[Effects of Yisui Jiedu Recipe on JAK2-STAT5 signal transduction pathway in bone marrow hematopoietic cells from patients with myelodysplastic syndrome-refractory anemia].

OBJECTIVE: To investigate the effect of Yisui Jiedu Recipe (YSJDR), a compound traditional Chinese herbal medicine, on cytokines and their corresponding just another kinase 2-signal transducers and activators of transcription 5 (JAK2-STAT5) signal transduction pathway in bone marrow hematopoietic cells from patients with myelodysplastic syndrome-refractory anemia (MDS-RA). METHODS: Fluorogenic quantitative polymerase chain reaction (FQ-PCR) method was established to detect the levels of JAK2, STAT5 and Bcl-xL mRNA expressions, and JAK2-STAT5 signal transduction pathway was activated by granulocyte-macrophage-colony stimulating factor (GM-CSF) in cultured bone marrow hematopoietic cells from 10 patients with MDS-RA. The levels of interleukin-2 (IL-2), interleukin-3 (IL-3), gamma-interferon (gamma-INF) and tumor necrosis factor-alpha (TNF-alpha) in the cultural supernatant of untreated control, AG490-treated and YSJDF-treated cells were measured by enzyme-linked immunosorbent assay. RESULTS: The levels of IL-2 and TNF-alpha in YSJDR-treated group were significantly lower than those in untreated control group and AG490-treated group (P<0.01, P<0.05), and IL-3 level in YSJDP-treated group was remarkably higher than that in the other two groups (P<0.01). There were no significant differences in the levels of IL-2 and IL-3 between AG490-treated group and untreated control group (P>0.05), while the TNF-alpha level in AG490-treated group was decreased obviously as compared with the untreated control group (P<0.01). There was no significant difference in gamma-INF level between YSJDR-treated group and AG490-treated group (P>0.05), while TNF-alpha level in the two groups were significantly lower than that in the untreated control group (P<0.01). The expressions of JAK2, STAT5 and Bcl-xL mRNAs were significantly down-regulated in the YSJDR-treated and the AG490-treated groups as compared with those in the untreated control group (P<0.05, P<0.01), while there were no differences in the expressions of JAK2, STAT5 and Bcl-xL mRNAs between YSJDR-treated group and AG490-treated group. CONCLUSION: YSJDR can modulate cytokine level in bone marrow hematopoietic cells of MDS-RA, suppress JAK2-STAT5 signal transduction, and inhibit the Bcl-xL mRNA expression.

[Effects of Jianpi Bushen Huoxue recipe on hematopoietic cell apoptosis in a mouse model of aplastic anemia].

OBJECTIVE: Adopting methods of cell culture to explore the effects and mechanisms of Jianpi Bushen Huoxue Prescription (JPBSHXP), a traditional Chinese compound herbal medicine for strengthening spleen, reinforcing kidney and activating blood circulation, in inhibiting hematopoietic cells apoptosis in a mouse model of aplastic anemia (AA). METHODS: Blood serum of AA mice was made from an AA mouse model. Blood serums containing different traditional Chinese compound herbal medicine were made from rats after intragastric administration of JPBSHXP and its related decoctions, respectively. Bone marrow cells of normal mice were incubated by these blood serums for 24 hours, respectively. The apoptosis of the bone marrow cells were assayed by flow cytometry and transmission electron microscopy (TEM). RESULTS: It was indicated that the bone marrow cells of normal mice incubated with blood serum of AA mice displayed typical apoptosis. The apoptosis rates of bone marrow cells of the AA mice incubated by blood serum containing different traditional Chinese herbal medicine were decreased. The effect of Bushen (reinforcing kidey) Recipe was better than Jianpi (strengthening spleen) Recipe and Huoxue (activating blood circulation) Recipe, while the effect of JPBSHXP was the best. TEM results showed that the effect of Bushen Recipe was better than that of the Jianpi Recipe and the Huoxue Recipe, while the effect of JPBSHXP was the best. CONCLUSION: JPBSHXP and its related decoctions can significantly decrease the apoptosis rate of bone marrow mononuclear cells of the AA mice. It is inferred that JPBSHXP can promote bone marrow hematogenesis.

[Impact of cyclosporine A on the expression of T-bet, GATA-3, relevant signal transduction molecules, cytokine and Th1/Th2 balance in patients with chronic aplastic anemia].

The study was aimed to explore the effects of T-bet (T-box expressed in T cell), GATA-3(GATA binding protein 3) and relevant signal transduction pathways on the immune-related pathogenesis of chronic aplastic anemia (CAA), and to investigate the immunological regulation mechanism in the treatment of CAA by using cyclosporine A (CsA) at the level of Th cell imbalance, transcriptional factors, and relevant signal pathways. The real-time fluorescent quantitative polymerase chain reaction (real-time FQ-PCR) was used to determine the mRNA expression of T-bet, GATA-3, signal transducers and activators of transcription 4 (STAT4) and signal transducers and activators of transcription 6 (STAT6) in peripheral blood mononuclear cell (PBMNC) of CAA patients before and after treatment with CsA; the flow cytometry (FCM) and enzyme linked immunosorbent assay (ELISA) were used to determine the Th1/Th2 proportion in peripheral blood, and levels of IFN-γ, IL-12 and IL-4 in PBMNC-cultured supernatant. Healthy people were included to test the above indexes. The results showed that the mRNA expression of PBMNC T-bet, STAT4, T-bet/GATA-3 ratio, Th1 proportion, Th1/Th2 ratio and levels of IFN-γ and IL-12 in PBMNC-cultured supernatant of CAA patients were significantly higher than those of healthy people (p < 0.01). After treating with CsA for 6 months of CsA treatment, expression of T-bet, STAT4, T-bet/GATA-3 ratio, Th1 proportion, IFN-γ and IL-12 levels were lower than before, however, the expression of T-bet, STAT4, T-bet/GATA-3 ratio, Th1 proportion and IFN-γ had not been reduced to normal state. Compared to healthy people, no significant difference existed in the mRNA expression of GATA-3, STAT6, Th2 proportion, as well as level of IL-4 before and after treatment (p>0.05). It is concluded that the abnormal activation of IFN-γ/T-bet and IL-12/STAT4 pathways, as well as Th balance deviating to Th1 excursion play vital roles in the immunological pathogenesis of AA. CsA lowers the abnormal activation of IFN-γ/T-bet and IL-12/STAT4 pathways to correct Th1 hyperpolarization, which may reduce the abnormally activated cell-mediated immunity and relax hematopoietic depression of AA patients.

[Study on the changes of IL-3 and its receptor in mice with immune-mediated aplastic anemia].

The aim of this study was to find new idea for clinical treatment of aplastic anemia. Immune-mediated aplastic anemia mice were developed, IL-3 in the supernatant with PHA stimulating splenic cells was detected by ELISA, semi-quantiting analysis of IL-3R was performed by point hybridization. The results showed that the IL-3 level in the supernatant with PHA stimulating splenic cells of immune-mediated aplastic anemia mice was higher than controls, difference between them was significant (P <0.001), while amount of IL-3 receptor by semi-quantiting analysis was lower than control significantly. In conclusion, the IL-3 receptor expression level is important for pathogenesis and treatment strategy of aplastic anemia.