾病有显著疗效。然而，JZ对SARS-CoV-2和HCoV-229 E的抗病毒作用及其机制尚不清楚。本研究旨在揭示JZ对SARS-CoV-2和HCoV-229 E的抗病毒作用，并进一步探讨其调节宿主免疫应答的机制。采用岛津分析型高效液相色谱仪和Alltech ELSD 2000 ES进行JZ的色谱分离。我们采用细胞病变效应（CPE）和空斑减少试验来评价JZ的抗病毒作用。建立了人血管紧张素转换酶2（hACE 2）转基因小鼠致死性SARS冠状病毒感染模型，观察了JZ对小鼠死亡率和肺病毒滴度的保护作用。采用实时定量PCR方法分析促炎细胞因子在体外和体内的表达。Western blotting检测其对核因子κ B（NF-κB）/MAPK通路的调节活性。最后，使用线粒体膜电位测定、流式细胞术分析和蛋白质印迹来评估针对HCoV-229 E感染的抗凋亡效力。结果表明，从黄芩中鉴定出13种化学成分，其中5个峰的含量分别为没食子酸1.97 mg/g、黄芩苷20.69 mg/g、甘草酸4.92 mg/g、猪去氧胆酸4.86 mg/g、胆酸4.07 mg/g。通过CPE和空斑减少实验发现JZ对SARS-CoV-2和HCoV-229 E具有体外抑制作用，并进一步发现JZ对SARS-CoV-2感染的小鼠具有保护作用和抑制肺部病毒滴度。JZ还显著降低炎性细胞因子（IL-1α、IL-6、CCL-5和MIP-1β）的诱导，与体外观察到的作用相似。JZ能抑制HCoV-229 E诱导的细胞因子释放，降低p-p38 MAPK、p-JNK、p-NF-κB p65和p-IκBα蛋白表达，增加IκBα蛋白表达。值得注意的是，JZ显着保护HCoV-229 E感染的Huh-7细胞免受线粒体损伤，并减少凋亡细胞。JZ抑制了caspase-9、caspase-3和p-PARP的表达。综上所述，JZ（没食子酸1.97 mg/g，黄芩苷20.69 mg/g，甘草酸4.92 mg/g，猪去氧胆酸4.86 mg/g，胆酸4.07 mg/g）通过调节NF-κB/MAPK途径和细胞凋亡途径对SARS-CoV-2和HCoV-229 E具有抗病毒活性。这些发现证明了JZ对CoV的疗效，并表明JZ治疗是COVID-19的一种新的临床治疗策略。
Although severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and human coronavirus 229E (HCoV-229E) pose a huge threat to human public health, no specific treatment is available. Jinzhen granule (JZ) is a traditional eight ingredients-Chinese medicine with prominent efficacy for treating viral-induced diseases. However, little is known about the antiviral effect and mechanism of JZ against SARS-CoV-2 and HCoV-229E. This study aimed to reveal the antiviral effects of JZ against SARS-CoV-2 and HCoV-229E, and to further explore the underlying mechanisms regulating the host immune response. The chromatographic separation of JZ was performed using a Shimadzu analytical high-performance liquid chromatograph with UV detection and Alltech ELSD 2000ES. We conducted cytopathic effect (CPE) and plaque reduction assays to evaluate the antiviral effect of JZ. A lethal human angiotensin converting enzyme 2 (hACE2) transgenic mouse model of SARS-CoV-2 was established to determine the protective effect of JZ on mortality and lung virus titers. Real-time quantitative PCR assays were used to analyze the expression of proinflammatory cytokines in vitro and in vivo. Western blotting was further performed to determine the activities on regulating the nuclear factor kappa B (NF-κB)/MAPK pathway. Finally, mitochondrial membrane potential assays, flow cytometry analysis and western blotting were used to assess the anti-apoptotic potency toward HCoV-229E infection. The results showed that 13 chemical components were identified and five peaks were determined and quantitated (gallic acid 1.97 mg/g, baicalin 20.69 mg/g, glycyrrhizic acid 4.92 mg/g, hyodeoxycholic acid 4.86 mg/g, cholic acid 4.07 mg/g). We found that JZ exerted inhibitory potency against SARS-CoV-2 and HCoV-229E in vitro by using CPE and plaque reduction assays, and it was further found that JZ protected mice infected by SARS-CoV-2 from death and inhibited lung virus titers. JZ also significantly decreased the induction of inflammatory cytokines (IL-1α, IL-6, CCL-5 and MIP-1β), similar to the observed in vitro effect. Moreover, JZ suppressed the release of inflammatory cytokines in vitro and it decreased the protein expression of p-p38 MAPK, p-JNK, p-NF-κB p65 and p-IκBα induced by HCoV-229E and increased the expression of IκBα. Notably, JZ significantly protected HCoV-229E-infected Huh-7 cells from mitochondrial damage and decreased apoptotic cells. The activation of the mitochondria-mediated apoptotic pathway was inhibited by JZ, as shown by the reduced expression of cleaved caspase-9, caspase-3 and p-PARP. In conclusion, JZ (gallic acid 1.97 mg/g, baicalin 20.69 mg/g, glycyrrhizic acid 4.92 mg/g, hyodeoxycholic acid 4.86 mg/g, cholic acid 4.07 mg/g) exhibited antiviral activities against SARS-CoV-2 and HCoV-229E by regulating the NF-κB/MAPK pathway and the mitochondria-mediated apoptotic pathway. These findings demonstrated the efficacy of JZ against CoVs and suggested JZ treatment as a novel clinical therapeutic strategy for COVID-19.
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