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

Abstract

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.

Figure 1

Total ion chromatograms (TIC) of MXSGD, SJZD, and MXSGD + SJZD in positive and negative ion modes were characterized using UPLC-Q-Orbitrap HRMS-MS. Positive ion mode and negative ion mode of (a, b) MXSGD, (c, d) SJZD, and (e, f) MXSGD + SJZD.

Figure 2

The effect of MXSGD + SJZD on symptoms, clinical signs, and pulmonary functions. (a) Mice physical characteristics. (b) The score of clinical signs was evaluated. 0: healthy; 1: barely ruffled fur; 2: ruffled fur, but active; 3: ruffled fur and inactive; 4: ruffled fur, inactive, hunched, and gaunt; 5: dead. Body weight and body temperature changes of mice were measured and evaluated. (c) The mean survival time, mortality, and prolong survival rate of mice. (d) Morphological changes of lung tissues. (e) Hematoxylin-eosin staining of lung tissues (20×), scale bar = 100 μm. (f) The lung index of mice. The FM1 group and treatment groups, n = 15; normal control (NC) groups, n = 10. ^∗P < 0.05 or ^∗∗P < 0.01 or ^∗∗∗P < 0.001 vs. NC group; ^#P < 0.05 or ^##P < 0.01 vs. FM1 group.

Figure 3

Effect of MXSGD + SJZD on TLR signaling at 4 and 7 dpi (n = 5). (a) TLR7, (b) MyD88, (c) NF-κB mRNA levels, and (d) p-NF-κB protein change in lung tissue. ^∗P < 0.05 or ^∗∗P < 0.01 or ^∗∗∗P < 0.001 vs. NC group; ^#P < 0.05 or ^##P < 0.01 vs. FM1 group.

Figure 4

Effect of MXSGD + SJZD on the NLRP3 inflammasome pathway at 4 and 7 dpi (n = 5). (a) ASC, (b) NLRP3, (c) caspase-1 mRNA, and (d) caspase-1 protein in lung tissues. (e) IL-1β mRNA, (f) IL-1β, (g) IL-18 mRNA, and the level of (h) IL-18 in lung tissues was measured. ^∗P < 0.05 or ^∗∗P < 0.01 or ^∗∗∗P < 0.001 vs. NC group; ^#P < 0.05 or ^##P < 0.01 vs. FM1 group.

Figure 5

Flow cytometry results of T cell and DC cell in lung tissues of each group on day 7 and day 10 (n = 3). CD4⁺ and CD8⁺ T-cell levels change on (a) day 7 and (b) day 10. CD11c⁺ and CD103⁺ DC cell levels change on (c) day 7 and (d) day 10. ^∗P < 0.05 or ^∗∗P < 0.01 or ^∗∗∗P < 0.001 vs. NC group; ^#P < 0.05 or ^##P < 0.01 vs. FM1 group.

Figure 6

The effect of MXSGD + SJZD on Th1/Th2 cytokines (n = 5). (a) IFN-γ, (b) TNF-α, (c) IL-4, and (d) IL-10 in lung tissues were measured. ^∗P < 0.05 or ^∗∗P < 0.01 or ^∗∗∗P < 0.001 vs. NC group; ^#P < 0.05 or ^##P < 0.01 vs. FM1 group.

Figure 7

Mechanism of MXSGD and SJZD conjugated for the treatment of influenza-infected mice.