The aim of the article was to study the mechanism of Lipoxin A4 (LXA4)-mediated p38 MAPK pathway protecting mice against collagen-induced arthritis (CIA). The impact of LXA4 (0, 5, 10, 15 nM) on synoviocytes proliferation of CIA mice was detected using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. CIA mice were treated with LXA4, SB203580 (a p38 inhibitor), and/or anisomycin (a p38 agonist), and the arthritis severity score in each mouse was determined. The gene or protein expressions were detected with Western Blotting, ELISA, or qRT-PCR. LXA4 inhibited the synoviocytes proliferation of CIA mice with decreased levels of TNF-α, IL-6, IL-1β, and IFN-γ and reduced p-p38/total p38 expression in synoviocytes in a dose-dependent manner. LXA4 levels were decreased in synovial tissues and plasma of CIA mice, but p-p38/total p38 expression was increased in synovial tissues. LXA4 could downregulate p-p38/total p38 expression in synovial tissues of CIA mice. Both LXA4 and SB203580 reduced arthritis severity score of CIA mice with the reduction of synovial tissue hyperplasia and inflammatory cell infiltration. CIA mice treated with LXA4 and SB203580 had lower levels of TNF-α, IL-6, IL-1β, and IFN-γ, accompanying decreased MDA as well as increased SOD, CAT,and GPx. However, anisomycin could reverse the protect effects of LXA4 on CIA mice regarding the abovementioned inflammatory factors and oxidative stress indexes. LXA4 protected mice against collagen-induced arthritis via inhibiting p38 MAPK signaling pathway, which may be a potential new therapeutic target for rheumatoid arthritis.

Coptidis alkaloids are the primary active components of Coptis chinensis Franch. Clinical and pharmacodynamic studies have confirmed that Coptidis alkaloids have multiple therapeutic effects including anti-inflammatory, antioxidant and antitumor effects, and they are usually used to treat various inflammatory disorders and related diseases. Mouse bone marrow cells (BMCs) were isolated from BALB/c mice. Immune-mediated destruction of BMCs was induced by interferon (IFN) -γ. High-performance liquid chromatography-electrospray ionization/ mass spectrometry was used to analyze the ingredients of the aqueous extract from Coptis chinensis Franch. The results confirmed that Coptidis alkaloids were the predominant ingredients in the aqueous extract from Coptis chinensis. The functional mechanism of Coptidis alkaloids in inhibiting immune-mediated destruction of BMCs was studied in vitro. After Coptidis alkaloid treatment, the percentages of apoptotic BMCs and the proliferation and differentiation of helper T (Th) cells and regulatory T (Treg) cells were measured by flow cytometry. The expression and distribution of T-bet in BMCs were observed by immunofluorescence. Western blotting analysis was used to assay the expression of key molecules in the Fas apoptosis and Jak/Stats signaling pathways in BMCs. We identified five alkaloids in the aqueous extract of Coptis chinensis. The apoptotic ratios of BMCs induced by IFN-γ were decreased significantly after Coptidis alkaloid treatment. The levels of key molecules (Fas, Caspase-3, cleaved Caspase-3, Caspase-8 and Caspase-8) in Fas apoptosis signaling pathways also decreased significantly after treatment with low concentrations of Coptidis alkaloids. Coptidis alkaloids were also found to inhibit the proliferation of Th1 and Th17 cells and induce the differentiation of Th2 and Treg cells; further, the distribution of T-bet in BMCs was decreased significantly. In addition, the levels of Stat-1, phospho-Stat-1 and phospho-Stat-3 were also reduced after Coptidis alkaloid treatment. These results indicate that Coptidis alkaloids extracted by water decoction from Coptis chinensis Franch could inhibit the proliferation and differentiation of T lymphocytes, attenuate the apoptosis of BMCs, and suppress the immune-mediated destruction of the BMCs induced by pro-inflammatory cytokines.

目的 探讨黄芪、当归、忍冬藤活性成分对白细胞介素1β(interleukin-1β,IL-1β)诱导大鼠T细胞增殖和分化的影响.方法 IL-1β诱导T细胞分化,IL-β抑制剂为阳性对照组,黄芪、当归、忍冬藤水提物冻干粉干预;流式细胞仪检测大鼠脾淋巴细胞T细胞亚群比率的改变,Western-Blot检测T辅助细胞(helper T cell,Th)特异性转录因子T-bet、GATA-3、ROR-γ表达差异.结果 黄芪、当归、忍冬藤水提物冻干粉组合能显著降低细胞毒T细胞比率,显著降低Th1和Th17、升高Th2和调节性T细胞(regulatory T cell,Treg)比率;升高GATA-3,并降低ROR-γ和T-bet的表达.结论黄芪、当归、忍冬藤水提物冻干粉组合可能通过抑制IL-1β诱导的T细胞异常增殖和分化,缓解炎症免疫应答,恢复T细胞免疫应答的动态平衡.

Radix Astragali and Radix Angelicae Sinensis are two herbs that compose Danggui Buxue Tang (an herbal formula for treatment of anemia diseases). In this study, we explored the molecular mechanism and effective targets to immune destruction of bone marrow (BM) cells treated with Radix Astragali, Radix Angelicae Sinensis or a combination of two agents. The potential synergic advantages of two herbs should also be explored. The constituents of Radix Astragali and Radix Angelicae Sinensis were analyzed by high performance liquid chromatography-electrospray ionization/mass spectrometer system BM cells were separated from limbs of BALB/c mice, and immune destruction was induced with IFN-γ. The percentages of hematopoietic stem cells (HSCs) and CD3+ T cells were detected by flow cytometry. The distribution of T-bet and changes in the combination of SAP and SLAM in BM cells were observed by immunofluorescence. Western blotting was used to assay the expression of key molecules of the eIF2 signaling pathway in BM cells. Seven constituents of Radix Astragali and six constituents of Radix Angelicae Sinensis were identified. The percentages of HSCs increased significantly after treatment with Radix Angelicae Sinensis, especially at high concentrations. The percentages of CD3+ T cells were significantly decreased after Radix Astragali and Radix Angelicae Sinensis treatment. However, the synergistic function of two-herb combinations was superior to that of the individual herbs alone. The distribution of T-bet in BM cells was decreased significantly after Radix Angelicae Sinensis treatment. The number of SLAM/SAP double-stained cells was increased significantly after Radix Astragali treatment at low concentrations. The phosphorylation levels of eIF2α were also reduced after Radix Astragali and Radix Angelicae Sinensis treatment. Radix Astragali and Radix Angelicae Sinensis could intervene in the immunologic balance of T lymphocytes, inhibit the apoptosis of BM cells induced by immune attack, restore the balance of the T cell immune response network and recover the hematopoietic function of HSCs. The synergistic effects of Radix Astragali and Radix Angelicae Sinensis were superior to those of each herb alone.

目的:应用补肾生血解毒复方干预治疗再生障碍性贫血(AA)小鼠模型,探讨该方及其拆方对AA小鼠脾脏T淋巴细胞增殖和分化的影响.方法:免疫介导制作小鼠AA动物模型,以环孢素作为阳性对照药物,补肾生血解毒法进行中药分组干预,流式细胞仪检测小鼠脾淋巴细胞中T辅助细胞(Th)各亚型的比率的改变.Western Blot检测T-bet、GATA-3和ROR γ在小鼠脾细胞中的表达的变化.结果:流式检测结果显示,补肾生血解毒复方可以显著降低AA小鼠脾脏中Th1、Th17的比率,提高Th2和Treg细胞比率(P＜0.05,P＜0.01),生血解毒组与模型组比较能显著降低Th17细胞的比率(P＜0.01),并能显著升高Treg细胞的比率(P＜0.01);Western Blot实验结果显示,补肾生血解毒复方能够降低T-bet和ROR γ含量,升高GATA-3,但差异无统计学意义.结论:补肾生血解毒复方能够通过调控AA小鼠脾脏T淋巴细胞的增殖和分化,维持自身免疫应答网络动态的平衡,来达到重建和恢复骨髓造血机能的治疗作用,这可能是该方治疗AA的关键作用机制.

Objective To investigate the effects of Bushen Shengxue Jiedu Fang (Kidney-tonifying blood-generating toxin-removing Formula,BSSXJDF) on lymphocyte differentiation and hematological parameters in mice with immune-mediated aplastic anemia (AA).Methods Mice in all groups except the normal group received 60Co-γirradiation combined with injection of allogeneic immune cells in the tail vein to establish bone marrow hematopoietic inhibition model.The immunosuppressive cyclosporine A was used as the positive control drug,whereas Bushen Shengxue Jiedu Fang (BSSXJDF) was given to mice in the treatment groups for 28 consecutive days.Changes of hematological parameters after medication in the peripheral blood of mice were measured with cytometer at day 7,14,21 and 28.The ratio of T,B and NK cells was detected with flow cytometer.Results The count of red blood cells (RBC),hemoglobin (HGB),platelets (PLT) and white blood cells (WBC) in peripheral blood of mice were significantly increased (P ＜ 0.05) in BSSXJDF group compared with the model group;the ratio of CD3+ CD4+ cells in creased(P ＜ 0.01),and the ratio of CD3-CD19+ cells and CD3-NK1.1+ cells decreased (P ＜ 0.01) with statistical significance in the cyclosporine and BSSXJDF groups compored with the model group.Conclusion Bushen Shengxue Jiedu Fang can repair the hematopoietic function of bone marrow by regulating the differentiation of lymphocytes in the treatment of AA.

Angelicae Sinensis, Radix Astragali and Rhizoma Coptidis are all herbs of modified Danggui Buxue Tang (DGBX) and are extensively applied herbs in traditional Chinese medicine for the treatment of anemia and inflammation. In this study, immune-induced AA mice were used as an animal model, and the immunosuppressive agent, Ciclosporin A (CsA), was used as a positive control. Multiple pro-inflammatory cytokines were examined by bead-based multiplex flow cytometry. The T-cell subsets were assessed using a fluorescence-activated cell sorter (FACS). Western blot analysis was used to estimate the protein expression levels of specific transcription factors for T helper cells (Th1, Th2 and Th17) and key molecules of the Janus-activated kinase (Jak)/signal transducer and activator of transcription (Stat3) signaling pathway. DGBX treatment could significantly increase the production of whole blood cells in peripheral blood (PB); inhibit the expansion of Th1 and Th17 cells; increase the differentiation of Th2 and Tregs cells; regulate the expression levels of T-bet, GATA-3, RORγ and proinflammatory cytokines; and decrease the expression levels of key molecules in the Jak/Stat signaling pathway. These results indicate that DGBX can regulate the differentiation of T lymphocytes, resulting in immunosuppressive and hematogenic functions on AA mice. DGBX might be a good candidate for inclusion in a randomized study for AA with more data on the possible side effects and doses used in humans. Ultimately, it may be used for applications of traditional medicine against AA in modern complementary and alternative immunosuppressive therapeutics.

A derivative formula, DGBX, which is composed of three herbs (Radix astragali, Radix Angelicae sinensis, and Coptis chinensis Franch), is derived from a famous Chinese herbal formula, Danggui Buxue Tang (DBT) (Radix astragali and Radix Angelicae sinensis). We aimed to investigate the effects of DGBX on the regulation of the balance between proliferation and apoptosis of hematopoietic stem cells (HSCs) due to the aberrant immune response in a mouse model of aplastic anemia (AA). Cyclosporine (CsA), an immunosuppressor, was used as the positive control. Our results indicated that DGBX could downregulate the production of IFNγ in bone marrow cells by interfering with the binding between SLAM and SAP and the expressions of Fyn and T-bet. This herbal formula can also inhibit the activation of Fas-mediated apoptosis, interferon regulatory factor-1-induced JAK/Stat, and eukaryotic initiation factor 2 signaling pathways and thereby induce proliferation and attenuate apoptosis of HSCs. In conclusion, DGBX can relieve the immune-mediated destruction of HSCs, repair hematopoietic failure, and recover the hematopoietic function of HSCs in hematogenesis. Therefore, DGBX can be used in traditional medicine against AA as a complementary and alternative immunosuppressive therapeutic formula.