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ObjectiveTo observe the effects of Fuzitang （FZT） on the proliferation of MH7A cells， the human rheumatoid arthritis synovial fibroblasts， and the expression of miR-155 and explore its anti-rheumatoid arthritis mechanism. MethodMH7A cells were cultured in vitro and divided into a blank group， high- （25 g·L-1） and low-dose （12.5 g·L-1） FZT groups， and a positive drug group （hydroxychloroquine， 0.006 25 g·L-1）. The cell proliferation was detected by cell counting kit-8（CCK-8） method， and the change in the MH7A cell cycle was detected by flow cytometry. The mRNA expression of miR-155 and its downstream genes， including SH2 domain-containing inositol 5-phosphatase-1（SHIP-1）， protein kinase B 3（Akt3）， and mammalian target of rapamycin（mTOR）， was detected by Real-time fluorescence quantitative polymerase chain reaction（Real-time PCR）， and the protein expression of phosphatidylinositol 3-kinase （PI3K）， Akt3， and mTOR was detected by Western blot. ResultFZT in vitro in a concentration of 6.25 g·L-1 above could inhibit the proliferation of MH7A cells in the significant dose- and time-effect manner. Compared with the blank group， the FZT groups showed increased proportions of cells in the G2/M phase （P<0.05）， and the high-dose FZT group showed a decreased proportion of cells in the G0/G1 phase （P<0.05）. The arresting effect of FZT on the cell cycle was in a significant dose-effect manner. Compared with the blank group， the FZT groups showed down-regulated miR-155 and mTOR mRNA expression （P<0.05）， and the high-dose FZT group showed up-regulated SHIP1 mRNA expression and down-regulated Akt3 mRNA expression （P<0.05）. Compared with the blank group， the FZT groups showed reduced protein expression of PI3K， Akt3， and mTOR （P<0.05）. ConclusionFZT can significantly inhibit the proliferation of MH7A cells， and the mechanism is related to the promotion of the expression of SHIP-1 and down-regulation of the gene expression of the PI3K/Akt3/mTOR signaling pathway by down-regulating the expression of miR-155.
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@# Objective: To observe the effect of SHIP1 on NSCLC cell proliferation. Methods: The CDS region of human SHIP1 gene was obtained by inquiring NCBI Gene database and was inserted into the vector pTSB-CMV-MCS-SBP-3Flag-EGFP to construct SHIP1 over-expression plasmid, which was further used to construct SHIP1 overexpression lentivirus. SHIP1 over-expressed lentiviruses were used to transfect A549, SPCA-1 and PC-9 cell lines to construct SHIP1 overexpressed NSCLC cell line. Western blotting and qRT-PCR were used to determine the protein and mRNAexpression of SHIP1. The MTT assay and Clone formation assay were used to examine the cell proliferation ability and clone formation ability of PC-9 cells overexpressed SHIP1; Western blotting was performed to examine the level of AP-1 proteins. Results: The sequencing result suggested that the SHIP1 eukaryotic over-expression plasmid was successfully constructed. A519, SPCA-1 and PC-9 cells with SHIP1 over-expression were observed to display uniform green fluorescence under fluorescent microscopy. Compared with negative control group, the mRNA and protein levels of SHIP1 were significantly increased in SHIP1 overexpressed cells (all P<0.01). The over-expression of SHIP1 suppressed the abilities of proliferation and clone formation in PC-9 cells (all P<0.01), and down-regulated the expression of p-c-Jun and FosB etc. Conclusion: The SHIP1 overexpressed NSCLC cell lines were successfully established, and the over-expression of SHIP1 suppressed the cell proliferation ability by inhibitingAP-1 proteins in NSCLC cell lines.
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Objective:To investigate the expression of SHIP1 in the patients with acute myeloid leukemia and its effect on the apoptosis of human leukemia cells.Methods:The expression of SHIP1 in the bone marrow of patients with acute myeloid leukemia was detected by Westem blot.U937 cells was transfected with SHIP1 expression vector (pEGFP-SHIP1 group) and empty vector control (pEGFP group) respectively,U937 cells without transfection were used as the control group.Flow cytometry was used to detect the apoptosis of the cells,the expression of SHIP1,Bcl-2,Bax,Akt,p-Akt were detected by western blot.Results:The expression of SHIP1 in the bone marrow of patients with acute myeloid leukemia was significantly lower than that of the normal human bone marrow SHIP 1 (P＜0.01).The SHIP1 and Bax expressions as well as the apoptotic rate ofpEGFP-SHIP1 group were significantly higher than those of the control group(P＜0.01),while the Bcl-2 and p-Akt expressions were significantly lower than those in the control group(P＜0.01).Conclusions:SH-P1 expression was down regulated in the bone marrow of patients with acute myeloid leukemia.SHIP1 could promote the apoptosis of human leukemia cells via Akt signaling pathway.
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The symptoms of interstitial cystitis (IC)/bladder pain syndrome (BPS) may have multiple causes and involve many contributing factors. Traditional treatments (intravesical instillations) have had a primary focus on the bladder as origin of symptoms without adequately considering the potential influence of other local (pelvic) or systemic factors. Systemic pharmacological treatments have had modest success. A contributing factor to the low efficacy is the lack of phenotyping the patients. Individualized treatment based on is desirable, but further phenotype categorization is needed. There seems to be general agreement that IC is a unique disease and that BPS is a syndrome with multiple pathophysiologies, but this has so far not been not been well reflected in preclinical research with the aim of finding new pharmacological treatments. Current research approaches, including anti-nerve growth factor treatment, anti-tumor necrosis factor-α treatment, activation of SHIP1 (AQX-1125), and P2X3 receptor antagonists, and α1-adrenoceptor antagonists are potential systemic treatments, implying that not only the bladder is exposed to the administered drug, which may be beneficial if the IC/BPS is a bladder manifestation of a systemic disease, or negative (adverse effects) if it is a local bladder condition. Local treatment approaches such as the antagonism of Toll-like receptors (which still is only experimental) and intravesical liposomes (with positive proof-of-concept), may have the advantages of a low number of systemic adverse effects, but cannot be expected to have effects on symptoms generated outside the bladder. Assessment of which of the treatment approaches discussed in this review that can be developed into useful therapies requires further studies.
Subject(s)
Humans , Cystitis, Interstitial , Liposomes , Necrosis , Nerve Growth Factor , Phenotype , Receptors, Purinergic P2X3 , Toll-Like Receptors , Tumor Necrosis Factor-alpha , Urinary BladderABSTRACT
Objective To explore the expression changes in SHIP1 gene in patients with chronic granulocytic leukemia (CGL), healthy volunteers and the K562 cells (one of human CGL cell lines), and to explore the effects of BCR/ABL expression in K562 cells on the SHIP1 gene by blocking the BCR/ABL expression in K562 cells with a specific siRNA. Methods The expression levels of SHIP1 mRNA in leukocytes of the patients with CGL, healthy controls and K562 cells were assessed with RQ-PCR. In addition, K562 cells were divided into three groups: control group (untreated), non-specific interference group (treated with non-specific siRNA), and specific interference group (treated with specific siRNA to block BCR/ABL gene). RQ-PCR and Western blotting were employed to examine the expression level of mRNA and protein (P210 and P145) of BCR/ABL and SHIP1 genes. Results The mRNA expression of SHIP1 in CGL patients and K562 cells was significantly lower than that of the healthy controls. The expression of BCR/ABL gene was down-regulated, while SHIP1 gene up-regulated, in the specific interference group. No statistical significant difference was found between the non-specific interference group and the control group. Conclusions The mRNA expression of SHIP1 is lower in patients with CGL than in the healthy control. The specific siRNA can block the expression of BCR-ABL and further suppress the expression of SHIP1.