Gene Expression Profiles Underlying Selective T-Cell-Mediated Immunity Activity of a Chinese Medicine Granule on Mice Infected with Influenza Virus H1N1.

A Chinese medicine granule, Shu-Feng-Xuan-Fei (SFXF), is critical for viral clearance in early phase of influenza virus infection. In this study, 72 ICR mice were randomly divided into six groups: normal control group, virus control group, Oseltamivir group, low-dose SFXF, medium-dose SFXF, and high-dose SFXF. Mice were anesthetized and inoculated with 4LD50 of influenza virus A (H1N1) except normal control group. Oseltamivir group received 11.375 mg·kg(-1) ·d(-1) Oseltamivir Phosphate. SFXF 3.76, 1.88 and 0.94 g·kg(-1) ·d(-1) were administrated to mice in all SFXF groups. Each group was in equal dose of 0.2ml daily for 4 consecutive days. Mice were sacrificed and then total RNA was extracted in lung tissue. Some genes involved in T-cell-mediated immunity were selected by DNA microarray. These candidate genes were verified by Real-Time PCR and western immunoblotting. Compared with virus control group, in Toll-like receptor signaling pathway, 12 virus-altered genes were significantly reduced following medium-dose SFXF treatment. Eighteen antigen processing presentation-associated genes were upregulated by medium-dose SFXF. In the process of T cell receptor signaling pathway, 19 genes were downregulated by medium-dose SFXF treatment. On exploration into effector T cells activation and cytokines, all of altered genes in virus control group were reversed by medium-dose SFXF. Real-time PCR and western immunoblotting showed that the regulation of medium-dose SFXF in IL-4, IFN-γ, TNF-α, IL-1ß, TLR7, MyD88, p38, and JNK was superior to Oseltamivir and high-dose SFXF group. Therefore, SFXF granules could reduce influenza infected cells and activation of T cells.

[Effect of shufeng xuanfei recipe and jiebiao qingli recipe on mRNA and protein expressions of TLR7, MyD88, and NF-kappaB in mice infected with influenza virus].

OBJECTIVE: To observe effect of Shufeng Xuanfei Recipe (SXR) and Jiebiao Qingli Recipe (JQR) on mRNA and protein expressions of Toll-like receptor 7 (TLR7), myeloid differentiation factor 88 (MyD88), and nuclear factor-kappaB (NF-kappaB) in mice infected with influenza virus FM1. METHODS: One hundred and eight mice were randomly divided into nine groups, i.e., the normal control group, the model group, the Oseltamivir group (at the daily dose of 2.5 g/mL), the high dose SXR group (at the daily dose of 3.762 g/kg), the middle dose SXR group (at the daily dose of 1.881 g/kg), the low dose SXR group (at the daily dose of 0.941 g/kg), the high dose JQR group (at the daily dose of 4.368 g/kg), the middle dose JQR group (at the daily dose of 2.184 g/kg), and the low dose JQR group (at the daily dose of 1.092 g/kg), 12 in each group. All mice were mildly anesthetized by ether. Mice in the normal control group were treated by nasal drop of 0.05 mL normal saline, while mice in the rest groups were infected by nasal drop of 0.05 mL influenza virus strain FM1 (LD50). The successful modeling rate was 100%. All medication was performed by gastrogavage 2 h after infection. Distilled water was given by gastrogavage to mice in the normal control group and the model group at the daily dose of 0.2 mL, each time per day for 4 successive days. mRNA expressions of TLR7, MyD88, and NF-kappaB in the lung tissue were determined by Western blot. RESULTS: Compared with the normal control group, mRNA expressions of TLR7, MyD88, and NF-kappaB increased in the model group (P < 0.01). Compared with the model group, mRNA and protein expressions of TLR7, MyD88, and NF-kappaB decreased in the Oseltamivir group, the high, middle, and low dose SXR groups (P < 0.05, P < 0.01); mRNA and protein expressions of TLR7 and NF-kappaB decreased in the high and middle dose JQR groups (P < 0.05, P < 0.01); mRNA expressions of MyD88 decreased in the high and middle dose JQR groups (P < 0.05); protein expressions of MyD88 decreased in the middle dose JQR group (P < 0.05); protein expressions of TLR7 and NF-kappaB decreased in the low dose JQR group (P < 0.05). Compared with the Oseltamivir group, protein expressions of MyD88 decreased in the low dose SXR group (P < 0.05); protein expressions of NF-kappaB decreased in the middle and low dose SXR groups (P < 0.01); mRNA and protein expressions of TLR7 (P < 0.05, P < 0.01), and protein expressions of MyD88 (P < 0.01) decreased in the high, middle, and low dose JQR groups; mRNA and protein expressions of NF-kappaB decreased in the low dose JQR group (P < 0.05, P < 0.01). CONCLUSIONS: Each dose SXR and middle dose JQR could down-regulating the activity of NF-kappaB through adjusting MyD88 dependent TLR signal pathway, thus fighting against influenza virus. SXR was more effective than JQR.

[Screening for genes associated with natural killer cell mediated cytotoxicity in pneumonia mice infected with influenza virus and regulation of two herbal anti-virus formulas].

OBJECTIVE: To investigate the regulation of two herbal anti-virus formulas on gene expression profile associated with natural killer cell (NK cell) mediated cytotoxicity in pneumonia mice infected with influenza virus. METHODS: According to random number table, 90 ICR mice were divided into nine groups with 10 mice in each group: normal group (N), model group (M), oseltamivir group (control group, C), low-dose, medium-dose and high-dose Shufeng Xuanfei formula groups (SL, SM, SH groups), and low-dose, medium-dose and high-dose Jiebiao Qingli formula groups (JL, JM, JH groups). The model of pneumonia was reproduced by nasal dropping influenza virus A (FM1) in mice. N group was given isotonic saline 0.05 ml in nasal drops. After 2 hours of model-building, C group was received 11.375 mg×kg⁻¹×d⁻¹ oseltamivir phosphate. Shufeng Xuanfei formula (mainly honeysuckle, forsythia and radix isatidis, etc.) with 3.76, 1.88 and 0.94 g×kg⁻¹×d were administrated to SH, SM and SL groups by gastric irrigation respectively. Jiebiao Qingli formula (mainly ephedra, gypsum, glycyrrhiza glabra, etc.) with 4.36, 2.18 and 1.09 g×kg⁻¹×d⁻¹ were administrated to JH, JM and JL groups by gastric irrigation respectively. In N and M groups, normal saline was administrated with gastric perfusion. Each group was in equal dose of 0.2 ml daily over a 4-day period. Total RNA in lung tissue of mice were extracted in each group, then gene chips were used to screen these RNA samples. Some genes involved NK cell mediated cytotoxicity were selected, with "I" representing of signal intensity. These candidate genes were verified by real-time fluorescent quantitation polymerase chain reaction (PCR) and Western blotting. RESULTS: In the pathway of NK cell mediated cytotoxicity, M group up-regulated 43 genes expression, and 36, 29, 22, 21, 20 and 10 genes showed down-regulation in SM, JM, SL, JH, SH and JL groups, respectively. Apart from gene co-expression network in SH, SL, JH, JM and JL, SM also expressed other differential genes which SH, SL, JH, JM and JL did not. So medium-does Shufeng Xuanfei formula had the most significant regulation in gene expression of NK cell mediated cytotoxicity. By real-time PCR and Western blotting experiments showed that compared with the M group, mRNA and protein expression of tumor necrosis factor-α (TNF-α) in these two formula groups were significantly down-regulated, especially prominent in SM group and JM group (TNF-α mRNA: 1.07 ± 0.19, 1.19 ± 0.14 vs. 3.20 ± 0.56, both P<0.01). CONCLUSIONS: Influenza viral replication in host cell, which means influenza antigens exposure in infected cells as target cells. NK cells recognize and exert cell mediated cytotoxic function against influenza antigens. Genes associated with NK cell mediated cytotoxicity in influenza infection were up-regulated. Shufeng Xuanfei and Jiebiao Qingli formulas could down-regulate these genes. The mechanism of down-regulated genes is that the number of influenza infected cells and NK cells activation decreases in treatment with two formulas.

Effect and mechanisms of Gong-tone music on the immunological function in rats with Liver (Gan)-qi depression and Spleen (Pi)-qi deficiency syndrome in rats.

OBJECTIVE: To investigate the effects and mechanisms of Gong-tone music on the immunological function in rats with the Chinese medicine syndrome of Liver (Gan)-qi stagnation and Spleen (Pi)-qi deficiency (LSSD). METHODS: Twenty five male Wistar rats of SPF grade were randomly divided into 5 groups: normal group, model group, Xiaoyao Powder () group, Gong-tone group and combined group (the combination of Gong-tone and Xiaoyao Powder), with 5 rats in each group. The rat model for the Chinese medicine syndrome of LSSD was induced by chronic bandage and irregular diet. The course of treatment was 21 days. After the treatment, the levels of serum gastrin and IgG were detected by enzyme-linked immunoabsorbent assay (ELISA). Phagocytosis of macrophages was detected by the neutral red uptake assay and T cell proliferation was investigated by 3-(4,5-dimethylthiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay. RESULTS: The serum gastrin, macrophage phagocytosis, IgG level and proliferation ability of T cells in the model group were significantly decreased compared with those in the normal group (P <0.05). Compared with those in the model group, the serum levels of gastrin, macrophage phagocytosis, IgG level and proliferation ability of T cells in Gong-tone, Xiaoyao Powder, and combined groups were significantly increased (P <0.05). The combined group was superior to either Gong-tone group or Xiaoyao Powder group. CONCLUSION: Gong-tone music may upregulate the immunological function and play a role in adjuvant therapy in the Chinese syndrome of LSSD.

[Antagonism of baicalin on cell cyclical distribution and cell apoptosis in A549 cells infected with influenza A (H1N1) virus].

To observe the inhibitive effect of Baicalin against influenza A H1N1 virus infection in epithelial cell line A549, the cell proliferation and cytotoxicity were assayed by MTT, the cell cycle and the apoptosis were analyzed by flowcytometer using PI staining, the morphology of cellular nucleolus was observed by Hoechst 33258 staining and the effects of activation on caspase 3 and caspase 8/9 were also detected by immunofluorescent staining with a fluorescence microscope. The results showed that Baicalin exerted an inhibitive effect on CPE after influenza A H1N1 virus infection. The FACS with PI staining showed that the cell cycle of the infected cell was arrested at S phase, the Baicalin-treated group decreased S phase cell ratio and subG0 phase peak in comparison with the control (P < 0.05) and significantly promoted cell proliferation (# P < 0.05). Hoechst33258 staining suggested that Baicalin protected the cellular nucleolus against the influenza virus-induced apoptosis. Observation under the immunofluorescent microscope suggested that the activities of caspase-8 and caspase-3 were enhanced at 36 h post the influenza virus infection, but 100 microg/mL Baicalin suppressing the activation of caspase-8 and caspase-3 rather than that of caspase-9. In summary, this research confirmed that Baicalin inhibited the influenza A H1N1 virus strain infection in vitro, the drug obviously protected cells from apoptosis damages through regulating cell cycle and suppressed the activation of caspase-8 and caspase-3. The down-regulation was significant and showed a dose-dependent relationship.

[Effects of Dureping Injection on the contents of MMP-9 and TIMP-1 in the lung tissue of mice with pneumonia of influenza virus infection].

OBJECTIVE: To observe the effects of Dureping Injection on the contents of matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) in the lung tissue of mice with pneumonia of influenza virus infection. METHODS: Sixty-six ICR mice were randomly divided into the normal group, the model group, the low, middle, and high dose Dureping Injection groups (0.435, 0.870, and 1.740 mg/d, respectively), and the positive control group (Ribavirin, 2.500 mg/d), 11 in each. The pneumonia of mice with influenza virus infection model was established using influenza virus strain FM1. Mice were intraperitoneally injected with 0. 3 mL FM1 starting from the infection day, once daily. Five days later mice were killed to calculate the lung index. The pathomorphological changes of the lung tissue were observed using routine HE stained sections. The contents of MMP-9 and TIMP-1 in the homogenate of the lung tissue were detected by ELISA double antibody sandwich method. RESULTS: Compared with the normal group, obvious inflammation occurred in the lung tissue of mice in the model group. The lung index, the content of MMP-9, and the value of MMP-9/TIMP-1 increased significantly in the model group (P < 0.01) , while the content of TIMP-1 was not significantly different (P > 0.05). Compared with the model group, the content of MMP-9 in the low and middle dose Dureping Injection groups, and the positive control group was significantly lowered (P < 0.01). The content of TIMP-1 in the low, middle, and high dose Dureping Injection groups, as well as the positive control group significantly increased (P < 0.01) and the value of MMP-9/TIMP-1 decreased (P < 0.01). CONCLUSION: Dureping Injection could alleviate the inflammatory injury of the lung tissue through decreasing the content of MMP-9, elevating the content of TIMP-1 in the lung tissue, and regulating the value of MMP-9/TIMP-1 of mice with pneumonia of influenza virus infection, thus alleviating the inflammatory injury of the lung tissue.

[Effect of dureping injection on T-cells function and their function in killing FM1 infected Mphi in vitro].

OBJECTIVE: To investigate the effect of Dureping Injection (DRP) on the T-cells function of mice and the function of T-cells in killing MF infected by influenza virus subtype A mice-lung adaptive strain FM1 in vitro. METHODS: Number of splenic normal and FM1 infected T-cells in mice were measured by MTT and double-antibody sandwich ELISA, after being treated with DRP at different concentrations (2.1, 8.5 and 17.0 microg/mL), and the effect of DRP on interferon-gamma (IFN-gamma) and interleukin-10 (IL-10) production as well as on splenic T-cell killing FM1 infected Mphi/Ana-1 function were detected. RESULTS: DRP inhibited the multiplication of normal spleen T cells induced by concanavalin A in vitro, suppressed Th2 cell factor IL-10 production, and maintained Th1 cell factor IFN-y at a definite level, moreover, it directly enhanced the power of T-cells in killing FM1 infected Mphi (P < 0.05, P < 0.01). CONCLUSION: DRP could act on mice T-cells to enhance the immune response for antiinfluenza viral FM1 in vitro.

[Experimental study on anti-arthritis effect of jiawei mufangji decoction in rats].

OBJECTIVE: To explore the therapeutic effect of Jiawu Mufangji Decoction (JMD) in treating rats with adjuvant arthritis (AA) and its mechanism. METHODS: AA model rats induced by Freund's complete adjuvant were treated with JMD by gastrogavage starting from 18 days after modeling. On the 39th day, body weight, spleen and thymus index, and swelling degree of paw of the AA rats were measured, pathological changes of the ankle joint tissue were observed using HE staining, and serum levels of interleukin-1beta (IL-1beta) and tumor necrosis factor a (TNF-alpha) were determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: JMD could relieve the symptoms of AA rats, decrease the paw swelling, improve the weight and spleen and thymus index, reduce the dropsy of joints and lymphocytes infiltration, inhibit the proliferation of synovium, and obviously lower the serum levels of interleukin-1beta and TNF-alpha. CONCLUSION: The therapeutic effect of JMD might be related to its action in down-regulating the serum levels of IL-1beta and tumor necrosis factor alpha.

[The study on the relationship between the tumor-like proliferation of synoviocytes and the expression of c-myc and ODC genes in adjuvant arthritis rats].

AIM: To investigate the mechanism of the tumor-like proliferation of synoviocytes in adjuvant arthritis (AA) rats. METHODS: Wistar rats were randomly divided into 2 groups, AA group and control group. Freund's complete adjuvant was injected into AA group rats to induce adjuvant arthritis. The knee synovial tissues of the rats were taken and synoviocytes were separated and cultured in vitro. The proliferation of cultured synoviocytes was measured by MTT colorimetry. Meanwhile, the mRNAs of C-myc and ODC genes in synovial tissues of the rats were detected by semi-quantitative RT-PCR. RESULTS: (1)Synoviocytes of AA rats proliferated more markedly than those of control rats (P<0.01), and exhibited a tumor-like proliferation. (2)The mRNAs of C-myc and ODC genes were obviously higher in synovial tissues of AA rats than those of control rats. CONCLUSION: The tumor-like proliferation of synoviocytes from AA rat joint might be related to the increased mRNAs of C-myc and ODC genes.

[Influence of bondage stress on Th1/Th2 cytokines and growth of tumors in mice bearing S180 tumor cells].

AIM: To observe the influence of bondage stress on Th1/Th2 cytokines and growth of tumor in mice bearing S180 tumor cells. METHODS: 2 x 10(6) S180 tumor cells were injected subcutaneously into the murine right oxter,and bondage stress was imposed on mice for 8 hours per day,ten days running. Then T lymphocyte proliferation was detected by MTT colorimetry. IFN-gamma and IL-2 secreted by splenocytes was measured by mitogen-activating lymphoblast assay and macrophage NO production assay. At the same time, the levels of IL-4 and IL-10 in serum were detected by ELISA, and the weight of the tumors and thymuses were measured. RESULTS: Compared with mice only injected with S180 tumor cells, tumor bearing mice suffered from bondage stress showed decreased T lymphocyte proliferation, thymus index, and levels of IFN-gamma and IL-2, but increased levels of IL-4 and IL-10(P<0.05), and tumor weight (P<0.01). CONCLUSION: Bondage stress could aggravate the suppression of cellular immunity in mice bearing S180 tumor and promote the T cells to shift towards Th2 type cells, which may be one of the mechanisms of promoting tumor growth by bondage stress.