In Silico and In Vitro studies of taiwan chingguan yihau (NRICM101) on TNF-α/IL-1β-induced human lung cells

Summary COVID-19 pandemic has been a global outbreak of coronavirus (SARS-CoV-2 virus) since 2019. Taiwan Chingguan Yihau (NRICM101) is the first traditional Chinese medicine (TCM) classic herbal formula and is widely used for COVID-19 patients in Taiwan and more than 50 nations. This study is to investigate in silico target fishing for the components of NRICM101 and to explore whether NRICM101 inhibits cytokines-induced normal human lung cell injury in vitro. Our results showed that network prediction of NRICM101 by a high throughput target screening platform showed that NRICM101 has multiple functions that may affect cytokine regulation to prevent human lung cell injury. In addition, NRICM101 revealed protective effects against TNF-α/IL-1β-induced normal human lung HEL 299 cell injury through JNK and p38MAPK kinase signaling. Next-generation sequencing (NGS) analysis of NRICM101 on TNF-α/IL-1β-injured HEL 299 cells indicated that inflammatory pathway, cell movement of macrophages, cellular infiltration by macrophages, and Th1/Th2 immuno-regulation pathways were included. Thus, NRICM101 is a therapeutic agent, and it can improve COVID-19 syndrome to confer beneficial effects through multiple targeting and multiple mechanisms.

In Silico and In Vitro studies of taiwan chingguan yihau (NRICM101) on TNF-a/IL-1b-induced human lung cells 1

A traditional Chinese medicine formula Taiwan
Chingguan Yihau (NRICM101) is the first traditional Chinese medicine (TCM) classic herbal formula that is widely used in more than 50 nations for COVID-19 patients [1]. NRICM101 consists of ten herbs (Fig. 1 [1]. All herbal preparations have been shown to exert potential for inhibiting SARS-CoV-2 of respiratory infection and immune-modulatory effect, indicated by the National Research Institute of Chinese Medicine (NRICM) of Taiwan after evaluating clinical symptoms. Scientific evidence based on a clinical perspective indicates that NRICM101 may disrupt COVID-19 disease progression via antiviral and anti-inflammatory effects to offer promise as a multi-target agent for the prevention and treatment [1]. NRICM101 is an anti-SARS-CoV-2 therapeutic agent as mentioned in COVID-19 treatment guidelines in Taiwan (https://www.mohw.gov.tw/ cp-16-60830-1.html). Patients with severe COVID-19 to cause a large number of pro-inflammatory macrophages were found in bronchoalveolar lavage fluid and released a large number of IL-1, TNF-a, IL-6 IL-8, CSF, and MCP-1 to induce cytokine storm [2]. Therapeutics with the potential to mitigate inflammatory cytokines may attenuate disease progression and mortality [3e5].
The manifestations of SARS-CoV-2 infection vary widely from asymptomatic diseases to severe pneumonia and life-threatening complications [6,7]. Abnormal lung function is the most common feature among patients of SARS-CoV-2 infection and can be complicated by acute respiratory distress syndrome (ARDS), particularly in elderly people with multiple comorbidities [8e10]. As the pandemic of COVID-19 continues which are with high levels of inflammatory markers, and are often accompanied by evidence of pulmonary fibrosis including interstitial thickening, coarse reticular patterns, and parenchymal bands lymphopenia [11e13]. Retrospective analyses have found that elevated inflammatory markers (such as erythrocyte sedimentation rate, Creactive protein, ferritin, TNF-a, IL-1, and IL-6) are higher in patients who died compared to survivors [14,15]. Cytokine-release syndrome (CRS), cytokine storm and post-COVID syndrome (PCS) are suggested as two of the major pathophysiological  . Laboratory analysis demonstrated that SARS-CoV-2 spike (S) protein was associated with the up-regulation of AT1 signaling which led to the induction of transcriptional regulatory molecules (NF-kB, c-Fos, and MAPK activation) [16]. The signal transduction may lead to secrete high levels of inflammatory cytokines that are observed in the lungs of COVID-19 patients like cytokine storm and pulmonary fibrosis due to macrophage activation and infiltration [17,18].
Our goal of this study is to understand whether NRICM101 could interact with other target proteins and inhibit cytokine-induced human lung cell injury. The study design and schematics were performed by in silico target fishing for the components of NRICM101, the workflow of in silico assay and in vitro bioactivity analysis are presented in Fig. 2 liquiritingenin, isoliquiritin, isoliquiritingenin, Magnolol, Honokiol, a-eudesmol, b-eudesmol, Menthol, Menthone, Glucoside, Apigenin, Chlorogenic acid, Caryophyllene, Pulegone, Menthone, a-Phytosterol, a-Tocopherolquinone) were sketched using BIOVIA Draw and prepared for generating the fitting compound for protonated isomers and tautomers at pH 7.4. A total of 16,035 target proteins by pharmacophore models in PharmaDB (BIOVIA Discovery Studio 2020 software; Dassault Syst emes) were then applied as screening targets for the 60 components of NRICM101 [19,20]. For network analysis, a goodness-of-fit value of >0.6 was considered to be potential compound target proteins. To generate a corresponding molecular network of those target proteins, all human target genes were set as focus molecules and analyzed using a core analysis tool in IPA (IPA 2020; Qiagen Sciences, Inc.). All presented pathways were deemed to be statistically significant according to Fisher's exact t-test (P < 0.05) [12].   Bark (M. officinalis, NK, 11.25 g), Peppermint Herb (M. haplocalyx, NL, 11.25 g), and Fineleaf Nepeta (N. tenuifolia, NR, 11.25 g). For a patient's daily dose, a full set of herbs and 1 L of water were placed in a boiler, boiled, and simmered for the decoction to reduce to 300 mL. The NRICM101 formulation was boiled with 1000 mL distilled water for 60 min into a 350 mL decoction and then concentrated under reduced pressure to 7.17 g by Rotary Evaporator (Ne1300VF/OSB-2200; EYELA, Japan).

Cell injury and DNA condensation by DAPI stain
HEL 299 cells were treated with TNF-a, IL-1b (50 ng/ mL, individual), and NRICM101 (50 and 100 mg/mL) for 24 h. Cell injury and DNA condensation were examined by DAPI staining (SigmaeAldrich and Merck KGaA). The cell fluorescent imaging was examined by Echo Revolve microscope (Echo Laboratories, San Diego, California, 200X) [24,25].

JNK and p38MAPK kinase activities assay
JNK and p38MAPK kinase activities assay were examined by phosphorylated protein kinase sandwich ELISA assay. p-p38 MAPK (Thr180/Tyr182) (cat. no. 7946), p-JNK (Thr183/Tyr185) (cat. no. #7325), and assays were performed according to the manufacturer's protocols (PathScan Sandwich ELISA kits; Cell Signaling Technology, Inc.). HEL 299 cells were treated with TNF-a, IL-1b (50 ng/mL, individual), and NRICM101 (50 and 100 mg/mL) for 12 h. Cells were harvested and total proteins were collected. Proteins were incubated in appropriate antibody-coated micro-wells overnight at 4 C. The 100 mL/well of the appropriate antibody was added for 1.5 h at 37 C, and an HRP-linked secondary antibody was added for 60 min at 37 C. Absorbance was measured by an ELISA reader (Anthos 2001) at 450 nm as previously described [26,27].

Network and signaling pathways analysis by ingenuity pathway analysis (IPA) and Kyoto Encyclopedia of Genes and Genomes (KEGG)
For network and signaling pathways analysis of the whole transcriptome sequencing database, a total of 331 human target genes were prepared and subsequently analyzed using a core analysis tool in IPA software (IPA 2021; Qiagen Sciences, Inc.) and Kyoto Encyclopedia of Genes and Genomes (KEGG) database. The network analysis rankings were calculated based on statistical significance using Fisher's exact t-test (p < 0.05) [28,29].

Statistical analysis
For each in vitro study, three independent experiments were conducted. Data are presented as the mean ± standard (SD) deviation. One-way analysis  of variance followed by Dunnett's test and Tukey's post hoc test was conducted to analyze the differences between two groups and among multiple groups by SPSS software version 25.0 (IBM, Corp.). ***P < 0.001 and ###P < 0.001 were considered to indicate a statistically significant difference.

Results
3.1. In silico study 3.1.1. Pathways and network prediction of NRICM101 by high throughput target screening platform To predict target proteins of NRICM101, we used high throughput target screening platform analysis. The canonical pathway of NRICM101 was showed in Fig. 3 . We summarized that molecule targets contribute to inflammation of the lung and respiratory systems (Fig. 4A) and COVID-19 (Fig. 4B)  on NRICM101. In addition, network analysis and visualization revealed pulmonary healing signaling pathways (Fig. 5), pulmonary fibrosis idiopathic (Fig. 6), and IL-1/TNF signaling pathway (Fig. 7) with a significant positive correlation. Our results suggest that NRICM101 has not only anti-SARS-CoV-2 activity but also pneumonic protection and immuneregulated effects. Here, it is hypothesized that NRICM101 has multiple functions that may affect cytokine regulation and prevent human lung cell injury.

In vitro study
3.2.1. NRICM101 revealed protective effects against TNF-a/IL-1b-induced normal human lung HEL 299 cell injury To confirm the results of in silico prediction, we further investigated the potential protective effects of NRICM101 on TNF-a/IL-1b-induced cell injury in HEL 299 cells. The results demonstrated that cell viability significantly decreased in TNF-a/IL-1b (50 ng/mL, individual) treatment (Fig. 8A) and increased dead cells (Fig. 8B) and DNA condensation (Fig. 8C). However, the treatment with TNF-a/ IL-1b (50 ng/mL, individual) and NRICM101 (50 and 100 mg/mL) showed increased viable cells (Fig. 8A) and decreased dead cells (Fig. 8B) and DNA condensation (Fig. 8C). These results suggest that NRICM101 might exert protective effects against TNF-a/IL-1b-induced cell injury in HEL 299 cells.
3.2.2. NRICM101 against TNF-a/IL-1b-induced HEL 299 cell injury was attenuated through JNK and p38MAPK kinase activities In order to further confirm the results of TNF-a/IL-1b-induced cell injury, both of JNK and p38MAPK kinase activity assays were examined in TNFa/IL-1b-treated HEL 299 cells after NRICM101 treatment. Our results in Fig. 9 showed that phosphorylation of JNK and p38MAPK significantly increased in TNF-a/IL-1b treatment, and NRICM101 significantly attenuated JNK and p38MAPK phosphorylation on TNF-a/IL-1b-treated HEL 299 cells. The abovementioned findings revealed the concentration-dependent effects of NRICM101 on the down-regulation of JNK and p38MAPK kinase in TNF-a/IL-1b-treated HEL 299 cells.

Next-generation sequencing analysis of NRICM101 on TNF-a/IL-1b-injured HEL 299 cells
To improve insight into the biological activity of NRICM101 in TNF-a/IL-1b-induced cell injury in HEL 299 cells, RNA sequencing transcriptional profile analysis was performed. As shown in Fig. 10A, normalized RNA-sequencing data from NRICM101treated samples and the control group (TNF-a/IL-1b) were clustered, indicating a significantly different gene expression analysis. In Fig. 10B, red dots mean significantly up-regulated genes, and green dots mean significantly down-regulated genes in MA plot. The 213 genes were up-regulated and 118 genes were down-regulated. Supplementary Table S1 (https:// www.biomedicinej.com/cgi/editor.cgi?article¼1378& window¼additional_files&context¼biomedicine)   Fig. 8. Effect of cell viability on TNF-a/IL-1b and NRICM101 co-incubation in HEL 299 cells. The cells (2.5 Â 10 5 cells/mL/well) were exposed to TNF-a/IL-1b and NRICM101 (50 and 100 mg/mL) for 24 h. (A) Cell viability was detected by the MTT assay. Data are presented as the mean ± standard deviation (n ¼ 3) and analyzed using one-way ANOVA followed by Tukey's post hoc test. ***P < 0.001 vs control; ###P < 0.001 vs TNF-a/IL-1b group. (B) Cell morphology was examined by phase-contrast microscopy. (C) Cell injury and DNA condensation were evaluated via DAPI stain. showed the raw sequencing data of NRICM101treated TNF-a/IL-1b-injured HEL 299 cells. To further determine the mechanism of action (MOA) of the genes and associated functions, the Ingenuity pathway analysis (IPA) database and KEGG database were used. The most significantly enriched pathways were selected and shown in Fig. 11 IL-1A,  IL-1B, TNFSF4, TNFSF18, IFI44L, IL32, and CCL2 genes were down-regulated of NRICM101 on TNF-a/ IL-1b-injured HEL 299 cells ( Fig. 12 and Supplementary Table S1 (https://www.biomedicinej.com/ cgi/editor.cgi?article¼1378&window¼additional_ files&context¼biomedicine)). In addition, those results suggest NRICM101 regulated TNF-a/IL-1b medicated inflammatory pathway (Fig. 12A), cell movement of macrophages, cellular infiltration by macrophages (Fig. 12B), and Th1/Th2 immunoregulation pathways (Fig. 13) in TNF-a/IL-1b-injured HEL 299 cells.

Discussion
Traditional Chinese medicine (TCM) formula, Taiwan Chingguan Yihau (NRICM101), has been administered orally to COVID-19 patients in Taiwan [1]. NRICM101 consisted of ten herbs [1], and it was demonstrated that Scutellaria Root (S.    [9,19]. In silico study showed quercetin and kaempferol of Heartleaf Houttuynia (H. cordata, HC.) had high binding affinities for 3C-like protease (3CL pro ) and RNAdependent RNA polymerase (RDRP) of SARS-CoV-2 [19]. Honokiol, isolated from Magnolia Bark (M. officinalis, NK.), exerted a high binding affinity on angiotensin-converting enzyme 2 (ACE2) [38]. Sinigrin and hesperetin, two active components in Indigowoad Root (I. indigotica, NE.), had high binding affinities for 3C-like protease (3CL pro ) of SARS-CoV-2 [42,43]. The mechanism studies on pharmacological activities of NRICM101 in the treatment of COVID-19 are limited to confirming the interaction of the formula with viral proteins and other structures according to identified pathogenic pathways of these remedies must be performed before further clinical trials. In our study, the results of NGS analysis indicated NRICM101 regulated SARS-CoV-2 infected pathways on COVID-19 (Supplementary Figure S1 (https://www. biomedicinej.com/cgi/editor.cgi?article¼1378& window¼additional_files&context¼biomedicine)). The experimental studies showed that all active components of NRICM101 may be a potent anti-SARS-CoV-2 agent for COVID- 19. It has been demonstrated that an association between poor outcome and cytokine-release syndrome (CRS) of COVID-19 [44,45]. In this study, we first analyze target proteins, biological pathways, disease, and function of bioactive compounds of NRICM101 using a high throughput target screening system. According in silico study results, we predicted that NRICM101 exerted multiple targets and signaling pathways (Fig. 3). Further analysis revealed that the main bioactive compounds contained within the cytokine-related pathways (IL-15 production, LPS/IL-1 Mediated Inhibition of RXR Function) (Fig. 7), oxidative stress response pathways (NRF2-mediated Oxidative Stress Response, HIF1a Signaling), pulmonary related signaling pathways (Pulmonary Fibrosis Idiopathic Signaling Pathway, Pulmonary Healing Signaling Pathway) (Figs. 4e6). NRICM101 potentially affected the inflammatory cytokine signaling, including TNFR2, TNFR1, IL-1, IL-2, IL-4, IL-6, IL-7, IL-12, IL-13, IL-17, and IL-23 Signaling (Fig. 3), all of which were previously identified in COVID-19 [46,47]. Through high throughput target screening and signaling pathway analysis on the inflammation of the lung, respiratory systems and SARS-CoV-2, these compounds contained within NRICM101 not only serve important roles on anti-SARS-CoV-2 agents but can also provide potential points for developing lung and respiratory system protection agents. The importance of nodes, the results in Fig. 7 found the MAP3K7, JNK, MKK3/6, p38MAPK, and MAPKAPK2 target proteins were involved in IL-1/ TNF signaling pathways.
The first purpose of this study was to investigate the effects of NRICM101 on TNF-a/IL-1b-induced normal human lung HEL 299 cell injury. Our results demonstrated the protective effects of NRICM101 against TNF-a/IL-1-induced injury (Fig. 8A), cytotoxicity (Fig. 8B), DNA condensation and damage (Fig. 8C), and JNK (Fig. 9A), p38MAPK kinase activities (Fig. 9B). Second, NGS analysis was conducted to investigate the molecular mechanisms and signaling pathway of NRICM101 on TNF-a/IL-1b-injured HEL 299 cells. Results for Fig. 10 and Supplementary  Table  S1 (https://www. biomedicinej.com/cgi/editor.cgi? article¼1378&window¼additional_files&context ¼biomedicine) showed a total of 213 genes were upregulated, and 118 genes were down-regulated. A network of the associations of different genes was generated following IPA analysis, as shown in Fig. 11, Which regulation of cytokines and immune cells (IL-15 production, Th1 and Th2 activation pathway, role of cytokines in immune cells, Th1 pathway, role of macrophages and fibroblasts, cytokine production in macrophages and T helper cells, differential regulation of cytokine production in IL-17, IL-10 signaling, role of IL-17F in allergic inflammatory airway diseases, IL-6 signaling; red lines), pulmonary related disease (airway pathology in chronic obstructive pulmonary disease, role of hypercytokinemia in the pathogenesis of influenza, wound healing signaling pathway; green lines), kinase (STAT3 pathway, p38 MAPK signaling; yellow lines), receptor (LXR/RXR activation, glucocorticoid receptor signaling; purple lines) and others (cardiac hypertrophy signaling, HMGB1 signaling, apelin cardiac fibroblast signaling pathway, atherosclerosis signaling, tumor microenvironment pathway, hepatic fibrosis, 3-phosphoinositide degradation, dmyo-inositol-5-phosphate metabolism, role of oct4 in mammalian embryonic stem cell pluripotency; blue lines) played main roles of NRICM101 on TNFa/IL-1b-injured HEL 299 cells. These results suggest the importance of the immune regulation of   Fig. 11, the ingenuity canonical pathways and major target proteins of NRICM101 were shown, including IL-1-mediated inflammatory signaling (Fig. 12A), cell movement of macrophages, cellular infiltration by macrophages (Fig. 12B) and Th1 and Th2 activation signaling (Fig. 13). Surprisingly, the results of NGS analysis demonstrated that IL-1A, IL-1B, TNFSF4, TNFSF18, IFI44L, IL32, and CCL2 genes were down-regulated of NRICM101 on TNF-a/IL-1b-injured HEL 299 cells (Supplementary  Table S1 (https://www.biomedicinej.com/cgi/editor. cgi?article¼1378&window¼additional_files&context ¼biomedicine)). In the present study, in vitro study results are not only support our in silico results but also suggest a protective role and immuneregulation function of the NRICM101 against normal human lung cell injury.
IL-15 is an immune-regulatory cytokine and plays an important role in anti-viral properties [48]. IL-15 is a T cell response to cytokine and is expressed in myeloid cells. IL-15 activates natural killer (NK) cells and then modulates inflammation when virus-infect host cells [48,49]. It has been reported that IL-15 expression increases innate immune responses through the induction of NK cells and CD 8þ T cells, and then decreased IL-4, IL-5, and IL-13. IL-15 inhibits viral replication, reduces viral loads and reduces SARS-CoV-2-induced inflammation and fibrosis [48e50]. Our in silico and in vitro studies showed that IL-15 production signaling is involved in TNF-a/IL-1b-induced normal human lung cell injury after NRICM101 treatment. Our results suggest that NRICM101 may affect T cell proliferation, activation, and NK cells activation. In future experiments, we will focus on IL-15 production signaling and design a series in vivo experiments of functional assays to include T cell proliferation, T cell activation, and NK cell activation and Th1, Th2 regulation.

Conclusion
Although most of the bioactive components of NRICM101 were selected for this study, the list of compounds in Table 1 was investigated to be not representative of all the chemical components in NRICM101. Finally, our study had several limitations in this study. All in silico and in vitro approaches require further in vivo and clinical experimental verification. Collectively, our results revealed the potential of NRICM101 as a therapeutic agent based on TCM that may confer beneficial effects on COVID-19 patients through multiple targeting and multiple mechanisms. Furthermore, the major bioactive compounds of NRICM101 are worthy of attention as striking candidates for anti-viral agent and protective agent discovery studies on COVID-19. It may be useful for further studies on the therapeutic properties of NRICM101 and its constituents on a molecular level, with the aim of findings that might contribute to improving care for COVID-19 patients.