Anti-inflammatory and autonomic effects of electroacupuncture in a rat model of diet-induced obesity.

OBJECTIVE: To study the effect of electroacupuncture (EA) on the cholinergic anti-inflammatory pathway (CAP) by measurement of vagal activity in rats with high-fat diet (HFD)-induced obesity. METHODS: Diet-induced obesity (DIO) was induced in 30 rats by feeding them a HFD for 12 weeks. A further 10 rats fed normal food comprised the lean diet (LD) control group. DIO rats were further subdivided into three groups that received a HFD only (HFD group, n=10), a HFD plus electroacupuncture (HFD+EA group, n=10) or a HFD plus minimal acupuncture (HFD+MA group, n=10). EA and MA treatments were continued for 8 weeks. Heart rate variability (HRV) was used to measure the function of the autonomic nervous system before and after treatment. ELISA was used to determine acetylcholine (ACh) and tumour necrosis factor (TNF)-α levels in the serum. Real-time PCR was used to assess the mRNA expression of α7-subtype nicotinic acetylcholine cholinergic receptors (α7nAChRs) and TNF-α in the mesenteric white adipose tissues (MWAT). RESULTS: EA but not MA significantly reduced rats' bodyweight. No difference was found in the low frequency (LF), high frequency (HF) and the balance between LF and HF (LF/HF) components of HRV before treatment. After the EA intervention, HF was elevated and LF/HF was reduced in the HFD+EA group comparedwith the HFD group. TNF-α in the serum and MWAT were increased in the HFD group, but were reduced in the HFD+EA group. Furthermore, EA promoted expression of α7nAChRs and ACh in the MWAT. There was no difference between the HFD and HFD+MA groups for any indices. CONCLUSIONS: EA enhanced vagal activity, promoted ACh release and activated α7nAChRs in the MWAT, leading to inhibition of proinflammatory cytokine production.

Downregulation of Sprouty homolog 2 by microRNA-21 inhibits proliferation, metastasis and invasion, however promotes the apoptosis of multiple myeloma cells.

The aim of the present study was to assess the effects of sprouty homolog 2 (SPRY2) gene regulation by miR-21 on the occurrence, development and tumor metastasis in multiple myeloma (MM). The miR-21 expression lentiviral vector (LV)-anti-miR-21 and a liposome transfection method were used to screen MM cell lines with stable silent SPRY2. Real-time quantitative polymerase chain reaction (PCR) and western blot analyses were used to detect SPRY2 expression and miR-21 protein expression levels. An MTT assay was used to assess cell proliferation. Flow cytometry was used for analysis of cell cycle. A scratch test/wound healing assay was used to detect the cell migration ability. A Transwell assay was used to detect the cell invasion ability. Real-time quantitative PCR and western blot analysis showed that in the MM cell lines with high endogenous miR-21 expression (RPMI8226 and KM3), SPRY2 expression was significantly lower. Conversely, in the U266 cell line with low endogenous miR-21 expression, SPRY2 expression was significantly higher, and the gray values of miR-21 and SPRY2 protein in the respective cell lines showed statistically significant differences (P<0.01). Following transfection of U266 cells, the expression of miR-21 in the U266/LV-anti-miR21 lentiviral multiplicity of infection (MOI) 20 group and -MOI 40 group decreased significantly compared with that in the untransfected U266 group (P<0.05). SPRY2 protein expression in U266 cells transfected with miR-21 mimics was significantly reduced compared with that in the non-transfected (untreated) group and the negative control-transfected group (P<0.01). An MTT assay showed that compared with the non-transfected and negative control groups, the cell growth rate as well as the proliferation rate were significantly decreased in the transfection group 48, 72 and 96 h after transfection (P<0.01). Flow cytometric analysis showed that 48 and 72 h after transfection of U266 cells with miR-21 mimics, the apoptotic rates were (24.7 ± 1.97 and 38.6 ± 1.56%) in the U266 group, (27.3 ± 1.72 and 37.3 ± 1.59%) in the siRNA group and (12.7 ± 1.27 and 22.1 ± 1.63%) in the U266/miR-21 group. Compared with the two control groups, the apoptotic rate in the U266/miR-21 group was significantly decreased and the G0/G1 phase cell population was significantly reduced (P<0.05). Scratch experiments showed that the cell migration ability was significantly reduced in the transfection group 24 and 48 h after transfection (P<0.05). A Transwell invasion assay confirmed that the number of U266 cells which migrated through a Matrigel-covered polyphosphate membrane significantly decreased in the transfection group 24 and 48 h after transfection. The cell-penetrating ability was also significantly decreased (P<0.05). In conclusion, the downregulation of SPRY2 gene expression mediated by miR-21 promotes the proliferation and invasion of MM cells in vitro, suggesting that miR-21 may be a novel potential molecular therapeutic target in the treatment of MM.

Correlation between microRNA­21 and sprouty homolog 2 gene expression in multiple myeloma.

The aim of the present study was to investigate the expression level of microRNA 21 (miR­21) in the peripheral blood of patients with multiple myeloma (MM) and to investigate the correlation between miR­21 and sprouty homolog 2 (SPRY2) gene expression levels in MM. A total of 30 patients with MM, 15 with monoclonal gammopathy of undetermined significance (MGUS) and 20 normal control (NC) outpatients were selected for the detection of miR­21 and SPRY2 expression using reverse transcription-quantitative polymerase chain reaction. In addition, western blot analysis was performed to detect the expression of miR­21 and SPRY2 in MM cell lines. The expression of miR­21 in U­266 cells following lipofectamine transfection of fluorescence­labeled miR­21 mimic/inhibitor was observed using a fluorescence microscope and the expression level of SPRY2 in the miR­21 mimic/inhibitor­transfected U­266 cells was detected using western blot analysis. The miR­21 expression level in the circulating serum of the MM patient group was significantly higher (P<0.01) than that of the MGUS and NC groups. The MM cell lines with high endogenous miR­21 expression exhibited an expression level of SPRY2 that was significantly lower than that in the MM cells with low endogenous miR­21 expression. The transfection efficiency of fluorescence­labeled miR­21 mimic/inhibitor was >90%. Compared with the miR­21 expression level in untreated U­266 cells (0.82±0.13), the expression level of miR­21 was increased by 120.2­fold in miR­21 mimic­transfected cells (98.6±14.2; P<0.001) and was decreased by 61.9% in the miR­21 inhibitor­transfected cells (0.37±0.06; P<0.05). The grayscale value of protein bands demonstrated that SPRY2 protein expression significantly decreased in miR­21 mimic­transfected U­266 cells compared with that in the inhibitor­transfected, siRNA­transfected and untreated cells (P<0.01). miR­21 may represent a negative regulator involved in the downregulation of SPRY2 in MM. miR­21 is closely associated with the pathogenesis, progression and prognosis of MM and may thus be used as an indicator of poor MM prognosis.