Effects of Sodium Chlorophyllin Copper on APO-1 Expression in Bone Marrow Mesenchymal Stem Cells of Rats with Aplastic Anaemia.

Background: Aplastic anaemia (AA) is a highly prevalent blood disorder in the East and Southeast Asian countries, and a proportion of the patients is poorly treated with immunosuppressive agents. This study is aimed at exploring the effects of sodium copper chlorophyllin (SCC) on rats with AA and at providing the theoretical basis for the treatment of AA using traditional Chinese medicine. Methods: A rat model of AA was induced by combining 5-fluorouracil with busulfan, and different groups were treated with 25 mg/kg cyclosporin A (CsA) and low-, medium-, and high-dose SCC (25-, 50-, and 100-mg/kg; L-, M-, and H-SCC, respectively). A comparative analysis of peripheral blood counts, T-cell subsets, cytokine levels, bone marrow pathology, and APO-1 expression in mesenchymal stem cells in each group was conducted. Results: SCC can increase the platelet count and haemoglobin concentration in the peripheral blood of AA rats, whereas bone marrow biopsies revealed that the number of nucleated cells and megakaryocytes of SCC-treated rats increased compared with the model group. This was particularly evident in the H-SCC group. As regards the correction of immune function, unlike CsA, which reduced the absolute CD8+ T-cell count, SCC corrected the imbalanced CD4/CD8 ratio by increasing the absolute CD4+ T-cell count, whereas SCC increased the number of regulatory T-cells and reduced the level of interferon-γ in AA rats. When comparing the expression of APO-1 in the MSCs, results of the reverse-transcriptase polymerase chain reaction and Western blot analysis showed that SCC can increase the expression of APO-1 both at the mRNA and protein levels. Conclusion: We found that SCC can improve haematopoietic function and regress immune disorders in AA rats, which enhanced the expression of APO-1 in bone marrow MSCs. This may be one of the mechanisms of SCC in treating AA.

Blood circulation activating effect of Sanqi (Radix Notoginseng) on venous thromboembolism rat.

OBJECTIVE: To explore the blood circulation activating effect and mechanism of Sanqi (Radix Notoginseng) in vivo, using a venous thromboembolism (VTE) rat model. METHODS: We established the VTE rat model, and then intervened with low molecular weight heparin (LMWH), as well as low, medium and high doses of Sanqi (Radix Notoginseng), to observe the blood circulation activating effect of Sanqi (Radix Notoginseng) on VTE rats. RESULTS: After the treatment with high concentrations of Sanqi (Radix Notoginseng), the pulmonary thromboembolism was alleviated, and the lower limb thrombosis was markedly improved. Moreover, the expression quantities of plasma activated partial thromboplastin time, prothrombin time and D-dimer, as well as endothelin, von Willebrand factor, and plasminogen activator inhibitor-1 in thrombosis segment tissues were markedly down-regulated; while those of nitric oxide and tissue-type plasminogen activator were up-regulated. After low and medium concentration Sanqi (Radix Notoginseng) treatment, no obvious improvement was observed in each index. Moreover, the high concentration Sanqi (Radix Notoginseng) showed comparable efficacy to the positive drug LMWH. CONCLUSION: This data suggests that high concentration of Sanqi (Radix Notoginseng) is effective in preventing and treating VTE.

Efficacy of Sanqi (Radix Notoginseng) in treating cerebral hemorrhage in rats with traumatic brain injury.

OBJECTIVE: To evaluate the protective efficacy of Sanqi (Radix Notoginseng) on cerebral hemorrhage in a rat model of traumatic brain injury (TBI) by investigating plasminogen activator inhibitor-1 (PAI-1), tissue-type plasminogen activator (t-PA), nuclear factor-κB (NF-κB, p-p65), nitric oxide (NO), endothelin (ET), cluster differentiation (CD61CD62), and coagulation. METHODS: The free-fall method was used to create a rat model of TBI. Forty-eight rats were randomly divided into six groups: the blank group, sham group, model group, low-dose Sanqi (Radix Notoginseng) group, middle-dose Sanqi (Radix Notoginseng) group, and high-dose Sanqi (Radix Notoginseng) group. At 24 h after the model was created, we investigated brain MRI, brain tissue morphology using HE staining, flow cytometry, and immunohistochemical changes. RESULTS: Cerebral hemorrhage was aggravated in TBI rats (observed in brain specimens, brain MRI, and brain tissue HE). Cerebral immunohistochemistry results demonstrated that the expression of t-PA, PAI-1 and p-p65 increased significantly in TBI rats, while t-PA/PAI-1 had a significant decrease. In addition, CD61CD62, D2D, and ET were significantly increased in TBI rats, and PT and APTT were significantly prolonged; in contrast, NO was significantly decreased. Sanqi (Radix Notoginseng) decreased cerebral hemorrhage in TBI rats (observed in brain MRI and brain tissue HE), and increased t-PA/PAI-1, CD61CD62 significantly. It also significantly decreased the expression of t-PA, PAI-1, and p-p65 in brain immunohistochemistry and significantly decreased PT, APTT, D2D, and ET. However, there were no differences in NO between the model group and the Sanqi (Radix Notoginseng) group. CONCLUSION: Sanqi (Radix Notoginseng) can decrease the expression of p-p65, increase t-PA/PAI-1, and stem traumatic intracranial hemorrhage in a TBI rat model.

Efficacy and advantages of modified Traditional Chinese Medicine treatments based on "kidney reinforcing" for chronic aplastic anemia: a randomized controlled clinical trial.

OBJECTIVE: To compare the efficacy of modified treatments based on "kidney reinforcing" in the management of chronic aplastic anemia (CAA), and explore their advantages and specialties. METHODS: One hundred and eleven patients with CAA were randomly divided into three groups: kidney reinforcing alone (KA), "kidney reinforcing and Qi tonifying" (KQ), and "kidney reinforcing and blood circulation invigorating" (KC). Normal and positive control groups were also formed. All patients were treated for 6 months (two courses). Hemograms, Traditional Chinese Medicine (TCM) syndrome scores, and therapeutic effects were assessed, and changes in T-lymphocyte subsets, regulatory T cells and cytokines were detected. RESULTS: The KQ and KC groups had lower TCM syndrome scores than the positive control group after 6 months (P < 0.05). The KQ group had a higher overall efficacy than the positive control group after 3 months (P < 0.05), while platelet counts increased in the KC group after 6 months (P < 0.05). CD3+ T-lymphocyte ratios decreased only in the KQ group, while CD3 + CD4 + CD8 − Tlymphocytes increased only in the KC group after 6 months (P < 0.05). Levels of interferon-γ, tumor necrosis factor tor-α, interleukin (IL)-2 and IL-6 decreased and levels of IL-4 and IL-10 increased in all treated groups after 6 months. Levels of IL-6 in the KQ and KC groups were lower than those in the positive control group (P < 0.05). CONCLUSION: Treatments based on kidney reinforcing have a rebalancing effect on cytotoxic and T helper cells, and regulate expression of interferon- γ, IL-2, IL-6 and IL-4. KQ may be more effective in treating CAA, and KC may have an advantage in platelet recovery.

The Peanut (Arachis hypogaea L.) Gene AhLPAT2 Increases the Lipid Content of Transgenic Arabidopsis Seeds.

Lysophosphatidic acid acyltransferase (LPAT), which converts lysophosphatidic acid (LPA) to phosphatidic acid (PA), catalyzes the addition of fatty acyl moieties to the sn-2 position of the LPA glycerol backbone in triacylglycerol (TAG) biosynthesis. We recently reported the cloning and temporal-spatial expression of a peanut (Arachis hypogaea) AhLPAT2gene, showing that an increase in AhLPAT2 transcript levels was closely correlated with an increase in seed oil levels. However, the function of the enzyme encoded by the AhLPAT2 gene remains unclear. Here, we report that AhLPAT2 transcript levels were consistently higher in the seeds of a high-oil cultivar than in those of a low-oil cultivar across different seed developmental stages. Seed-specific overexpression of AhLPAT2 in Arabidopsis results in a higher percentage of oil in the seeds and greater-than-average seed weight in the transgenic plants compared with the wild-type plants, leading to a significant increase in total oil yield per plant. The total fatty acid (FA) content and the proportion of unsaturated FAs also increased. In the developing siliques of AhLPAT2-overexpressing plants, the expression levels of genes encoding crucial enzymes involved in de novo FA synthesis, acetyl-CoA subunit (AtBCCP2) and acyl carrier protein 1 (AtACP1) were elevated. AhLPAT2 overexpression also promoted the expression of several key genes related to TAG assembly, sucrose metabolism, and glycolysis. These results demonstrate that the expression of AhLPAT2 plays an important role in glycerolipid production in peanuts.

Effects of sodium copper chlorophyllin on mesenchymal stem cell function in aplastic anemia mice.

OBJECTIVE: To investigate the effects of sodium copper chlorophyllin (SCC) on the proliferation, differentiation and immunomodulatory function of mesenchymal stem cells (MSCs) from mice with aplastic anemia. METHODS: A mouse model of aplastic anemia was established by exposure of BALB/c mice to sublethal doses of 5.0 Gy Co60 γ radiation, followed by transplantation of 2×10(6) lymph node cells from DBA/2 donor mice within 4 h after radiation. Aplastic anemic BALB/c mice were randomly divided into six groups: the treated groups, which received 25, 50, or 100 mg/kg/day SCC, respectively; a positive control group treated with cyclosporine A (CsA); and an untreated model control group (model group); while, the non-irradiated mice as the normal control group. SCC or CsA were administered by gastrogavage for 20 days, starting on day 4 after irradiation. Peripheral blood cells were counted and colony-forming fibroblasts (CFU-F) in the bone marrow were assayed. The ability of MSCs to form calcium nodes after culture in osteoinductive medium was also observed. The immunosuppressive effect of MSCs on T lymphocytes was analyzed by enzyme-linked immunosorbent assay and flow cytometry, to evaluate the efficacy of SCC in mice with aplastic anemia. RESULTS: Peripheral blood white cell and platelet counts were increased by medium and high SCC doses, compared with the untreated control. CFU-Fs were also increased compared with the untreated control, and the numbers of calcium nodes in MSCs in osteoinductive medium were elevated in response to SCC treatment. The percentage of Forkhead box protein 3 (FOXP3(+)) T cells was increased in T cell-MSC cocultures, and the cytokine transforming growth factor ß1 was up-regulated in SCC-treated groups. CONCLUSION: The results of this study suggest that SCC not only promotes the proliferation and differentiation of MSCs, but also improves their immunoregulatory capacity in mice with aplastic anemia.

Abundant microsatellite diversity and oil content in wild Arachis species.

The peanut (Arachis hypogaea) is an important oil crop. Breeding for high oil content is becoming increasingly important. Wild Arachis species have been reported to harbor genes for many valuable traits that may enable the improvement of cultivated Arachis hypogaea, such as resistance to pests and disease. However, only limited information is available on variation in oil content. In the present study, a collection of 72 wild Arachis accessions representing 19 species and 3 cultivated peanut accessions were genotyped using 136 genome-wide SSR markers and phenotyped for oil content over three growing seasons. The wild Arachis accessions showed abundant diversity across the 19 species. A. duranensis exhibited the highest diversity, with a Shannon-Weaver diversity index of 0.35. A total of 129 unique alleles were detected in the species studied. A. rigonii exhibited the largest number of unique alleles (75), indicating that this species is highly differentiated. AMOVA and genetic distance analyses confirmed the genetic differentiation between the wild Arachis species. The majority of SSR alleles were detected exclusively in the wild species and not in A. hypogaea, indicating that directional selection or the hitchhiking effect has played an important role in the domestication of the cultivated peanut. The 75 accessions were grouped into three clusters based on population structure and phylogenic analysis, consistent with their taxonomic sections, species and genome types. A. villosa and A. batizocoi were grouped with A. hypogaea, suggesting the close relationship between these two diploid wild species and the cultivated peanut. Considerable phenotypic variation in oil content was observed among different sections and species. Nine alleles were identified as associated with oil content based on association analysis, of these, three alleles were associated with higher oil content but were absent in the cultivated peanut. The results demonstrated that there is great potential to increase the oil content in A. hypogaea by using the wild Arachis germplasm.

Expressed sequence tags in cultivated peanut (Arachis hypogaea): discovery of genes in seed development and response to Ralstonia solanacearum challenge.

Although an important oil crop, peanut has only 162,030 expressed sequence tags (ESTs) publicly available, 86,943 of which are from cultivated plants. More ESTs from cultivated peanuts are needed for isolation of stress-resistant, tissue-specific and developmentally important genes. Here, we generated 63,234 ESTs from our 5 constructed peanut cDNA libraries of Ralstonia solanacearum challenged roots, R. solanacearum challenged leaves, and unchallenged cultured peanut roots, leaves and developing seeds. Among these ESTs, there were 14,547 unique sequences with 7,961 tentative consensus sequences and 6,586 singletons. Putative functions for 47.8 % of the sequences were identified, including transcription factors, tissue-specific genes, genes involved in fatty acid biosynthesis and oil formation regulation, and resistance gene analogue genes. Additionally, differentially expressed genes, including those involved in ethylene and jasmonic acid signal transduction pathways, from both peanut leaves and roots, were identified in R. solanacearum challenged samples. This large expression dataset from different peanut tissues will be a valuable source for marker development and gene expression analysis. It will also be helpful for finding candidate genes for fatty acid synthesis and oil formation regulation as well as for studying mechanisms of interactions between the peanut host and R. solanacearum pathogen.