Objective To investigate the effects of astragaloside Ⅳ(AS-Ⅳ)on axon growth inhibitory factor A(Nogo-A)and its downstream pathway protein RHO-associated coiled spiral kinase 2(ROCK2)in experimental autoimmune encephalomyelitis(EAE)mice,and to explore the mechanism by which it promotes axon repair and regeneration.Methods EAE model was induced in C57BL/6 female mice by subcutaneous injection of myelin oligodendrocyte glycoprotein 35-55(MOG35-55).Mice were randomly divided into EAE group and AS-Ⅳ group(n=8 per group).EAE group received intraperitoneal injection of PBS on the 3rd day post-immunization,while AS-Ⅳ group was administered AS-Ⅳ at a dosage of 30mg/(kg.d)once daily,0.2 ml per injection,for 25 consecutive days.On the 28th day post-immunization,the expression levels of growth-associated protein 43(GAP-43),neuronal core antigen(NeuN),microtubule associated protein 2(MAP-2),glial fibroacidic protein(GFAP),and Iba1 in the spinal cord were detected using immunofluorescence assay.Real-time fluorescence quantitative PCR(qRT-PCR)was conducted to detect mRNA expression levels of GAP-43,Nogo-A,and Nogo receptor(NgR)genes.Western blotting was utilized to determine the expression levels of GAP-43,Nogo-A,ROCK2,phosphorylated myosin phosphatase(p-MYPT1),B-lymphoblastoma-2(Bcl-2),and Bcl-2 associated X protein(Bax).Results Compared with EAE group,AS-Ⅳ treatment significantly reduced the positive cell expression rates of Iba1 microglia and GFAP astrocyte in spinal cord(P<0.01 and P<0.001,respectively),while it also increased the positive expression rates of NeuN and MAP-2(P<0.001 and P<0.05,respectively).The treatment also upregulated the expression level of anti-apoptotic factor Bcl-2(P<0.001)and downregulated the expression level of pro-apoptotic factor Bax(P<0.05),leading to an increase in Bcl-2/Bax ratio(P<0.05).Furthermore,AS-Ⅳ enhanced the expression of GAP-43 protein(P<0.05)and decreased the mRNA expression levels of neuroregeneration inhibitor Nogo receptor(NgR)and ROCK2 gene(P<0.001,P<0.05,respectively);as well as decreased the expression levels of Nogo-A,ROCK2 and p-MYPT1 proteins(P<0.05,P<0.001).Conclusion AS-Ⅳ may inhibit the activation of microglia and astrocytes and neuronal apoptosis in EAE mice by inhibiting Nogo-A and downstream pathway ROCK 2,thereby promoting the expression of GAP-43,NeuN and MAP-2,alleviating neuronal damage,and facilitating axon repair and regeneration.

Spontaneous remission (SR) of leukemia is a rare event in clinic, which possibly correlated with severe infection and sepsis, but its exact mechanism has not been confirmed. Plasmacytoid dendritic cells (pDC) and myeloid dendritic cells (mDC) play a key role in innate and adaptive immunity respectively. A patient with severe infection of staphylococcus aureus acquired completely spontaneous remission (SR), moreover a increased number of pDC were observed, suggesting that bacteria-activated pDC may play an important role in SR. This study was purposed to explore if the bacteria can stimulate pDC successfully and get a functional pDC. Both pDC and mDC were isolated from freshly collected, leukocyte-rich buffy coats from healthy blood donor and leukemic patient with SR by using MACS and FACS. The pDC were cultured in RPMI 1640 medium and were stimulated with different kinds of bacteria and the expression of CD40, CD86 and HLA-DR on the cell surface was analyzed by flow cytometry. The cytokine (IFN-α, IL-12, IFN-γ, IL-2, IL-4, IL-10) production was measured by using ELISA kits. The results showed that the stimulation with staphylococcus aureus and pseudomonas aeruginosa resulted in the maturation of pDC, which secrete a large number of IFN-α and promote the differentiation of naive CD4⁺ T cells to Th1 cells. The activated pDC expressed high level of CD40 and CD86 and showed higher T cell stimulatory capacities. It is concluded that staphylococcus aureus and pseudomonas aeruginosa can activate pDC, the activated pDC secrete high quantity of IFN-α. This result suggests that bacteria stimulated pDC may play a key role in SR of leukemia following severe infections.
CD4-Positive T-Lymphocytes ; Dendritic Cells ; immunology ; microbiology ; Humans ; Interferon-alpha ; Interleukin-10 ; Interleukin-12 ; Interleukin-2 ; Interleukin-4 ; Leukemia ; diagnosis ; immunology ; microbiology ; Remission, Spontaneous ; Staphylococcus aureus

OBJECTIVE: To study the mechanism of Shengmai Injection (SMI, ) on anti-sepsis and protective activities of intestinal mucosal barrier. METHODS: The contents of 11 active components of SMI including ginsenoside Rb1, Rb2, Rb3, Rd, Re, Rf, Rg1, Rg2, ophioposide D, schisandrol A and schisantherin A were determined using ultra-performance liquid chromatography. Fifty mice were randomly divided into the blank, the model, the low-, medium- and high-dose SMI groups (0.375, 0.75, 1.5 mL/kg, respectively) by random number table, 10 mice in each group. In SMI group, SMI was administrated to mice daily via tail vein injection for 3 consecutive days, while the mice in the blank and model groups were given 0.1 mL of normal saline. One hour after the last SMI administration, except the blank group, the mice in other groups were intraperitoneally injected with lipopolysaccharide (LPS) saline solution (2 mL/kg) at a dosage of 5 mL/kg for development of endotoxemia mice model. The mice in the blank group were given the same volume of normal saline. Inflammatory factors including interferon-γ (INF-γ), tumor necrosis factor-α (TNF-α), interleukin (IL)-2 and IL-10 were measured by flow cytometry. Myosin light-chain kinase (MLCK), nuclear factor κB (NF-κB) levels, and change of Occludin proteins in jejunum samples were analyzed by Western blot. RESULTS: The decreasing trends of INF-γ, TNF-α and IL-2 were found in serum of SMI treatment groups. In SMI-treated mice, the content of Occludin increased and MLCK protein decreased compared with the model group (P<0.05 or P<0.01). The content of cellular and nuclear NF-κB did not change significantly (P>0.05). CONCLUSION SMI may exert its anti-sepsis activity mainly through NF-κB-pro-inflammatory factor-MLCK-TJ cascade.
Animals ; Drug Combinations ; Drugs, Chinese Herbal ; Mice ; NF-kappa B/metabolism* ; Occludin ; Saline Solution ; Sepsis/drug therapy* ; Tumor Necrosis Factor-alpha/metabolism*

Prohibitin (PHB), an evolutionarily conserved mitochondrial inner membrane protein, is highly expressed in cells that require strong mitochondrial function. Recently, we demonstrated that the deletion of Phb in spermatocytes results in impaired mitochondrial function. In addition, PHB expression in the mitochondrial sheath of human sperm has a significantly negative correlation with mitochondrial reactive oxygen species levels, but a positive one with mitochondrial membrane potential and sperm motility. These results suggest that mitochondrial PHB expression plays a role in sperm motility. However, the mechanism of PHB-mediated regulation of sperm motility remains unknown. Here, we demonstrate for the first time that PHB interacts with protein kinase B (AKT) and exists in a complex with phospho-PHB (pT258) and phospho-AKT in the mitochondrial sheath of murine sperm, as determined using colocalization and coimmunoprecipitation assays. After blocking AKT activity using wortmannin (a phosphatidylinositol 3-kinase [PI3K] inhibitor), murine sperm have significantly ( P < 0.05) decreased levels of phospho-PHB (pT258) and the total and progressive motility. Furthermore, significantly ( P < 0.05) lower levels of phospho-PI3K P85 subunit α+γ (pY199 and pY467) and phospho-AKT (pS473; pT308) are found in sperm from infertile asthenospermic and oligoasthenospermic men compared with normospermic subjects, which suggest a reduced activity of the PI3K/AKT pathway in these infertile subjects. Importantly, these sperm from infertile subjects also have a significantly ( P < 0.05) lower level of phospho-PHB (pT258). Collectively, our findings suggest that the interaction of PHB with AKT in the mitochondrial sheath is critical for sperm motility, where PHB phosphorylation (pT258) level and PI3K/AKT activity are key regulatory factors.

OBJECTIVE: To investigate the clinical characteristics, laboratorial and bone marrow pathological features of primary thrombocytopenia (ET) patients with different mutations of CALR, JAK2 and MPL genes. METHODS: The chinical data of 120 cases of ET in Jiangsu provincial people's hospital/ The First Affiliated Hospital of Nanjing Medical University from January 2015 to December 2017 were collected and analyzed, including 76 cases with JAK2 gene mutation, 40 cases with CALR gene mutation, 2 cases with MPL gene mutations, 2 cases without gene mutation. RESULTS: Among the ET patients, compared with the JAK2 gene mutation, CALR gene mutation showed statistically significant deareament of white blood cells and hemoglobin (P=0.001, P=0.01) and the male platelets in CALR group showed significant increament (P=0.04). Fourthermore, the average number of megakaryocytes and its cluster numbers in each hight power field of vision showed statistically significant decreament in CALR group as compared with JAK2 group (P=0.001, P=0.001), and thrombotic events in CALR group were signicantly lower than those in JAK2 group (7.5% vs 18.4%) (P=0.03). CONCLUSION Mutations of CALR, JAK2 have different clinical characteristics and blood pathological changes of Chinese ET patients, and their clinical significance is worth to explore.
Bone Marrow ; Calreticulin ; genetics ; China ; Humans ; Janus Kinase 2 ; genetics ; Male ; Mutation ; Receptors, Thrombopoietin ; genetics ; Thrombocythemia, Essential