在新型冠状病毒肺炎（corona virus disease 2019，COVID-19）的病理机制中，过度的炎症反应、补体活化和氧化应激发挥着核心作用。新冠病毒及其关键组件作为病原相关分子模式（pathogen-associated molecular patterns，PAMPs），通过特定的Toll样受体（Toll-like receptors，TLRs）激活免疫细胞；同时，病毒或感染引发的组织损伤也能激活NOD样受体热蛋白结构域相关蛋白3（NOD-like receptor thermal protein domain associated protein 3，NLRP3）炎症小体，这两种机制共同介导了炎症反应，引起细胞因子风暴。此外，过度炎症促使免疫细胞产生大量活性氧（reactive oxygen species，ROS），加剧氧化应激，而持续的氧化应激又会进一步激化炎症。新冠病毒的刺突蛋白（spike protein，S蛋白）和核衣壳蛋白（nucleocapsid protein，N protein）还能直接激活补体系统，加重炎症与组织损伤。炎症因子、补体活化及氧化应激标志物被认为是评估COVID-19严重程度的关键指标。针对这三大因素的干预，成为降低重症率和改善患者预后的关键策略。中医药在治疗传染病方面有着悠久的历史和丰富的经验。在中国抗击COVID-19疫情中，中医药“三药三方”发挥了重要作用，其中化湿败毒方在改善肺部CT表现、提高血氧饱和度、降低炎症水平及缓解流感样症状方面均有显著效果。然而该方治疗COVID-19的药理作用与机制尚不明确。本研究围绕COVID-19相关病理对化湿败毒方的作用与机制进行探索。

本研究按照临床煎药法制备了化湿败毒方水煎液，并通过醇沉法除去无法经肠道吸收的大分子，获得化湿败毒方提取物（extract of Huashi Baidu Fang，HSBD）。首先本研究运用多种经典抗氧化实验模型评估了HSBD的体外抗氧化应激能力，结果显示：在非细胞体系，HSBD的总抗氧化能力、1,1-二苯基-2-三硝基苯肼（1,1-diphenyl-2-picrylhydrazyl，DPPH）和超氧阴离子清除、以及抗脂质过氧化能力随浓度升高而增强；在细胞体系，HSBD能对抗胞质和线粒体活性氧ROS累积，且其减少胞质ROS生成的作用与抑制NADPH氧化酶活性相关。由于有色受试物对可见光测定法的干扰较大，本研究还利用丙二醛与β-硫代巴比妥酸的产物三甲川的荧光特性，建立了更适合测定有色受试物且对有机溶剂耐受度更好的羟自由基荧光检测方法，以此评价有色受试物HSBD的作用。结果显示，HSBD还能剂量依赖地清除羟自由基。

补体活化在COVID-19从轻症向重症转变的关键病理过程有重要贡献。补体系统可以经三条不同途径活化即经典途径、替代途径和凝集素途径，以介导关键成分补体片段3a（complement component 3a，C3a）、补体片段5a（complement component 5a，C5a）以及末端补体复合物（terminal complement complex，C5b-9）的生成。本研究运用ELISA法评估了HSBD体外抗补体活化的作用。结果表明HSBD对补体三条常规途径的激活均有显著的抑制作用。鉴于新冠S蛋白和N蛋白能够直接激活补体系统，本研究进一步测定了HSBD对原始毒株S蛋白及N蛋白活化补体的作用。结果显示HSBD对两种蛋白所致的补体活化均有明显的抑制作用；相较于位于病毒内部的N蛋白，其对位于病毒表面的S蛋白的抑制能力更强。由于新冠病毒不断变异，尤其S蛋白的变异程度极大，因此本研究将原始毒株S蛋白与当时正在大流行的Omicron B1.1.529变异株S蛋白（有多达32处突变）进行了比较。结果发现两种S蛋白的补体活化能力相当，提示新冠变异株Omicron感染仍然存在着机体补体被活化的病理状况。

TLRs在COVID-19细胞因子风暴中发挥了关键作用。新冠病毒的RNA可活化细胞内体RNA感受器TLR7/8和TLR3，而病毒表面的S蛋白本身也是一种PAMP。然而，在本研究进行之时学界对于新冠S蛋白向胞内传导信号所借助的模式识别受体（pattern recognition receptor，PRR）到底是TLR4还是TLR2尚存争议。为此，本研究采用TLR4与TLR2各自的阻断剂进行甄别。尽管新冠S蛋白在活化的巨噬细胞上表现出TLR2信号被激活的特征—髓样分化因子88（myeloid differentiation primary response 88，MyD88）偏向，但最后本研究确认新冠S蛋白的PRR是TLR4而非TLR2。随后，本研究围绕新冠病毒感染可能活化的TLR7/8、TLR3及TLR4展开HSBD对巨噬细胞生成促炎细胞因子的作用研究。结果发现HSBD对这些新冠相关TLRs相应配体活化的巨噬细胞释放促炎介质一氧化氮（nitric oxide，NO）、白介素6（interleukin-6，IL-6）、肿瘤坏死因子α（tumor necrosis factor α，TNF-α）和单核细胞趋化蛋白1（monocyte chemoattractant protein 1，MCP-1）均有明显的抑制作用，尤其对IL-6的抑制效果最为显著。当时正值Omicron第一代变异株B1.1.529流行，部分临床证据显示Omicron致病力减弱。对此本研究比较了其S蛋白与原始毒株S蛋白致炎能力的差异。结果显示Omicron S蛋白激活巨噬细胞内核因子κB（nuclear factor kappa B，NF-κB）炎症信号通路及诱导促炎因子释放的能力均明显弱于原始毒株S蛋白，且在相同浓度下未能诱导IL-6的产生。随后，本研究在原始毒株S蛋白活化的巨噬细胞模型中进一步考察了HSBD的作用。结果显示，HSBD也能显著抑制原始毒株S蛋白刺激巨噬细胞释放促炎介质NO、IL-6、TNF-α和MCP-1。此外，本研究还发现HSBD也会抑制TLR3或TLR4配体诱导的抗病毒因子干扰素β（interferon-β，IFN-β）生成，提示化湿败毒方在治疗I型干扰素反应严重受损的重症COVID-19患者时，应与I型干扰素联用。

大量的临床证据显示NLRP3炎症小体活化在COVID-19的发生发展中也起着极为关键的作用，因此本研究还考察了HSBD对其活化的影响。结果显示，HSBD不仅能抑制NLRP3活化的巨噬细胞上清液中白介素1β（interleukin-1β，IL-1β）和caspase-1 p20的释放，HSBD（i.g.，高剂量为成人一日的临床等效剂量）还能剂量依赖地降低尿酸盐（monosodium urate，MSU）结晶诱导的腹膜炎小鼠腹腔灌流液中的IL-1β，表明HSBD在体内外均能抑制NLRP3炎症小体的激活。机制研究显示，HSBD可抑制凋亡相关斑点样蛋白（apoptosis-associated speck-like protein containing a CARD，ASC）寡聚化和斑点形成，进而阻止NLRP3炎症小体组装，但不影响其上游事件胞内K⁺外流和线粒体ROS产生。由于环磷酸腺苷（cyclic adenosine monophosphate，cAMP）可直接与NLRP3结合，以抑制炎症小体组装，本研究进一步聚焦于此考察HSBD的作用。结果显示HSBD能对抗胞内cAMP水平的下降，且这一作用是通过抑制磷酸二酯酶（phosphodiesterase，PDE）活性实现的。进一步，本研究采用基因沉默技术将巨噬细胞中PDE存在的主要亚型PDE4B表达下调，结果显示：在PDE4B缺陷巨噬细胞中，HSBD对NLRP3活化的抑制效果大幅减弱。一致地，选择性PDE4抑制剂咯利普兰也展现了与HSBD类似的效果。这些结果均表明HSBD通过抑制PDE4B，对抗胞内cAMP下降，抑制ASC寡聚化和斑点形成，从而起到抑制NLRP3炎症小体活化的作用。

最后，本研究采用脂多糖（lipopolysaccharides，LPS）诱导急性肺损伤（acute lung injury，ALI）模型来模拟COVID-19肺部炎症与损伤状态。结果显示：HSBD（i.g.，高剂量为成人一日临床等效剂量）能剂量依赖地显著抑制ALI小鼠支气管肺泡灌流液中炎症因子IL-6、TNF-α、IL-1β和趋化因子（C-X-C基序）配体1（C-X-C motif chemokine ligand 1，CXCL-1）水平，有效减轻肺组织氧化应激（抗氧化指标超氧化物歧化酶、过氧化氢酶活性和谷胱甘肽含量升高、氧化指标髓过氧化物酶活性和丙二醛含量降低），改善肺组织的病理损伤（肺间隔增厚、间质水肿、肺泡出血和中性粒细胞浸润）。

本研究围绕COVID-19的相关病理—氧化应激、补体活化和炎症反应考察了HSBD的作用。实验结果显示：HSBD在体外非细胞体系与细胞体系均具有抗氧化作用，能抑制补体三条常规途径的激活及新冠病毒S蛋白和N蛋白所致的补体活化，以及对抗新冠病毒相关TLRs活化介导的炎症反应。在抗炎方面，HSBD还能通过抑制PDE4B，对抗cAMP下降，减少ASC寡聚化和斑点，起到抑制NLRP3炎症小体活化的作用。与临床效果一致，HSBD能改善LPS诱导小鼠ALI的炎症反应、氧化应激和病理状况。这些发现能为解读化湿败毒方在COVID-19治疗中可能的作用机理提供可供参考的药理学依据。

Excessive inflammatory responses, complement activation, and oxidative stress play central roles in the pathological mechanisms of corona virus disease 2019 (COVID-19). SARS-CoV-2 and its key components act as pathogen-associated molecular patterns (PAMPs), activating immune cells through specific Toll-like receptors (TLRs); concurrently, virus or infection-induced tissue damage can also activate the NOD-like receptor thermal protein domain associated protein 3 (NLRP3) inflammasome. These two mechanisms together mediate inflammatory responses, leading to a cytokine storm. Furthermore, excessive inflammation prompts immune cells to produce a large amount of reactive oxygen species (ROS), exacerbating oxidative stress, which in turn further intensifies inflammation. The spike (S) protein and nucleocapsid (N) protein of SARS-CoV-2 can directly activate the complement system, aggravating inflammation and tissue damage. The markers of inflammatory responses, complement activation, and oxidative stress are considered key indicators for assessing the severity of COVID-19. Interventions targeting these three factors have become crucial strategies for reducing morbidity and improving patient prognosis. Traditional Chinese Medicine has a long history and rich experience in treating infectious diseases. In the battle against COVID-19 epidemic in China, “three TCM drugs and three herbal formulas” played an important role, among which Huashi Baidu Fang has shown significant effects in improving lung CT manifestations, increasing blood oxygen saturation, reducing inflammation levels, and alleviating flu-like symptoms. However, the pharmacological effects and mechanisms of this formula in treating COVID-19 remain unclear. This study explores the effects and mechanisms of Huashi Baidu Fang on COVID-19-related pathogenesis.

The extract of Huashi Baidu Fang, called HSBD, was obtained by preparing the water decoction of Huashi Baidu Fang according to traditional clinical decoction methods and removing the large molecules that cannot be absorbed through the gastrointestinal tract using the alcohol precipitation method. First, various classical antioxidant experimental models were used to assess the in vitro antioxidative stress capability of HSBD. The results showed that in non-cellular systems, the total antioxidant capacity of HSBD, as well as its 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and superoxide anion scavenging activities and lipid peroxidation inhibition properties, concentration-dependently enhanced. In cellular systems, HSBD could counteract the accumulation of cytosolic and mitochondrial ROS, and its effect in reducing cytosolic ROS production was associated with the inhibition of NADPH oxidase activity. Due to the interference of colored test substances with visible light detection methods, a new hydroxyl radical fluorescence detection method was established based on the fluorescence characteristics of the product of malondialdehyde and β-thiobarbituric acid—3,5,5-trimethyloxazolidine-2,4-dionetone. This new fluorescence method was more suitable for colored test substances and more tolerant to organic solvents, thereby could evaluate the effect of HSBD (colored test substance). The results showed that HSBD could also scavenge hydroxyl radicals in a concentration-dependent manner.

Complement activation plays a crucial role in the pathological process of COVID-19 from mild to severe cases. The complement system can be activated through three distinct pathways: the classical pathway, the alternative pathway, and the lectin pathway, leading to the generation of key components such as complement component 3a (C3a), complement component 5a (C5a), and terminal complement complex (C5b-9). The in vitro anti-complement activation effects of HSBD were evaluated using the ELISA method. The results indicated that HSBD effectively inhibited the activation of the complement system through all three activation pathways. Given that the S and N protein of SARS-CoV-2 can directly activate the complement system, the effects of HSBD on the complement activation induced by S and N protein of the original strain were further assessed. The findings showed that HSBD could significantly inhibit complement activation triggered by both proteins, with a stronger inhibitory effect on the S protein located on the virus surface compared to the N protein inside the virus. Considering the continuous mutation of SARS-CoV-2, especially the extensive variations in the S protein, the complement activation induced by the S protein of the original strain was compared with that of the widely prevalent Omicron B1.1.529 variant strain at that time, which featured up to 32 mutations. The results showed that the complement activation capabilities of both S proteins were comparable, indicating that complement activation still exists in Omicron variant infections.

TLRs play a crucial role in the cytokine storm associated with COVID-19. SARS-CoV-2 RNA can activate intracellular endosomal RNA sensors TLR7/8 and TLR3, while the surface S protein also acts as a PAMP. However, at the time of this study, there was academic debate over whether the pattern recognition receptor (PRR) of S protein for intracellular signaling was TLR4 or TLR2. To address this, the antagonist specific to both TLR4 and TLR2 were used for identification. Despite the S protein displaying characteristics of activated TLR2 signaling—myeloid differentiation primary response 88 (MyD88) bias on activated macrophages, the PRR of S protein was TLR4, not TLR2, which was confirmed by the antagonist experiment. Subsequently, RAW264.7 macrophages were used to investigate the effects of HSBD on the pro-inflammatory cytokines induced by SARS-CoV-2 related TLRs ligands, as well as SARS-CoV-2 S protein, in vitro. The results showed that HSBD significantly inhibited the release of pro-inflammatory mediators—nitric oxide (NO), interleukin (IL)-6, tumor necrosis factor α (TNF-α), and monocyte chemoattractant protein 1 (MCP-1) by macrophages activated by ligands for these SARS-CoV-2-related TLRs, especially demonstrating a most significant inhibitory effect on IL-6. At the time, the first-generation Omicron variant strain B1.1.529 was prevalent, with some clinical evidence suggesting reduced virulence of Omicron. Accordingly, the pro-inflammatory ability of its S protein was compared with that of the original strain. The results indicated that the ability of Omicron S protein to activate the nuclear factor kappa B (NF-κB) signaling pathway and induce pro-inflammatory mediators release in macrophages was significantly weaker than that of the original strain one, and it failed to induce IL-6 production at the same concentration. Furthermore, the anti-inflammatory effect of HSBD on macrophages activated by the S protein of original strain was investigated. The findings indicated that HSBD also significantly inhibited the release of pro-inflammatory mediators NO, IL-6, TNF-α, and MCP-1 stimulated by the S protein of original strain. Additionally, the results showed that HSBD also suppressed the production of the antiviral factor interferon-β (IFN-β) induced by TLR3 or TLR4 ligands, suggesting that Huashi Baidu Fang should be combined with Type I interferons when treating severe COVID-19 patients with a significantly impaired Type I interferon response.

Substantial clinical evidence has indicated that the activation of the NLRP3 inflammasome plays a crucial role in the onset and progression of COVID-19; hence, we also explored the effects of HSBD on its activation. The results demonstrated that HSBD could inhibit the release of interleukin-1β (IL-1β) and caspase-1 p20 in the supernatant of NLRP3-activated macrophages. Furthermore, HSBD (i.g., with the high dose being the clinical equivalent dose for an adult per day) could reduce the levels of IL-1β in the peritoneal lavage fluid of monosodium urate crystal (MSU)-induced mouse peritonitis in a dose-dependent manner, indicating that HSBD could inhibit NLRP3 inflammasome activation both in vitro and in vivo. Mechanism studies revealed that HSBD could inhibit apoptosis-associated speck-like protein containing a CARD (ASC) oligomerization and speck formation, thereby preventing the assembly of the NLRP3 inflammasome, but not affecting its upstream events—intracellular K⁺ efflux and mitochondrial ROS production. Given that cyclic adenosine monophosphate (cAMP) can directly bind to NLRP3 to inhibit inflammasome assembly, we further focused on investigating the effect of HSBD on cAMP. The results showed that HSBD could counteract the reduction in intracellular cAMP levels, which was attributed to its inhibition on phosphodiesterase (PDE) activity. Moreover, gene silencing technique was used to downregulate the expression of PDE4B, the predominant subtype present in macrophages. The results showed that the inhibitory effect of HSBD on NLRP3 activation was significantly weakened in PDE4B-deficient macrophages. Consistently, the selective PDE4 inhibitor, rolipram, exhibited similar effects to HSBD. Collectively, these findings indicated that HSBD inhibited NLRP3 inflammasome activation by counteracting the decline in intracellular cAMP levels through the inhibition of PDE4B, thereby preventing ASC oligomerization and speck formation.

LPS-induced acute lung injury (ALI) model was used to mimic the pulmonary inflammation and damage associated with COVID-19. The results showed that HSBD (i.g., with the high dose being the clinical equivalent dose for an adult per day) could significantly inhibit the levels of inflammatory cytokines IL-6, TNF-α, IL-1β, and C-X-C motif chemokine ligand 1 (CXCL-1) in the bronchoalveolar lavage fluid of ALI mice in a dose-dependent manner. It effectively reduced pulmonary oxidative stress (increased activities of antioxidant markers such as superoxide dismutase, catalase, and levels of glutathione, and decreased activities of oxidative markers like myeloperoxidase and levels of malondialdehyde), and ameliorated pathological lung damage characterized by thickened alveolar septa, interstitial edema, alveolar hemorrhage, and neutrophil infiltration.

This study comprehensively investigated the effects of HSBD on the COVID-19-related pathogenesis—oxidative stress, complement activation, and inflammatory responses. The results demonstrated that HSBD exhibited antioxidative effects in both non-cellular and cellular systems in vitro, inhibited the activation of the three conventional complement pathways and the complement activation induced by the SARS-CoV-2 S and N proteins, and counteracted the inflammatory response mediated by SARS-CoV-2-related TLRs activation. In terms of anti-inflammatory effects, HSBD also suppressed NLRP3 inflammasome activation by inhibiting PDE4B, countering the decrease in cAMP, thereby reducing ASC oligomerization and speck formation. Consistent with clinical outcomes, HSBD could improve the inflammatory response, oxidative stress, and pathological conditions in LPS-induced ALI mice. These findings provide pharmacological evidence that may elucidate the potential mechanisms of Huashi Baidu Fang in the treatment of COVID-19.