CN118105495A - Method for enhancing broad-spectrum antiviral effect of bile acid medicine - Google Patents

Abstract

A method of enhancing the broad-spectrum antiviral effect of a bile acid drug comprising administering to a subject an FXR inhibitor or a pharmaceutical composition comprising an FXR inhibitor. The method can obviously enhance bile acid and biliary traditional Chinese medicine to inhibit RNA virus infection. The invention demonstrates that FXR can inhibit the antiviral effect of CDCA by inhibiting the transcription activity of IRF3, and the inhibitor of FXR can obviously enhance the effect of bile acid and bile traditional Chinese medicine on resisting RNA virus, thereby providing a new strategy and method for enhancing the clinical effect of bile acid and bile traditional Chinese medicine on inhibiting RNA virus infection and widening the application of bile acid and bile traditional Chinese medicine in clinical treatment.

Description

Method for enhancing broad-spectrum antiviral effect of bile acid medicine

Technical Field

The invention relates to the technical field of traditional Chinese medicine immunopharmacology, in particular to a method for enhancing broad-spectrum antiviral effect of a bile acid medicine.

Background

Bile Acids (BAs) are important metabolites in the human body, and are synthesized from cholesterol in the liver, and together with cholesterol, phospholipids and bilirubin constitute the major component of Bile (PMID: 18670431). Bile acids produced by the human body include primary bile acids (primary bile acid, PBA), i.e., cholic acid (cholic acid, CA) and chenodeoxycholic acid (chenodeoxycholic acid, CDCA), and corresponding secondary bile acids (RESPECTIVE SECONDARY BILE ACID, SBA), i.e., deoxycholic acid (deoxycholic acid, DCA) and lithocholic acid (lithocholic acid, LCA).

In recent years, bile acids have been widely studied by scientists as multifunctional signaling molecules. Bile acid receptors are largely classified into G protein-coupled receptors such as the wuta G protein receptor 5 (TGR 5, takeda G-protein receptor), and nuclear hormone receptors including the farnesol X receptor (FXR, farnesoid X receptor) (PMID: 18670431), the pregnane X receptor (PXR, pregnane X receptor) (PMID: 11248085), the constitutive androstane receptor (CAR, constitutive androstane receptor) (PMID: 11981033), and the vitamin D receptor (VDR, vitamin D receptor) (PMID: 12016314). By activating receptors and downstream signaling pathways, bile acids are involved in a variety of biological processes, particularly those associated with immune responses, effectively linking metabolism to immunity. The innate immunity of the gut to chikungunya virus infection requires the involvement of bile acids (PMID: 32668198). Specifically, LCA effectively inhibits replication of porcine Delta coronavirus in vitro by activating GPCR-interferon-ISG 15 signaling pathway (PMID: 33933854). With respect to the regulation of host cell antiviral activity, early studies showed that CDCA inhibited transcription of target genes in the interferon signaling pathway (PMID: 10347128). In terms of inflammation, bile acids effectively inhibit activation of NLRP3 inflammasome by activating the TGR5-cAMP-PKA signaling pathway (PMID: 27692610). The FXR agonist acetocholic acid (INT-747) can improve the function of the ileal barrier by reducing intestinal inflammation. Thus, bile acids can directly affect the cellular immune response.

Immune function of bile acids depends on the signaling pathway mediated by bile acids and their corresponding receptors, mainly TGR5 and FXR. TGR 5-perceived bile acids have been shown to be involved in the innate immune response of cells (PMID: 30651583). Inhibition of FXR with Z-Guggulsterone (GUGG) blocked HCV replication, indicating that FXR had an inhibitory effect on the antiviral response of hepatocytes (PMID: 18096266). A recent study has shown that ursodeoxycholic acid (ursodeoxycholic acid, UDCA), an FDA approved drug for the treatment of primary cholangitis, can reduce ACE2 expression by inhibiting the co-transcriptional activity of FXR and prevent SARS-CoV-2 invasion of cells (PMID: 36470304). This finding suggests a potential role for bile acid receptors in RNA viral infection and interferon signaling. However, there is very limited evidence concerning the specific role and detailed mechanism of FXR in RNA viral infection.

The biliary traditional Chinese medicine is a traditional Chinese medicine with long history in China, such as Bear Bile Powder (BBP), pig Bile Powder (PBP), cow Bile Powder (CBP) and the like, and the biliary powder traditional Chinese medicine for treating diseases is recorded in Chinese herbal medicine classical works such as materia medica outline, chinese great pharmacopoeia, materia medica channel concentrated injection, typhoid treatises and the like. The main active ingredient of the gall traditional Chinese medicine is bile acid, and has wide clinical foundation and research prospect in the aspect of treating diseases. However, the application of the biliary traditional Chinese medicine in clinical antivirus is very few, and the research of the main active ingredient bile acid thereof in antivirus is widely focused by people, so that people want to explore possible reasons for limiting the antiviral curative effect of the biliary traditional Chinese medicine.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention provides a method for enhancing the broad-spectrum antiviral effect of bile acid medicines, which solves the problems in the prior art. The invention combines the practical demands of the current society for virus infection epidemic, aims at important scientific problems (specificity of antiviral interferon signal channel 'brake') of molecular immunology and metabolism related virus infection, explores the deep mechanism of activated FXR for weakening bile acid/bile traditional Chinese medicine cell antiviral immune response, and fully considers the frontier of the subject research level. The research based on the cross regulation of bile acid receptor and natural antiviral immunity is very original in the research stage both internationally and domestically. Through the researches, in the aspect of basic theory, the understanding of human beings on the interaction deep molecular mechanism of bile acid receptor-virus infection is enhanced, the bile acid receptor is related with the cell antiviral reaction, the existing natural immunoregulation network theoretical knowledge is enriched, and a new theoretical basis is provided for further understanding the relationship between bile acid metabolism and virus infection; in the aspects of traditional Chinese medicine research and application, the feasibility of targeting FXR inhibition to enhance the antiviral activity of the biliary traditional Chinese medicine is explored, a solid theoretical support is provided for exploring the compatibility activity of the biliary traditional Chinese medicine prescription, and a new direction and thinking can be provided for the antiviral accurate treatment and medicine development of the traditional Chinese medicine.

The invention is realized by adopting the following technical scheme:

a method of enhancing the broad-spectrum antiviral effect of a bile acid drug comprising:

administering to the subject an FXR inhibitor or a pharmaceutical composition comprising an FXR inhibitor.

The principle of the invention is as follows:

The research shows that FXR can be used as negative regulator of interferon signal to reduce the effect of bile acid (CDCA/DCA) and biliary Chinese medicine in inhibiting RNA virus infection. Inhibition of FXR in modulating the interferon signaling pathway depends on its ability to inhibit DNA binding and transcriptional activity of IRF 3. FXR inhibitors or FXR gene deletions may reduce infection with various RNA viruses in hepatocytes. Thus, by administering an FXR inhibitor or a pharmaceutical composition comprising an FXR inhibitor to a subject, bile acids and biliary agents can be significantly enhanced to inhibit RNA viral infection.

Further, the FXR inhibitor is GUGG.

Among the various types of bile acids in the invention, UDCA is an approved clinical drug for the treatment of primary cholangitis and plays an important role in clinical treatment. Our studies focused mainly on the antiviral efficacy of CDCA, which has not been approved for clinical use. Therefore, we cannot verify the therapeutic effect of CDCA in clinic for the treatment of viral infectious diseases. However, we have performed detailed experiments at the animal level and found that GUGG in combination with CDCA has an anti-RNA viral effect superior to CDCA alone. Therefore, our research results suggest GUGG can be used as a potential drug to enhance the antiviral efficacy of bile acids.

Further, the pharmaceutical composition containing the FXR inhibitor comprises a pharmaceutical composition GUGG and bile acid CDCA, a pharmaceutical composition GUGG and bile acid DCA, a pharmaceutical composition GUGG and biliary BBP, a pharmaceutical composition GUGG and biliary PBP, and a pharmaceutical composition GUGG and biliary CBP.

Further, the FXR inhibitor or the pharmaceutical composition containing the FXR inhibitor is a drug for improving the curative effect of bile acid or biliary traditional Chinese medicine in resisting RNA viruses by inhibiting activation of FXR.

Further, the FXR inhibitor or the pharmaceutical composition containing the FXR inhibitor is a preparation prepared by taking the FXR inhibitor as an active ingredient and adding pharmaceutically acceptable auxiliary materials or auxiliary ingredients.

Further, the medicament is an oral preparation.

Further, the oral preparation is granule, solution, pill, paste or tablet.

Further, a suitable dosage of the oral formulation should be an amount of the compound effective to produce the lowest dosage of therapeutic effect, such as 0.00001mg/kg to 0.1mg/kg.

Compared with the prior art, the invention has the beneficial effects that:

1. The methods of the invention can significantly enhance bile acid and biliary traditional Chinese medicine inhibition of RNA viral infection by administering FXR inhibitors or pharmaceutical compositions comprising FXR inhibitors to a subject.

2. In the study of the invention, FXR can be used as a negative regulator of interferon signals, and the efficacy of bile acid (CDCA/DCA) and biliary traditional Chinese medicines in inhibiting RNA virus infection is reduced. Inhibition of FXR in modulating the interferon signaling pathway depends on its ability to inhibit DNA binding and transcriptional activity of IRF 3. FXR inhibitor GUGG or FXR gene deletion can reduce infection of various RNA viruses in hepatocytes. At the animal level, the use of FXR inhibitor GUGG in combination with CDCA is superior to CDCA alone in treating RNA viral infection. In summary, the present invention has been studied to demonstrate that FXR inhibits the antiviral effects of CDCA by inhibiting IRF3 transcriptional activity, GUGG as an inhibitor of FXR can significantly enhance the anti-RNA virus effects of bile acids and biliary traditional Chinese medicines. The research provides a new strategy and method for improving the clinical effect of enhancing bile acid and gall traditional Chinese medicine in inhibiting RNA virus infection, and widens the application of the enhanced bile acid and gall traditional Chinese medicine in clinical treatment.

3. The invention discloses the function of FXR as signal brake in inherent immunity, which is an inhibiting factor for weakening the antiviral effect of bile traditional Chinese medicines and bile acid, and provides FXR inhibitor GUGG which can be used as a compatible medicine for enhancing the antiviral effect of bile acid and bile traditional Chinese medicines, provides theoretical basis and pharmacological evidence for clinical application of bile acid and bile traditional Chinese medicines as antiviral medicines, provides firm theoretical support for exploring the compatibility activity of bile traditional Chinese medicines, and also provides new direction and thought for antiviral accurate treatment and medicine development of traditional Chinese medicines.

Drawings

FIG. 1 is a graph showing the results of detecting the positive rate of VSV-GFP in THP1 cells cultured in complete culture solution (CM) containing fetal bovine serum and basal culture solution (BM) without fetal bovine serum in example 1, infected with VSV-GFP, and treated with bear gall powder (BBP)/pig gall powder (PBP)/ox gall powder (CBP) for 12 hours;

FIG. 2 is a graph showing the results of the detection of the cytotoxic effect of BBP/PBP/CBP on THP1 cells by CCK8 in example 1;

FIG. 3 is a graph showing the results of qPCR analysis of mRNA expression of VSV, IFNB1 and ISG56 of THP1 cells infected with CM/BM for 12h and treated with different types of bile acids (40. Mu.M) in example 1;

FIG. 4 is a graph showing the results of detecting the positive rate of VSV-GFP in THP1 cells by flow cytometry, wherein the THP1 cells in CM/BM were infected with VSV-GFP for 12h and treated with DCA/CDCA at various concentrations for 12h in example 1;

FIG. 5 is a graph showing the results of qPCR detection of mRNA expression of VSV and NR0B2 in THP1 cells infected with CM/BM for 12h and treated with DCA (40. Mu.M)/CDCA (40. Mu.M) in example 1;

FIG. 6 is a graph showing the results of qPCR detection of mRNA expression of FXR in hepatocytes and immune cells in example 2;

FIG. 7 is a graph showing the results of WB detection of protein expression of FXR in hepatocytes and immunocytes in example 2;

FIG. 8 is a graph of the results of CCK8 in example 2 for detecting the cytotoxic effect of DCA/CDCA on AML12 cells;

FIG. 9 is a graph showing the results of qPCR analysis of VSV mRNA expression by treating AML12 cells cultured in CM/BM for 12h with DCA/CDCA (40. Mu.M) in example 2;

FIG. 10 is a graph showing the results of qPCR analysis mIfnb and mIsg of mRNA expression of AML12 cells cultured in CM/BM in example 2 infected with VSV 12h, treated with DCA/CDCA (40 μM);

FIG. 11 is a graph showing the results of qPCR analysis mNr b2 mRNA expression from AML12 cells cultured in CM/BM in example 2 infected with VSV 12h, treated with DCA/CDCA (40. Mu.M);

FIG. 12 is a graph showing the results of qPCR analysis of mRNA expression of mIfnb and mNr b2 of AML12 cells transfected with Poly (I: C) for 6h and then treated with GW4064 (10. Mu.M) in example 2;

FIG. 13 is a graph showing the results of analysis of the positive rate of VSV-GFP by flow cytometry, obtained by infecting Huh7 cells with VSV-GFP for 16h and treating with GUGG (10. Mu.M) in example 3;

FIG. 14 is a graph showing the results of a flow cytometer analyzing the positive rate of VSV-GFP in example 3 in which Huh7 cells were infected for 16h with VSV-GFP and treated with GUGG at no concentration;

FIG. 15 is a graph showing the results of qPCR detection of mRNA expression of EMCV, H1N1 and SEV or NR0B2 in example 3 when Huh7 cells were infected for 12H with or without GUGG treatment;

FIG. 16 is a graph showing the fluorescence amount of VSV-GFP obtained by subjecting FXR-siRNA Huh7 cells to 12h after VSV-GFP infection in example 3, wherein the ratio is: 100 μm;

FIG. 17 is a graph showing the results of flow cytometry analysis of the positive rate of VSV-GFP after 12h of VSV-GFP infection of FXR-siRNA Huh7 cells in example 3;

FIG. 18 is a graph showing the results of flow cytometry analysis of the positive rate of VSV-GFP after 12h of VSV-GFP infection of FXR-siRNA Huh7 cells in example 3;

FIG. 19 is a graph showing the results of qPCR analysis of mRNA expression of H1N1/EMCV and FXR after 12H infection of FXR-siRNA Huh7 cells in example 3;

FIG. 20 is a graph showing the results of qPCR analysis of the expression of IFNB1 and FXR genes in Huh7 cells before and after VSV infection in example 4, and the detection of protein levels of FXR in Huh7 cells before and after VSV infection by WB;

FIG. 21 is a graph showing the results of qPCR analysis of mRNA expression of VSV and IFNB1 in FXR-KO Huh7 cells before and after 12h of VSV infection and WB detection of protein levels of FXR in FXR-KO Huh7 cells before and after 12h of VSV infection in example 4;

FIG. 22 is a graph showing the results of qPCR analysis of the mRNA expression of IFNB1 in FXR-siRNA Huh7 cells 12H before and after H1N1/EMCV infection in example 4;

FIG. 23 is a graph showing the results of qPCR detection of mRNA expression of IFNB1 in Huh7 cells before and after 12H and GUGG (10/20. Mu.M) treatment of H1N1 virus infection in example 4;

FIG. 24 is a graph showing the effect of dual luciferase reporter gene assay of example 4 on IFNB1/ISRE transcriptional activity of RAD-I CARD, MAVS, TBK1 or IRF3 after treatment with over-expressed FXR and GUGG;

FIG. 25 is a graph showing the results of WB assay of protein levels of P-TBK1 and P-IFR3 before and after 12h infection of FXR-siRNA Huh7 cells by VSV in example 4;

FIG. 26 is a graph showing the results of WB assay of protein levels of cytoplasmic and nuclear IFR3 before and after 12h infection of FXR-siRNA Huh7 cells by VSV in example 4;

FIG. 27 is a graph showing the results of Co-IP assay of FXR interaction with innate immunity molecules following Co-transfection of HEL293T cells into different expression vectors as described in example 5;

FIG. 28 is a graph showing the results of Co-IP assay of interaction between FXR and IRF3 after 12H of infection of Huh7 cells transfected with Flag-FXR and HA-IRF3 with VSV/H1N1 in example 5;

FIG. 29 is a graph showing the results of detection of endogenous interactions of FXR and IRF3 after WB detection of Huh7 cells infected with VSV virus for 12h and treatment with GUGG (10. Mu.M) in example 5;

FIG. 30 is a graph showing the results of confocal immunofluorescence observation of the co-localization of FXR and IRF3 after 12h of VSV infection of Huh7 cells transfected with IRF3-mCherry plasmid 24h in example 5, followed by fixation and FXR staining, in which the scale bar: 10 μm;

FIG. 31 is a graph showing the results of Co-transfection of HA-FXR with Flag-IRF3 deletion mutants in HEK293T cells, IRF3 deletion protein domain structure as schematically shown, and Co-IP detection of FXR interaction with IRF3 deletion mutants in example 5;

FIG. 32 is a graph showing the results of the ChIP assay to detect the binding of IRF3 to the mIfnb gene promoter after the mFxr-siRNA AML12 cells of example 5 are infected with VSV;

FIG. 33 is a graph showing the results of qPCR analysis of mRNA expression of VSV and mIfnb1 in mIrf-KO AML12 cells 12h after VSV infection in example 5;

FIG. 34 is a graph showing the results of immunofluorescence detection of the fluorescence intensity of VSV-GFP in example 6 after GUGG (10. Mu.M)/CDCA (40. Mu.M) treatment of AML12 cells infected with VSV-GFP 12 h;

FIG. 35 is a graph showing the results of WB detection of GFP levels in GUGG (10. Mu.M)/CDCA (40. Mu.M)/DCA (40. Mu.M) treated AML12 cells infected with VSV-GFP 12h in example 6;

FIG. 36 is a graph showing the results of qPCR analysis of the mRNA levels of VSV/H1N1/EMCV after GUGG (10. Mu.M)/CDCA (40. Mu.M) treatment of AML12 cells infected with VSV/H1N1/EMCV 12H in example 6;

FIG. 37 is a graph showing the results of qPCR analysis of mRNA expression of mFxr from mFxr-KD AML12 cells infected with VSV 12h treated with CDCA (40. Mu.M) in example 6;

FIG. 38 is a graph showing the results of qPCR analysis of mRNA expression of VSV/H1N1/EMCV and mIfnb1 after CDCA (40. Mu.M) treatment of mFxr-KD AML12 cells infected with VSV 12H in example 6;

Fig. 39 is a graph showing the results of monitoring survival rate (n=6) of mice infected with VSV virus in example 6 after GUGG/CDCA treatment;

fig. 40 is a graph of the results of H & E staining of mouse lung sections in example 6, wherein scale = 100 μm;

FIG. 41 is a graph showing the results of qPCR analysis of mRNA levels of EMCV/mIfnb1/mIsg after GUGG (10. Mu.M)/BBP (50. Mu.M) treatment of AML12 cells infected with EMCV 12h in example 7;

FIG. 42 is a graph showing the results of qPCR analysis of mRNA levels of H1N1/mIfnb1/mIsg56 in AML12 cells infected with H1N 1H in example 7 after GUGG (10. Mu.M)/BBP (50. Mu.M) treatment;

FIG. 43 is a schematic diagram showing the mechanism of GUGG in enhancing the antiviral activity of biliary traditional Chinese medicine (BBP) in example 7.

Detailed Description

The present invention will be further described with reference to the following specific embodiments, and it should be noted that, on the premise of no conflict, new embodiments may be formed by any combination of the embodiments or technical features described below.

EXAMPLE 1 study of serum supplementation to impair the antiviral Effect of biliary traditional Chinese medicine/bile acid

The gall traditional Chinese medicine is a traditional Chinese medicine taking bear gall powder (BBP), pig gall powder (PBP), ox gall powder (CBP) and the like as main active ingredients. These traditional biliary agents have a variety of pharmacological actions, such as improving cholestasis, inhibiting neuroinflammation, improving type 2 diabetes and alleviating liver fibrosis. Previous reports suggest that bile acids can inhibit viral infection and enhance IFNB1 expression by activating TGR5 bile acid receptor, and that viral infection can promote absorption of bile acids in serum by cells. Therefore, the antiviral effect of the biliary traditional Chinese medicine is studied in the embodiment. To exclude the effect of serum bile acids on host cell antiviral, we studied with Complete Medium (CM) containing Fetal Bovine Serum (FBS) and Basal Medium (BM) without FBS, the experiments are shown in fig. 1 and 2.

The results showed that BBP can significantly inhibit viral infection at high concentrations (100 μm) under BM culture conditions compared to CM culture conditions (see fig. 1). While the three biliary drugs have little cytotoxicity (< 500 μm) to THP1 cells (see fig. 2). These results indicate that removal of serum can enhance the antiviral efficacy of BBP. Under CM conditions, BBP has a weaker antiviral effect, suggesting that bile acids in BBP hinder the antiviral effect of BBP.

Next, in this example, screening experiments were performed on bile acid as an active ingredient in BBP, as shown in fig. 3 to 5.

The results showed that 40. Mu.M DCA/CDCA promoted IFNB1 expression and inhibited viral replication under BM culture conditions (FIG. 3). In CM, DCA/CDCA was found to be effective at 80. Mu.M (FIG. 4) to inhibit viral replication. In BM-cultured cells, CDCA (40. Mu.M) had a reduced effective antiviral concentration (FIG. 4). Thus, the observed differences in BBP antiviral effects in CM/BM-cultured cells may be due to differences in antiviral effects of DCA/CDCA under CM/BM conditions, suggesting that the antiviral effects of DCA/CDCA may be antagonized by serum components added to the culture medium. Since FXR inhibitor GUGG is resistant to the HCV virus, we next examined the activity of DCA/CDCA receptor FXR. FXR, as a transcription factor, can induce the expression of its target gene (e.g., NR0B 2). NR0B2 is a typical marker for FXR activation. As a result, it was found that the expression of NR0B2 was enhanced after DCA/CDCA treatment in CM-cultured THP1 cells, whereas the expression of NR0B2 was not increased in BM (see FIG. 5). Thus, we speculate that activation of FXR may be responsible for the impaired antiviral effects of DCA/CDCA.

EXAMPLE 2 investigation of FXR expression Difference in hepatocytes and immune cells

Whereas the difference in cellular FXR activity under conditions of Complete Medium (CM) containing FBS and Basal Medium (BM) without FBS may be a factor affecting the antiviral effect of CDCA, the present example examined the expression of FXR in hepatocytes and immune cells, and experiments as shown in FIGS. 6 and 7 were performed.

The results showed that the mRNA expression level of FXR was higher in human hepatocytes (Hep 3B, huh and HepG 2) compared to human immune cells (THP 1, LX 2). The same experimental results were also found in mouse cells, i.e. higher expression in AML12 cells (mouse hepatocytes) but lower expression levels in J774 or iBMDM cells (see fig. 6). The same results were observed for WB experiments (see fig. 7). Thus, these results indicate that FXR expression in hepatocytes is significantly higher than in immune cells.

Next, the present example examined the cytotoxicity of DCA/CDCA on hepatocytes, and the results are shown in fig. 8 to 12.

The results showed that DCA/CDCA was almost non-toxic to AML12 cells at a concentration of 100 μm (fig. 8). Due to the safety of DCA/CDCA against hepatocytes, we examined the antiviral effects of DCA/CDCA in BM/CM. After 12h of VSV infection with AML12 cells, low concentration (40 μm) of DCA/CDCA failed to inhibit VSV replication in CM, but under BM conditions, the effect of DCA/CDCA at 40 μm concentration was very pronounced against VSV (fig. 9). Furthermore, under BM conditions, CDCA/DCA at a concentration of 40 μm promoted activation of interferon signals, i.e. enhanced transcriptional activity of mIfnb and mIsg genes in AML12 cells (fig. 10), which we speculated to be due to differences in FXR activation in AML12 cells under CM/BM conditions. Consistent with our hypothesis, CDCA/DCA increased the transcriptional activity of mNr b2 (a marker of FXR transcriptional activity) in CM-cultured AML12 cells, but had no significant effect under BM conditions (fig. 11). FXR agonist GW4064 significantly inhibited Poly (I: C) -induced transcription of mIfnb1 gene, mIfnb1 gene being a marker of antiviral interferon signaling activation, suggesting that FXR has a negative regulatory effect in cellular antiviral response (fig. 12).

Taken together, these results suggest that FXR may inhibit the antiviral effects of bile acids.

EXAMPLE 3 investigation that inhibition of FXR reduces viral infection in hepatocytes

To investigate whether FXR impairs the role of bile acids in antiviral innate immunity, the present example used the FXR inhibitor Z-Guggulsterone (GUGG) to investigate the role of FXR in cellular viral infection. Previous studies have demonstrated GUGG to inhibit hepatitis c virus replication. Thus, the present example examined GUGG for antiviral ability, and a study was performed as shown in FIGS. 13-19.

The results show that GUGG treatment significantly reduced the infection rate of Huh7 cells VSV (fig. 13), indicating that inhibition of FXR can enhance antiviral capacity of the cells. Furthermore, GUGG blocked VSV infection in a dose-dependent manner (fig. 14), while significantly inhibiting EMCV, H1N1 or SeV infection and inhibiting FXR activity in Huh7 cells (fig. 15), suggesting that GUGG has a broad spectrum of effects of inhibiting RNA viral infection.

To demonstrate the role of FXR in cellular antiviral responses, this example uses small interfering RNAs (sirnas) to reduce FXR expression. The fluorescence results showed that the VSV positive rate was significantly reduced in FXR-siRNA Huh7 cells (fig. 16). Flow cytometry detection found a decrease in the infection rate of VSV in FXR-siRNA Huh7 cells (FIGS. 17-18). Meanwhile, qPCR results showed that EMCV and H1N1 virus replication was reduced in FXR-silenced Huh7 cells (fig. 19). Taken together, these findings reveal that targeted inhibition of FXR may promote antiviral innate immune responses, but the mechanism of which remains unclear.

EXAMPLE 4 investigation of FXR downregulated interferon signaling pathways

The interferon signaling pathway is critical for the antiviral response of cells and is considered as a major target for the virus to evade the cell's innate immunity. Therefore, this example next investigated whether negative regulation of FXR in cellular antiviral response is associated with the interferon signaling pathway, and experiments were performed as shown in FIGS. 20-26.

The results show that this example first examined the dynamic activity level of FXR during the viral infection phase, as GUGG was shown to enhance the antiviral response of the cells in example 3 (fig. 15). mRNA expression levels of FXR increased significantly in Huh7 cells at early stages (< 12 h) of VSV infection, but decreased in late stages of VSV infection (fig. 20).

To determine the role of FXR in antiviral immune response and interferon signaling, this example constructed FXR knockout Huh7 cell lines using the CRISPR/Cas9 system. Replication of VSV was significantly reduced in FXR gene deficient Huh7 cells compared to control cells. The FXR gene knockout significantly enhanced IFNB1 expression in VSV infected cells (fig. 21). Similarly, FXR gene knockout also upregulated mRNA expression of IFNB1 in H1N 1/EMCV-infected cells (fig. 22). In addition, GUGG treated Huh7 cells significantly up-regulated mRNA expression of IFNB1 after infection with a variety of RNA viruses including EMCV, H1N1, and SEV (fig. 23). Thus, the results indicate that FXR may promote viral replication by compromising the interferon signaling pathway.

In addition, this example also discusses the detailed mechanism by which FXR inhibits the interferon signaling pathway under viral infection conditions. The results show that: RIG-I CARD, MAVS, TBK1 and IRF3 overexpression significantly inhibited the activation of both IFNB 1-luciferase and ISRE-luciferase by FXR overexpression, but was complemented by GUGG treatment (FIG. 24). The interferon signaling pathway is derived from RIG-I, which transmits signals through its CARD domain to MAVS, and MAVS-TBK1-IRF3. Given the inhibitory activity of FXR on IRF 3-initiated IFNB 1-luciferase, it can be inferred that FXR inhibits interferon signaling by targeting IRF3. WB results indicate that FXR gene knockout did not affect phosphorylation of TBK1 and IRF3 (fig. 25), suggesting that FXR acts on the interferon signaling pathway at a later stage following IRF3 activation. FXR deletion also did not affect IRF3 entry into the nucleus (fig. 26). Thus, the results indicate that FXR has a negative regulatory effect on the activation of the interferon signaling pathway under RNA virus infection after IRF3 enters the nucleus.

EXAMPLE 5 investigation of FXR inhibition of the transduction of the interferon signalling pathway by impairing IRF3 transcriptional Activity

Considering the negative regulation of FXR in the interferon signaling pathway, this example studied the molecular mechanism by which FXR inhibits activation of interferon signaling and conducted the study experiments as shown in FIGS. 27-33.

The above screening experiments for interactions of FXR with the interferon signaling pathway indicate that there is a strong interaction between FXR and the transcription factor IRF3/STAT1 (FIG. 27), and that VSV/H1N1 infection enhances the interaction of FXR and IRF3 (FIG. 28). Under VSV infection conditions GUGG inhibited the endogenous interaction of FXR with IRF3 (fig. 29), indicating that FXR interaction with IRF3 occurred under post-viral infection conditions. Confocal imaging also showed significant co-localization of FXR and IRF3 within the nucleus following VSV infection (figure 30). These results indicate that the interaction of FXR and IRF3 may be a key mechanism for FXR to modulate interferon signaling following viral infection.

To explore the specific mechanism of interaction between FXR and IRF3, this example constructed IRF3 site mutants or domain deletion mutants. IRF3-5D is a mutant in which 5 phosphorylated serine sites of the IRF3 regulatory domain are mutated to aspartic acid to mimic the phosphorylation activation of IRF 3. The results indicate that the interaction of phosphorylated IRF3 with FXR is increased. This example also found that DP and DNI deletion mutants resulted in reduced interaction of FXR and IRF3, indicating that the RD domain of IRF3 is necessary for FXR-IRF3 binding (fig. 31), suggesting that the regulatory domain of IRF3 is critical for the interaction between FXR and IRF 3. These results indicate that the interaction of FXR with intranuclear IRF3 is dependent on activation and phosphorylation of IRF3 regulatory domains, suggesting that FXR may directly affect IRF3 transcriptional activity in the nucleus. Thus, this example next examined whether the genomic DNA binding of IRF3 is under FXR control. ChIP experiments showed that GUGG-mediated inhibition of FXR or silencing of FXR gene could enhance FXR binding to FXR target mIfnb gene promoter in VSV infected cells (fig. 32). Furthermore, FXR deletion did not inhibit VSV replication nor enhance mRNA expression of mIfnb1 in mIrf-KO AML12 cells (fig. 33). Taken together, these results indicate that FXR-IRF3 interactions impair IRF3 transcriptional activity, thereby impeding the activation of interferon signaling pathways and antiviral responses of cells.

EXAMPLE 6 investigation of inhibiting FXR to enhance antiviral Capacity of CDCA

Since inhibition of FXR blocks viral replication and promotes interferon signaling, this example next investigated whether FXR impaired bile acid efficiency in cellular antiviral immunity, and experiments were performed as shown in fig. 34-40.

As a result, it was found that low concentration (40 μm) of CDCA alone did not inhibit VSV infection in cells, but GUGG in combination with CDCA/DCA significantly inhibited VSV infection in cells (fig. 34-35). At the same time, the combined use of GUGG and CDCA also inhibited replication of several RNA viruses, including VSV, H1N1, and EMCV (FIG. 36). Furthermore, FXR gene knockout was used to determine the role of FXR deletion in CDCA antiviral activity (fig. 37). The results of the study indicate that FXR deletion enhances the antiviral effect of CDCA under VSV, EMCV or H1N1 infection, while enhancing the mRNA expression of mIfnb a (fig. 38).

Finally, this example examined the effect of the combination of FXR inhibitor GUGG and CDCA on VSV-infected mice, and found that GUGG-CDCA combination significantly prolonged the survival of VSV-infected mice (FIG. 39). Whereas H & E staining of mouse lung tissue also showed that GUGG and CDCA combined application reduced pneumonic lesions and inflammatory cell infiltration in VSV infected mice (fig. 40). Combining these results, this example demonstrates that FXR inhibition can enhance antiviral activity in CDCA and biliary traditional Chinese medicine. Therefore, targeted inhibition of FXR may provide a new idea for enhancing related applications of bile acid and biliary traditional Chinese medicine antiviral.

EXAMPLE 7GUGG study of enhancing antiviral efficacy of biliary traditional Chinese medicine (BBP)

In order to further study GUGG the antiviral efficacy of the bile-based traditional Chinese medicine (BBP), a study experiment as shown in fig. 41-43 was performed, and the results show that: GUGG can also improve antiviral efficiency of biliary traditional Chinese medicine such as BBP (see figure 41-figure 43).

In the above experiments, the data are mean ± standard deviation. * P <0.05, P <0.01, P <0.001, ns = no statistical significance.

The above embodiments are only preferred embodiments of the present invention, and the scope of the present invention is not limited thereto, but any insubstantial changes and substitutions made by those skilled in the art on the basis of the present invention are intended to be within the scope of the present invention as claimed.

Claims (8)

1. A method of enhancing the broad-spectrum antiviral effect of a bile acid drug comprising:

administering to the subject an FXR inhibitor or a pharmaceutical composition comprising an FXR inhibitor.

2. The method of enhancing the broad-spectrum antiviral effect of a bile acid drug of claim 1, wherein said FXR inhibitor is GUGG.

3. The method of claim 1, wherein the pharmaceutical composition comprising FXR inhibitor comprises GUGG pharmaceutical composition of bile acid CDCA, GUGG pharmaceutical composition of bile acid DCA, GUGG pharmaceutical composition of bile medicament BBP, GUGG pharmaceutical composition of bile medicament PBP, and GUGG pharmaceutical composition of bile medicament CBP.

4. The method for enhancing the broad-spectrum antiviral effect of a bile acid drug according to claim 1, wherein said FXR inhibitor or a pharmaceutical composition comprising the FXR inhibitor is a drug for enhancing the therapeutic effect of a bile acid or a bile traditional Chinese medicine in an anti-RNA virus by inhibiting activation of FXR.

5. The method for enhancing a broad-spectrum antiviral effect of a bile acid drug according to claim 1, wherein the FXR inhibitor or the pharmaceutical composition containing the FXR inhibitor is a preparation prepared by adding pharmaceutically acceptable auxiliary materials or auxiliary components to the active ingredient of the FXR inhibitor.

6. The method of enhancing the broad-spectrum antiviral effect of a bile acid drug as claimed in claim 5, wherein said drug is an oral formulation.

7. The method of enhancing the broad-spectrum antiviral effect of a bile acid drug as claimed in claim 6, wherein said oral formulation is a granule, a solution, a pill, a paste or a tablet.

8. The method of enhancing the broad antiviral effect of a bile acid drug according to claim 6, wherein the suitable dose of said oral formulation is an amount of the compound effective to produce the lowest dose of therapeutic effect.