The herbal decoction modified Danggui Buxue Tang attenuates immune-mediated bone marrow failure by regulating the differentiation of T lymphocytes in an immune-induced aplastic anemia mouse model.

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.

Decreased expression of vitamin D receptor may contribute to the hyperimmune status of patients with acquired aplastic anemia.

Acquired aplastic anemia (AA) is an immune-mediated bone marrow failure syndrome. 1α,25-Dihydroxyvitamin D3 [1,25(OH)2 D3 ], the biologically active metabolite of vitamin D, is a critical modulator of immune response via binding with vitamin D receptor (VDR). Previous studies have established that 1,25(OH)2 D3 and VDR were involved in the pathogenesis of some autoimmune diseases. In this study, we evaluated the involvement of 1,25(OH)2 D3 and VDR on T-cell responses in AA. Plasma 25(OH)D3 levels were comparable between patients with AA and healthy controls. Surprisingly, VDR mRNA was significantly lower in untreated patients with AA than in healthy controls. Subsequent in vitro experiments revealed that 1,25(OH)2 D3 treatment suppressed the proliferation of lymphocytes and inhibited the secretion of interferon-γ, tumor necrosis factor-α, and interleukin-17A, meanwhile promoting the production of transforming growth factor-ß1 in patients with AA. Moreover, 1,25(OH)2 D3 inhibited the differentiation of type 1 and Th17 cells but induced the differentiation of type 2 and regulatory T cells. Interestingly, VDR mRNA was elevated in healthy controls after 1,25(OH)2 D3 treatment, but not in patients with AA. In conclusion, decreased expression of VDR might contribute to the hyperimmune status of AA and appropriate vitamin D supplementation could partly correct the immune dysfunction by strengthening signal transduction through VDR in patients with AA.

Role of heteroplasmic mutations in the mitochondrial genome and the ID4 gene promoter methylation region in the pathogenesis of chronic aplastic anemia in patients suffering from Kidney yin deficiency.

OBJECTIVE: To analyze changes in gene amplification in the mitochondrial genome and in the ID4 gene promoter methylation region in patients with chronic aplastic anemia (CAA) suffering from Kidney (Shen) yin deficiency or Kidney yang deficiency. METHODS: Bone marrow and oral epithelium samples were collected from CAA patients with Kidney yin deficiency or Kidney yang deficiency (20 cases). Bone marrow samples were collected from 20 healthy volunteers. The mitochondrial genome was amplified by polymerase chain reaction (PCR), and PCR products were used for sequencing and analysis. RESULTS: Higher mutational rates were observed in the ND1-2, ND4-6, and CYTB genes in CAA patients suffering from Kidney yin deficiency. Moreover, the ID4 gene was unmethylated in bone marrow samples from healthy individuals, but was methylated in some CAA patients suffering from Kidney yin deficiency (positive rate, 60%) and Kidney yang deficiency (positive rate, 55%). CONCLUSIONS: These data supported that gene mutations can alter the expression of respiratory chain enzyme complexes in CAA patients, resulting in energy metabolism impairment and promoting the physiological and pathological processes of hematopoietic failure. Functional impairment of the mitochondrial respiration chain induced by gene mutation may be an important reason for hematopoietic failure in patients with CAA. This change is closely related to maternal inheritance and Kidney yin deficiency. Finally, these data supported the assertion that it is easy to treat disease in patients suffering from yang deficiency and difficult to treat disease in patients suffering from yin deficiency.

Complete sequence analysis of mitochondrial DNA and telomere length in aplastic anemia.

The present study was primarily undertaken to examine the hypothesis that mitochondrial DNA (mtDNA) mutations and telomere length may be associated with aplastic anemia (AA). Our study included a single institution analysis of 40 patients presenting with AA first diagnosed at the Affiliated Hospital of Shandong, University of Traditional Chinese Medicine between 2010 and 2013. Bone marrow and oral epithelial samples were collected from patients with AA (n=40) for mtDNA mutation and telomere length determinations. Bone marrow specimens were collected from 40 healthy volunteers as controls for the examination of telomere length. The mitochondrial genome was amplified by polymerase chain reaction (PCR), and the products were used for sequencing and analysis. We detected 146 heteroplasmic mutations in 18 genes from 40 patients with AA, including 39 silent mutations and 28 frameshift mutations. We used the gamma globin gene (HBG) as the control gene in real-time PCR to survey the relative telomere length measurements of the patients with AA and the healthy volunteers. Telomere length was expressed as the relative T/S value. We observed a negative correlation between the mtDNA non-silent mutation and the white blood cell (WBC) count, hemoglobin and platelet count. Of note, there was a positive correlation between the relative T/S value and WBC count, hemoglobin and platelet count, and a negative correlation between the non-silent mutation and the relative T/S value. We conclude that the functional impairment of the mitochondrial respiratory chain induced by mutation and telomere length shortening may play an important role in the process of hematopoietic failure in patients with AA. Additionally, mtDNA mutations and telomere length shortening influenced each other.

Effect of kidney-reinforcing, blood-activating and stasis-removing recipes on adhesion molecule expression of bone marrow mesenchymal stem cells from chronic aplastic anemia patients.

OBJECTIVE: To explore the effect of kidney-reinforcing, blood-activating and stasis-removing recipes on adhesion molecule expression of bone marrow mesenchymal stem cells (MSCs) from patients with chronic aplastic anemia (CAA). METHODS: We used three Traditional Chinese Medicine recipes, namely a kidney-reinforcing recipe (KRR), blood-activating and stasis-removing recipe (BASRR), and kidney-reinforcing, blood-activating and stasis-removing recipe (KRBASRR), and a normal saline control to prepare herbal medicine serum in Sprague Dawley rats. Thirty CAA patients were enrolled in the experimental group, including 17 kidney-Yang deficient patients and 13 kidney-Yin deficient patients. Ten healthy individuals were included in the control group. MSCs were isolated from bone marrow samples, and the cell density was observed to measure their proliferation ability by microscopy on days 2, 7, and 14 after isolation. In addition, the expression of adhesion molecules of bone marrow MSCs (CD106, CD49d, CD31 and CD44) were detected by flow cytometry after 48 h of treatment with the four different herbal medicine serums. RESULTS: The proliferation of MSCs from kidney-Yang deficient and kidney-Yin deficient patients was weaker than that of MSCs from the control group. The expression of all adhesion molecules of bone marrow MSCs from CAA patients was obviously lower than that in the control group (P < 0.01). The expression of CD49d and CD31 in MSCs from patients with a kidney-Yin deficiency was lower than in those with a kidney-yang deficiency (P < 0.05 and P < 0.01, respectively). For kidney-Yang deficient patients, CD31 expression in the KRBASRR group was significantly higher than that in the BASRR group (P < 0.01), while CD44 in the KRBASRR group was significantly higher than that in both KRR and BASRR groups (P < 0.01). For kidney-Yin deficient patients, CD106 and CD49d expression in the KRBASRR group was obviously higher than that in the KRR group (P < 0.05), while CD31 and CD44 expression in the KRBASRR group was significantly higher than that in both KRR and BASRR groups (P < 0.05 and P < 0.01, respectively). CONCLUSION: The bone marrow microenvironment in CAA patients is abnormal. The effect of KRBASRR may be better than that of KRR and BASRR for kidney-Yang deficient and kidney-Yin deficient patients by improving the expression levels of MSC adhesion molecules.

[Gene expression profile analysis of T lymphocytes involved in pathogenesis of severe aplastic anemia by using bioinformatics method as a novel way of drug screening].

This study was aimed to explore the gene expression profile characteristics of T lymphocytes involved in pathogenesis of severe aplastic anemia (SAA) and to predict putative curative drugs for SAA by using biological principle of similarity contrast of gene expression profiles between drugs and diseases. SAA and T lymphocyte were used as key words to search gene expression datasets related to pathogenesis of SAA in public Gene Expression Omnibus (GEO) of NCBI. After significance test, gene expression profiling involved in pathogenesis of SAA were screened and applied to cluster analysis. And then SAA-related gene expression profiles were thrown into pharmacological gene expression datasets of 3000 candidate drugs for similarity analysis and significantly negative correlation was used as a screening criterion for selecting putative curative drugs of SAA. The results showed that only one gene expression dataset was found out, i.e. GSE3807. Computational bioanalysis identified a total of 515 candidate genes of T lymphocyte involved in pathogenesis of SAA, whose expression level exceeded more than 2-fold. Among them, 202 genes were upregulated and 313 genes were downregulated. Cluster analysis showed that those genes belonged to different pathways, including nucleic acid metabolic process, ubiquitin-dependent protein catabolic process, Golgi apparatus protein transport, protein phosphorylation and immunoglobin/major histocompatibility complex. Similarity analysis of gene expression profiles of SAA and drugs showed that hydroxycamptothecin and metformin might have a potential therapeutic efficacy on SAA. It is concluded that by means of novel bioinformatics method, gene expression profiling combined with similarity analysis between disease-related gene expression and pharmacological gene expression profiles may be a novel way of drug screening for SAA.

[Changes in T-cell receptor repertoire in aplastic anemia and effects of Shengxue Mixture].

OBJECTIVE: To explore the immune pathogenesis of aplastic anemia (AA) and the therapeutic effects of Shengxue Mixture (SM) through the gene expressions of subfamilies of T-cell receptor variable region beta (TCR Vbeta) using immunologic and molecular biologic technology. METHODS: Gene expressions of TCR Vbeta sub-families in peripheral blood mononuclear cells from 20 AA patients were detected before and after treatment with SM using RT-PCR and gene scanning method. RESULTS: TCR Vbeta gene repertoire of the 24 subfamily genes deviated in AA patients, and the oligoclonal gene expressions increased obviously compared with those in healthy people (P < 0.01), including Vbeta2, 5, 6, 15, 16, 22, and 23 were found in 30%-50% AA patients, and Vbeta8, 21 were in more than 50% patients. These oligoclonal genes reduced significantly after treatment with SM compared with those before treatment (P < 0.05). CONCLUSION: Multiple TCR Vbeta subfamilies of clonal proliferation participate in the pathogenesis of AA. SM can rectify the deviation of TCR Vbeta gene repertoire, reduce the abnormal clonal proliferation of T cells, thus to alleviate the immune injury to hematopoietic tissue, and thus to benefit the recovery of hematopoiesis of bone marrow.