Synergistic Therapeutic Effects and Immunoregulatory Mechanism of Maxing Shigan Decoction Combined with Sijunzi Decoction on Viral Pneumonia in Mice

Influenza is defined in traditional Chinese medicine (TCM) as an epidemic febrile illness and is usually treated with herbal compound formulas under the guidance of the “Qu Xie and Fu Zheng” theories. Ma Xing Shi Gan Tang (MXSGD) is a prominent remedy for clearing heat and detoxifying toxins in the clinical treatment of influenza in TCM, playing the role of “Qu Xie.” Si Jun Zi Tang (SJZD) is recognized as one of the “Fu Zheng” formulas for strengthening the spleen and nourishing the stomach, with immunomodulatory effects. In this study, we followed the principles of “Qu Xie and Fu Zheng” to explore the effects of MXSGD combined with SJZD on viral pneumonia and its mechanism. Results showed that the couse of MXSGD and SJZD was effective in reducing the mortality rates and severity of lung pathology in lethally infected FM1 mice compared to the use of either drug alone. Moreover, further research demonstrated that the combined use suppressed TLRs and NLRP3 inflammatory signaling pathways at 4 dpi while promoting them at 7 dpi. At 10 dpi, there was a significant increase in CD11c+ and CD103+ DCs in the lungs. Together, SJZD improved the therapeutic effectiveness of MXSGD in treating influenza virus pneumonia than when used alone. MXSGD and SJZD exhibit synergistic effects in the treatment of influenza, as evidenced by the inhibition of TLR7 and NLRP3 inflammatory pathways early in the infection and facilitation of the response later. They also increase CD11c+ and CD103+ DC levels, as well as balancing Th1/Th2 cytokines.


Introduction
Severe viral infections frequently lead to serious complications or death from severe viral pneumonia.Viral pneumonia has been well described in immunocompromised patients with immune dysfunction, suggesting that the host immune plays a crucial role in the development of viral pneumonia [1].Recent evidence showed that excessive infammation, labeled as cytokine storm, as well as insufcient antiviral immunity, such as lymphopenia, coexisted in severe infuenza A viruses (IAV) infection, and thus, a balanced immune response is required for a good clinical outcome [2,3].Resistance and side efects to conventional treatment drugs, including antiviral drugs such as oseltamivir, make the treatment of viral pneumonia a challenge [4].Meanwhile, antiviral therapy for infuenza viruses alone is less efective in treating the immune dysregulation caused by severe viral infection [5].Studies are also working on immunomodulators to reduce viral-mediated infammation fame and restore host immunity to balance [6].
Immune dysregulation is the main cause of lung injury in viral pneumonia.Te coexistence of an excessive infammatory response (infammatory cytokine storm) and repressed antiviral immune response (e.g., delayed interferon (IFN) production or inactivated T-cell response) can exacerbate infammation and promote virus replication leading to respiratory failure.In addition, the virus has evolved various strategies to interfere with the antiviral response in the host contributes to the poor outcome.In traditional Chinese medicine (TCM), infuenza is one of the epidemic febrile diseases that were treated under the guidance of "Quxie and Fuzheng."From the TCM point of view, "Quxie" refers to the elimination of pathogens and removal of toxins and the "Fuzheng" is interpreted as a therapy to strengthen immunity and restore damaged immunity.Te roles of the two are mutually reinforcing.
Maxing Shigan decoction (MXSGD) is a classic TCM formula composed of Ephedrae Herba (Ephedra sinica Stapf ), Armeniacae Semen Amarum (Prunus armeniaca L. var.anus Maxim.),Gypsum Fibrosum, and Glycyrrhizae Radix Et Rhizoma (Glycyrrhiza uralensis Fisch.).It is a representative formula in the therapeutic strategy of traditional Chinese medicine (TCM) for the treatment of COVID-19 infection and community-acquired pneumonia (CAP) [7][8][9][10].Tus far, several drugs, including MXSGD, have demonstrated efectiveness in fghting infuenza [11] and widely applied in the treatment for viral pneumonia to decrease body temperature, ameliorate headache, cough, and pharyngula [12][13][14].It plays the function of heat clearing and exterior releasing, which is consistent with the therapeutic principle and method of "Quxie" in TCM.According to the protocol for diagnosis and treatment of infuenza (2018), MXSGD is recommended as one of the TCM treatment strategies for infuenza treatment [15].MXSGD showed obvious anti-infammatory efects, improved clinical symptoms, and increased the total efective rate in the patients with pneumonia.It also inhibited the invasion of pathogenic bacteria such as Mycoplasma pneumoniae or Staphylococcus aureus to protect against worsening infammation [10,[16][17][18].Animal evidence suggests that MXSGD protects LPS-induced pulmonary microvascular hyperpermeability and infammatory response in rat models by regulating Toll-like receptors-4 (TLR4), Src, and nuclear factor kappa-B (NF-κB).During viral entry, MXSGD targeted viral entry by disrupting the viral surface, inhibited IAV surface glycoprotein neuraminidase (NA) activity, and regulated phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling against the infection [19,20].
Host utilizes various mechanisms to combat viruses, while both intrinsic viral pathogenicity and an excessive host innate immune response contribute to the infuenzamediated damage of the lungs [24].Tus, the balance between tissue tolerance and immune resistance is critical to viral challenge.Toll-like receptor-7 (TLR7) signaling has stimulated the expression of proinfammatory cytokines and type I interferons (IFNs) against the virus by activating NF-κB and interferon-regulatory factor 7 (IRF7) via the adaptor myeloid diferentiation primary response gene 88 (MyD88) [25].NOD-like receptor thermal protein domain-associated protein 3 (NLRP3) infammasome induced the proteolytic processes of pro-interleukin (IL)-18 and pro-IL-1β and protected mice against infuenza virus via triggering pyroptosis and IL-1β-mediated neutrophil recruitment [26][27][28].In adaptive immunity, CD4 + T cells and CD8 + T cells play an important role in the immune response and virus clearance.After infection with infuenza virus (A/ Puerto Rico/8/1934 H1N1, PR8), the levels of CD4 + T and CD8 + T cells were decreased [29].Te migration of CD11c + DC cells from lungs to mediastinal lymph nodes was reduced, and the level of CD103 + DC cells was lower than that in the control group.Te balance of cytokines derived from T helper 1 (T1) cells and T helper 2 (T2) cells also plays an important role in maintaining moderate immunoreactions [30].Liu et al. described that the contents of tumor necrosis factor α (TNF-α) and interferon-c (IFN-c) were obviously upregulated, while the level of IL-4 was not signifcantly changed in infuenza virus H1N1 (A/FM/1, FM1)-infected mice and the pathological process of FM1 infection might be correlated with excessive infammation mediated by proinfammatory cytokines [31].
Te fu or fu-like illness is often accompanied by cough, runny nose, sore throat, headache, fever, diarrhea, muscle or joint pain, or fatigue.When infected, mice show loss of appetite, inactivity, rufed fur, a hunched posture, hypothermia, disturbance of the intestinal fora, and respiratory distress, which is consistent with the syndrome of qi defciency in TCM and should adopt the treatment strategy of "Fuzheng."Sijunzi decoction (SJZD) is one of the most accepted "Fuzheng" formulae for strengthening and nourishing the spleen.Terefore, we attempted to combine MXSGD and SJZD based on TCM theories to investigate whether their conjunction could enhance the efcacy.Terefore, we aimed to investigate how MXSGD combined with SJZD would afect efcacy in FM1-infected mice.Te physical sign changes and lung tissue damage conditions in the mice were evaluated.Te pulmonary mRNA and protein levels of TLRs and NLRP3 infammasome signal pathways were determined.Te number of T cells and DCs cells and the cytokine levels of IL-18, IL-1β, TNF-α, IFN-c, IL-4, and IL-10 were measured.Tese results would provide evidence for applying the theory of "Quxie" and "Fuzheng" in the treatment of viral pneumonia, and clarify the mechanism of the anti-infammation and immunoregulatory efect of MXSGD combined with SJZD.To discuss the application of TCM theory in drug use and treatment is of great signifcance to clinical diagnosis and rational treatment.

Preparation of MXSGD and SJZD.
All herbs were provided by Beijing Tongrentang Pharmaceutical Co., Ltd., China.For the preparation of MXSGD, Armeniacae Semen Amarum (22.5 g), Gypsum Fibrosum (60 g), and Glycyrrhizae Radix Et Rhizoma (15 g) were soaked in distilled water for 1 h and boiled for 15 min (100 °C); then, Ephedrae Herba (15 g) was added and boiled for 5 min; and then, the residue was fltered and re-extracted.Te fltrate was concentrated to 1.5 g/ml.For the preparation of SJZD, Codonopsis Radix (25 g), Atractylodis Macrocephalae Rhizoma (25 g), Glycyrrhizae Radix Et Rhizoma Praeparata Cum Melle (25 g), and Poria (25 g) were extracted twice at 100 °C for 20 min and then concentrated to 1.5 g/ml.Teir combination was prepared by mixing 30 ml MXSGD and SJZD 30 ml.Te mixture was then concentrated to 30 ml.Decoction samples were dried by rotary evaporator and stored in a cool and dark place.Te detail information of the herbs is shown in Table 1.

Animals and Viruses.
Six-to eight-week-old specifc pathogen free (SPF) male BALB/c mice (16-20 g) were purchased from Beijing Vital River Laboratory Animal Technology Company (SCXK20070001, Beijing, China) and housed in an SPF laboratory animal room with 12-hr light/dark cycle.All animal treatment and experiments were overseen and approved by the experimental animal ethics committee of the Beijing University of Chinese Medicine (Permit number: BUCM-4-2021030701-1102) and Liuzhou people's hospital (Permit number: LRYIA-CUC2024015).To euthanize animals, CO 2 inhalation was used at the end of the experiment before cervical dislocation.
To evaluate the antivirus therapeutic efcacy, 85 mice were randomly divided into six groups (n � 15 per group, except normal control group): normal control group (NC, n � 10), FM1 group, MXSGD group, SJZD group, MXSGD + SJZD group, and oseltamivir group.Mice were challenged with a lethal dose of FM1, and the survival rate, animal bodyweight change, temperature, etc., were recorded and lung tissues were collected.
For mechanism research, 90 mice were randomly divided into six groups (n � 15 per group): the abovementioned group setting.Mice were challenged with virus (a dose of LD 50 ), and qPCR analysis of mRNA extracted from mouse lung tissue (n � 5 mice/group) was conducted at 4 and 7 dpi.Te fow cytometric analysis was performed in 7 and 10 dpi.
Te FM1 virus strain was provided by the Institute of Virology, Chinese Academy of Preventive Medicine (Beijing, China), and kindly donated by Prof. Cui Xiaolan from the Institute of Chinese Materia Medica China Academy of Chinese Medical Sciences (Beijing, China).Te virus was propagated in the allantoic cavities of 9-day-old SPF embryonic chicken eggs at 37 °C for 48 h followed by preservation at −20 °C for 1 h and transfer at 4 °C overnight.Te virus-containing allantoic fuid was harvested [32].Te LD 50 of FM1 in mice determined by the Reed-Muench method was calculated to be 10 −4.4 /0.02 ml.Virus stocks were collected and stored at −80 °C.

Survival Rate Analysis.
Mice were anesthetized and inoculated intranasally with 25 ul of virus solution (lethal dose of FM1 strain).Te survival rates of all mice were evaluated, and the weight changes, body temperature, clinical signs of mice, and the number of deaths were recorded.

Lung Histology.
Lungs from mice uninfected or infected with FM1 were dissected, fxed in 10% phosphate-bufered formalin, embedded in parafn, sectioned, stained with hematoxylin and eosin, and then observed by light microscopy to observe morphologic changes.

Establishment of FM1 Pneumonia Mouse Model.
Te mice were infected intranasally with LD 50 of FM1.Infection mice were randomly divided into four groups: MXSGD, SJZD, MXSGD + SJZD, and oseltamivir (2.5 mg/ml, 0.9% NaCl), which was given twice a day through the intragastrical at 1 h after the virus infection following the treatment schedule for each group.

Western Blot (WB) Assay. Te expression of p-NF-κB
(#3031, Cell Signaling Technology) and cysteinyl aspartate specifc proteinase-1 (caspase-1) (SAB4503271, SIGMA) was detected by WB.Proteins from lung tissues were extracted with lysis bufer and quantitated by bicinchoninic acid (BCA) assay according to the manufacturer's protocol.Lysate samples were separated by SDS-polyacrylamide gels (SDS-PAGE) and transferred to polyvinylidene difuoride (PVDF) membranes, blocked with 5% defatted milk on shaker for 1 h at room temperature, and incubated with diluted antibodies at 4 °C overnight.Te corresponding secondary antibody (1 : 2000 diluted) was incubated at room temperature for 1 h, prior to detection with enhanced chemiluminescence (ECL) technique, and analyzed by Quantity One imaging system.2. Te relative quantifcation of expressive genes was processed using the 2 −ΔΔCt method.

Canadian Journal of Infectious Diseases and Medical Microbiology
2.10.Flow Cytometry.100 mg lung tissues from each mouse were minced, digested, and centrifuged for single-cell isolation.45 µL microspheres were plated into a 96-well plate.Diluent and 45 µL of cells were then added and allowed to shake for 1 h at room temperature in the dark.25 µL of 1 × Biotin-dAb (BD Pharmingen, USA) of IL-1β, TNF-α, IFN-c, IL-4, or IL-10 was then added and allowed to shake for 30 min in the dark and quantifed by fow cytometer.CD4 + , CD8 + T cells and CD11c + , CD103 + DC cells in lung tissues collected in 7 and 10 dpi were detected in the same procedure.Te antibody of CD4, CD8, CD11c, and CD103 was purchased from BD Pharmingen (BD, USA).

Statistical Analysis.
All data analyses and the results are expressed as the mean ± standard deviation (SD) by using GraphPad software (version 9.0).Multiple group comparisons were performed using one-way analysis of variance (ANOVA), followed by Dunnett's test to determine signifcant diferences from the control.Diferences were considered signifcant with a p value <0.05.

Identifcation of the Chemical Composition of MXSGD, SJZD, and Teir Combination.
Te base peak chromatograms of MXSGD, SJZD, and their combination in positive and negative ion mode are shown in Figures 1(a), 1(b), 1(c), 1(d), 1(e), and 1(f ), respectively.Preliminary characterization of 16, 16, and 24 compounds was carried out in combination with the plots and based on standard database matches and literature information (Tables S1-S3).

MXSGD + SJZD Improved Symptoms and Alleviated
Clinical Signs, Attenuated Pathological Changes, and Ameliorated FM1 Infection.We compared the clinical signs and pneumonia lesions of each group.Te normal control group mice had no change in the general appearance.FM1-infected mice showed signifcant fu-like symptoms, such as fur puckering, immobility, slow movement, hunching, emaciation, hunched back, and even death.Te symptoms of the MXSGD or SJZD group were slightly better than those of the FM1 group, while MXSGD + SJZD and oseltamivir treatment could restore milder clinical symptoms after infection (Figure 2(a)).Te infection mice also lost signifcant weight and became hypothermic.Te MXSGD + SJZD combination was efective in alleviating the fu-like symptoms and the mice showed signifcant recovery in body weight and body temperature compared to mice in the FM1 group (Figure 2(b)).Compared to FM1-infected mice, treatment with the MXSGD + SJZD combination had lower mortality rates, longer mean survival times, and higher prolonged survival rates (Figure 2(c)).
Infuenza A virus (IAV) is a major causative agent of lower respiratory tract infections, and infection with FM1 can lead to a severe infammatory response in the lungs.Lung tissue from mice in the NC group appeared bright pink with a smooth surface and a soft, elastic texture.FM1infected mice showed marked lung lobe swelling and hemorrhage (Figure 2(d)).Pathological sections also showed irregular and disrupted alveoli, thickened alveolar septa, infltration of infammatory cell aggregates and abundant secretions, and congestion in the bronchial lumen of FM1infected mice (Figure 2(e)).Tat was accompanied by a marked upregulation of the lung index (Figure 2(f )).In contrast, the combination of MXSGD + SJZD efectively improved congestion and hemorrhage in lung tissue, reduced infammatory cell infltration, inhibited plasma exudation in the bronchial lumen, and reduced damage to alveolar or capillary endothelial cells.To some extent, it also ameliorated pathological changes in the lungs and downregulated the lung index (Figures 2(d) and 2(f )).

TLR and NLRP3 Infammasome Signal Pathways in FM1-
Infected Mice Were Inhibited by MXSGD + SJZD at 4 dpi and Promoted at 7 dpi.After establishing the mouse FM1 infection model with LD 50 , the expression of the TLR7 pathway was altered.Te pulmonary mRNA expression of TLR7, MyD88, and NF-κB was abnormally upregulated in FM1-infected mice at 4 dpi and was decreased at 7 dpi (Figure 3).MXSGD + SJZD inhibited these mRNAs and protein expression on day 4 and promoted them on day 7. Te TLR7 pathway might be activated that p-NF-κB showed an increased trend after treatment with MXSGD + SJZD.
Te disruption of NLRP3 pathways was observed by FM1 infection at 4 dpi and inhibited at 7 dpi.Te disruption of pathway proteins was signifcantly regulated by MXSGD + SJZD (Figure 4).Te levels of IL-1β and IL-18 in the supernatant confrmed the same change, and MXSGD + SJZD had the most signifcant efcacy (Figures 4(f ) and 4(h)).

Levels of CD11c + and CD103 + DC in Lung Tissue
Were Signifcantly Upregulated by MXSGD Combined with SJZD at 10 dpi.To investigate the immune cell response in the lungs of infected mice, we detected changes in the number of T cells and DCs cells in lung tissue using fow cytometry.Compared to the normal group, the number of CD4 + and CD8 + cells in the lung tissue of infected mice reduced at 7 dpi and increased at 10 dpi (Figures 5(a     Canadian Journal of Infectious Diseases and Medical Microbiology signifcantly higher than that in the virus group.Terefore, MXSGD + SJZD cannot directly increase the number of T cells, and its mechanism may involve increasing the antigen presentation and enhancing the activity of T cells.

Discussion
Flu is one of the epidemic febrile diseases in traditional Chinese medicine, which was treated under the guidance of "Quxie" and "Fuzheng."MXSGD, frst created by Zhang, Zhongjing, is a classic traditional Chinese formula for relieving cough and asthma, eliminating phlegm and clearing heat.SJZD, a formula associated with "Fuzheng" with the efcacy of "invigorating spleen and replenishing qi," could be used in spleen-qi defciency syndrome to improve immunity.FM1-infected mice showed wrinkled fur, inactivity, slowness, and hunchback, which were consistent with the description of lung qi defciency syndrome in TCM.According to the theory of TCM, in the treatment of external contraction diseases, while "Quxie" to eliminate pathogens, we should also pay attention to "Fuzheng" to strengthen healthy qi.Terefore, MXSGD + SJZD was used to treat infected mice in this study.
In our study, SJZD did not have a signifcant efect on pneumonia, but it was a signifcant enhancer of the therapeutic efect of MXSGD.When infected with a lethal dose of FM1 virus, MXSGD reduced mortality and prolonged mean survival in mice when combined with SJZD, and improved prolonged survival more efectively than the drug alone.Moreover, the combination of MXSGD and SJZD reduced lung indices and markedly improved the extent of lung tissue damage.Taken together, these results suggest a signifcant synergistic efect of MXSGD in combination with SJZD.We then investigated the mechanism underlying the efect of MXSGD combined with SJZD in viral pneumonia.Evidence showed that excessive infammation, referred to as cytokine storm, and insufcient antiviral immunity, such as lymphopenia, coexist in severe IAV infection, and thus, a balanced immune response is required for a good clinical outcome.Excessive or prolonged activation of the TLRs pathway is strongly associated with acute lung injury and harmful infammation in the early stages of viral infection [33,34].Tis leads to the release of infammatory factors.Tus, regulation of the infammatory responses of the TLRs signaling pathway may alleviate acute lung injury induced by infuenza virus and may be an efective therapy for infuenza infection.In this study, we provide evidence that MXSGD + SJZD signifcantly downregulated the TLR7 pathway at 4 dpi and activated it at 7 dpi in response to FM1 infection.At the early stage of immune infection, MXSGD + SJZD can control the infammatory damage caused by infection by inhibiting the TLR7 pathway.On day 7, MXSGD combined with SJZD can activate the TLR pathway during viral replication and promote an antiviral adaptive immune response in vivo.
Te NLRP3 infammasome is an essential component of the host immune response to infuenza virus infection.However, excessive activation of the NLRP3 infammasome pathway may have pathological implications in infuenza virus infection [35].Terefore, the immunological responses of the NLRP3 infammasome pathway must be strictly modulated to avoid hyperinfammatory or immunocompromised states during infuenza infection.Our results showed that MXSGD + SJZD could inhibit the hyperinfammatory state mediated by the NLRP3 infammasome pathway at 4 dpi and maintain the activity of the pathway and the release of certain infammatory factors conducive to virus clearance at 7 dpi.In contrast, infuenza mice treated with MXSGD and SJZD alone were unable to regulate the dysregulation of this pathway.CD4 + and CD8 + T cells have an important role to play in the protective immune response to an infuenza infection.Under attack by infuenza virus, CD4 + T cells are activated after recognizing viral epitopes associated with MHC class II molecules and interacting with costimulatory molecules on APC.Initial CD8 + T cells are activated after recognizing viral epitopes associated with MHC class I molecules on APC in draining lymph nodes and then diferentiate into CTL cells that migrate to the site of infection and play a role in recognizing and removing infected cells.Timely immune response plays a crucial role in the process of viral infection.Te results showed that MSXGD and SJZD did not afect the number of CD4 + and CD8 + in FM1-infected mice (Figure 5).DCs are the most efcient antigen-presenting cells.After viral infection, CD11c + DC was transported and aggregated in draining lymph nodes, where they progressively matured and promoted the diferentiation of primary lymphocytes.CD103 + DC, a migratory DC subset, can secrete complements C3 and C5, resulting in the induction of T-cell immune responses and virus clearance after infuenza infection [36,37].It is highly sensitive to infuenza infection and highly efective at activating lymph node cells, which is critical for initiating CD8 + T-cell responses early in infection [38][39][40][41].Interestingly, although the drug had no efect on T-cell numbers, MXSGD and MXSGD + SJZD could gradually promote DC cell antigen presentation in the lung tissue of infuenza-infected mice.Te combination of SJZD can enhance the efect of MXSGD in enhancing antigen presentation and promoting T-cell immunity.In healthy people, the frequencies of T1/T2 cells are relatively balanced, which is important for maintaining normal immune function, and the delicate balance of T1/T2 responses is disrupted in IAV infection [42,43].T1 cell polarization predominates, IFN-c secretion predominates, and macrophage infltration is excessive in FM1-treated mice [31].IL-4 production is signifcantly reduced in the serum and bronchoalveolar lavage fuid of infected mice, while IFNgamma release is increased [44].On the other hand, T1 cells modulate cellular immunity, while T2 cells regulate humoral immunity [45].Isotype switching to IgE has been induced by IL-4 in B cells [46].However, T2 cell-mediated allergic airway disease can be induced by a T1-mediated antiviral response to infuenza virus infection [47].Terefore, the balance of T1/T2 cell cytokines should be carefully regulated during infuenza virus infection.In the present study, MXSGD + SJZD may enhance the antiviral efect by modulating the immune response by promoting the expression of T1 cell-derived TNF-α and IFN-c and T2 cell-derived IL-4 at a late stage of FM1 infection.Te upregulated expression of T2 cell-derived IL-10 may suppress the immunological dysfunction of T1 cells in MXSGD combined with SJZD-treated mice at a late stage of FM1 infection (Figure 7).Infuenza is one of the epidemic febrile diseases.Te treatment principle of "Quxie" and "Fuzheng" is applied throughout the process of infuenza prevention and treatment to improve the prognosis of patients.Te infected mice show the symptoms of "qi defciency," such as loss of appetite, inactivity, and rufed fur, which are treated by SJZD based on the "Fuzheng" strategy.However, the symptoms of viral infection in the host will be more complex.Terefore, the method of "Fuzheng" should be adopted according to diferent clinical manifestations.
In modern medicine, oseltamivir, one of the classic antiviral drugs, is widely used to treat infuenza virus pneumonia and was selected as the positive drug in this study.Early treatment of viral pneumonia is critical, as our data show that in a mouse model of lethal dose infection, mice receiving antiviral therapy at the time of virus entry (1 dpi) still have high survival rates.However, the reality is more complicated because antiviral treatment is often not available in the early stages of the virus.It is therefore necessary to consider combination treatment with antivirals and other drugs.Our results will provide clues and ideas for drug therapy of viral pneumonia in the clinic.

Conclusion
In conclusion, our study showed that the combination of MXSGD and SJZD was more efective than either MXSGD or SJZD alone in FM1 infuenza infection.Te synergistic efects include improvement in clinical signs, weight gain, and body temperature in mice.In addition, MXSGD combined with SJZD plays a benefcial role in activating a balanced infammatory response in the host to limit immunopathological damage and improve clinical and survival outcomes.Tey may facilitate antiviral immunological responses in the late stage of FM1 infection by regulating TLRs and NLRP3 infammasome pathways, potentiating the regulation of CD11c + and CD103c + and maintaining the balance of T1/T2 cytokines.Te theory of "Quxie and Fuzheng" is refected in the combination of MXSGD and SJZD and confrmed to improve the curative efect of antiviral pneumonia, which may provide an efective strategy for future clinical treatment.
) and 5(b)).Virus infection increased the number of T cells, but MSXGD or Canadian Journal of Infectious Diseases and Medical Microbiology SJZD treatment has no signifcant efect.On the contrary, the levels of CD11c + and CD103 + DC increased in infected mice from day 7 to day 10 (Figures 5(c) and 5(d)).After the treatment with MXSGD + SJZD, CD11c + levels were signifcantly increased at 10 dpi.Te number of CD103 + DC cells in the MXSGD and combination groups was

3. 5 .
Te Balance of T1/T2 Cell Cytokines Was Modulated by MXSGD Combined with SJZD.Te proinfammatory factors of IFN-c and TNF-α were upregulated in FM1infected mice at 4 dpi by fow cytometry detection, while suppressed at 7 dpi (Figures 6(a) and 6(b)), while the antiinfammatory cytokines (IL-4 and IL-10) change in the opposite direction (Figures 6(c) and 6(d)).MXSGD + SJZD has the best efect of inhibiting infammation in the early stage and promoting an inadequate response in the late stage by regulating the T1/T2 ratio.