A study on the mechanism of PP2A in the recovery of SCI in rats through downregulation of MMP-9 via MAPK signaling pathway.

OBJECTIVE: The aim of this study was to investigate the mechanism of action of protein phosphatase 2A (PP2A) in the recovery of spinal cord injury (SCI) in rats by downregulating matrix metalloproteinase 9 (MMP-9) via the mitogen-activated protein kinase (MAPK) signaling pathway. MATERIALS AND METHODS: A model of SCI was first successfully established in rats. A total of three groups were set, including: sham operation group (A group), SCI group (B group) and PP2A group (C group). The Basso, Beattie and Bresnahan (BBB) motor function score and inclined plane test were adopted to evaluate the motor ability and limb muscle strength of rats in each group. The water content in spinal cord tissues was detected as well. Quantitative Polymerase Chain Reaction (qPCR) assay was performed to analyze the messenger ribonucleic acid (mRNA) expression levels of MAPK, MMP-2, and MMP-9 in spinal cord tissues. The expressions of inflammatory factors tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß) and IL-6 in each group of rats were determined via enzyme-linked immunosorbent assay (ELISA). Western blotting (WB) was employed to measure the protein expression levels of MAPK, MMP-2 and MMP-9 in each group of rats. Additionally, the apoptosis of nerve cells in spinal cord tissues was analyzed through terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. RESULTS: The BBB score was 8.8 points in C group at 5 d after operation, which was significantly different from that in B group (p<0.05). The slope in B and C groups was clearly lower than that in A group at each time point (p<0.001). Meanwhile, it was significantly higher in C group than that in B group at 5, 7 and 9 d (p<0.05). The edema rate rose notably in B group compared with A group (p<0.001). However, spinal cord edema was remarkably relieved after treatment with FRY720 (p<0.01), suggesting that PP2A agonist could treat SCI in rats. The levels of cytokines TNF-α, IL-1ß and IL-6 were markedly higher in B group than those in A group (p<0.01). However, they were significantly reduced after treatment with PP2A agonist (p<0.01). In comparison with A group, B group exhibited remarkably decreased mRNA expression of MAPK and elevated mRNA expressions of MMP-2 and MMP-9 (p<0.01). However, C group exhibited an upregulated mRNA expression of MAPK (p<0.05), a downregulated mRNA expression of MMP-9 (p<0.01), and an undifferentiated mRNA expression of MMP-2 (p>0.05). Compared with B group, the protein expression level of MAPK significantly increased (p<0.05), while that of MMP-9 evidently decreased in C group (p<0.05). Besides, no statistically significant difference was observed in the protein expression level of MMP-2 between C group and B group (p>0.05). Compared with that in A group, the apoptosis rate significantly increased in B group (p<0.001). In addition, the apoptosis rate was significantly lower in C group than that in B group, showing a statistically significant difference (p<0.01). CONCLUSIONS: PP2A downregulates MMP-9 through the MAPK signaling pathway, thereby conducing to the recovery of SCI in rats.

MiR-34a regulates cell apoptosis after myocardial infarction in rats through the Wnt/ß-catenin signaling pathway.

OBJECTIVE: To explore the molecular mechanism of micro ribonucleic acid-34a (miR-34a) in promoting the apoptosis of myocardial cells in the rat model of myocardial infarction (MI). MATERIALS AND METHODS: Sprague-Dawley (SD) rats were ligated with a left anterior descending branch to construct the MI model. The rats were randomly divided into four groups: sham operation group (Sham group), MI group, MI + miR-34a inhibitor group (MI + miR-34a antagomir group) and MI + miR-34a inhibitor negative control group (MI + antagomir NC group). Echocardiography (ECG) and magnetic resonance imaging (MRI) were adopted to detect the ejection fraction [EF (%)] and fraction shortening [FS (%)] of SD rats. Polymerase chain reaction (PCR) and Western blotting were used to detect expression levels of the apoptotic marker Caspase-3 and genes in Wnt/ß-catenin signaling pathway. Hematoxylin and eosin (H&E) staining was applied to detect cardiac injury. In in vitro experiments, the rat-derived myocardial cell line H9C2 was selected to simulate myocardial ischemia and hypoxia at the time of MI with an anoxic and serum-free injury model. C59, the Wnt/ß-catenin signaling pathway inhibitor was applied in MI + miR-34a antagomir + C59 group, and the effect of miR-34a on the apoptosis of myocardial cells through regulating the Wnt/ß-catenin pathway was measured with Real-Time quantitative PCR (qPCR) and 3-(4,5)-dimethylthiazol(-z-y1)-3,5-diphenyltetrazolium bromide (MTT) cell activity detection kits, respectively. RESULTS: It was found that after miR-34a antagomir reversed FS (%) and EF (%) in MI rats, the messenger RNA (mRNA) and protein levels of Caspase-3 in Sham group and MI + miR-34a antagomir group were significantly lower than those in the MI group (p < 0.05), indicating that the addition of miR-34a antagomir inhibited myocardial cell apoptosis after infarction, while the mRNA and protein levels of Wnt/ß-catenin were both higher than those in the MI group. Besides, H&E staining proved that miR-34a reversed the myocardial injury after MI. Similarly, in vitro experiments showed that, compared with those in Hypoxia group, the level of Caspase-3 decreased in Hypoxia + miR-34a inhibitor group and Sham group, while the apoptosis level in Hypoxia + miR-34a inhibitor + C59 group increased (p < 0.05). The results of the MTT assay were consistent with those of PCR. MiR-34a affects myocardial cell apoptosis by regulating the activation and inactivation of the Wnt/ß-catenin signaling pathway.

Vasoactive intestinal peptide is required in the maintenance of immune regulatory competency of immune regulatory monocytes.

Dysfunction of the immune regulatory system plays an important role in the pathogenesis of rheumatoid arthritis (RA). Vasoactive intestinal peptide (VIP) has multiple bioactivities. This study aims to investigate the role of VIP in the maintenance of the immune regulatory capacity of monocytes (Mos). Human peripheral blood samples were collected from RA patients and healthy control (HC) subjects. Mos and CD14+ CD71- CD73+ CD25+ regulatory Mos (RegMos) were isolated from the blood samples and characterized by flow cytometry. A rat RA model was developed to test the role of VIP in the maintenance of the immune regulatory function of Mos. The results showed that RegMos of HC subjects had immune suppressive functions. RegMos of RA patients expressed less interleukin (IL)-10 and showed an incompetent immune regulatory capacity. Serum levels of VIP were lower in RA patients, which were positively correlated with the expression of IL-10 in RegMos. In-vitro experiments showed that the IL-10 mRNA decayed spontaneously in RegMos, which could be prevented by the presence of VIP in the culture. VIP suppressed the effects of tristetraprolin (TTP) on inducing IL-10 mRNA decay in RegMos. Administration of VIP inhibited experimental RA in rats through restoring the IL-10 expression in RegMos. RegMos have immune suppressive functions. VIP is required in maintaining IL-10 expression in RegMos. The data suggest that VIP has translational potential in the treatment of immune disorders such as RA.

Two unilateral puncturation comparative analyses of multiple-level fresh osteoporotic vertebral body compression fractures treated with percutaneous vertebroplasty guided by C-arm fluoroscopy or in senile patients.

OBJECTIVE: To compare the curative effects of two unilateral puncturation percutaneous vertebroplasty (PVP) for the pain caused by multiple-level osteoporotic vertebral body compression fractures (OVCF) in senile patients. PATIENTS AND METHODS: From June 2008 to November 2014, eighty-nine cases suffering from fresh multiple-level OVCF were randomly divided into experimental group (n=51) and control group (n=38). Patients underwent PVP guided by C-arm fluoroscopy in the prone position. We monitored and recorded the visual analgesic scale (VAS) at pre-operation and 2 days post-operation, operation time, exposure duration, bone cement injection amount and extraosseous cement leakages. RESULTS: PVP procedures were successful in both groups without serious complications. The VAS scores in both two groups at 2 days post-operation were significantly lower than VAS scores at pre-operation (p<0.05). The operation time and exposure duration in the observational group were significantly lower than those in the control group (p<0.05). However, bone cement injection amount and extraosseous cement leakages in the observing group were similar to those in control (p>0.05). CONCLUSIONS: The curative effects of two unilateral puncturation PVPs were satisfactory. However, puncturation method had lower operation time and lower X-ray exposure dose. We concluded that puncturation method was a suitable method to be considered for clinical application.

Establishment of heterotopic ossification via sharp instrument injury in rats.

OBJECTIVE: To establish an animal model for heterotopic ossification (HO) induced by sharp instrument injury in Sprague-Dawley (SD) rat and to investigate its possible mechanism. MATERIALS AND METHODS: A total of 48 male SD rats were divided into 3 groups (n=16). In sham group, incision and suture were performed only in the left leg. Partial tenotomy was performed in the left Achilles tendons in the PAT group. In Achilles' tenotomy (AT) group, tenotomy was performed in the left Achilles tendons to establish animal model of EO. X-ray and histological examinations were made at 6 and 10 weeks after operation. RESULTS: No HO occurred in the sham and PAT groups. In AT group, X-ray results on 4 rats showed cartilage and bone formation while the remaining 4 rats showed chondrification in histological examination at 6 weeks after operation. At 10 weeks all rats showed bone formation with trabecular bone. This kind of HO usually develops through a process of endochondral ossification. CONCLUSION: Tenotomy is a simple, effective and feasible method to induce HO.

Effects of oncostatin M on cell proliferation and osteogenic differentiation in C3H10T1/2.

OBJECTIVE: To explore the effects of protein factor Oncostatin M (OSM), a member of the Interleukin-6 (IL-6) family on cell proliferation, osteogenic differentiation and mineralization. MATERIALS AND METHODS: Basal nutrient solutions of different concentrations of OSM (0, 5, 10, 20, 40, 80 ng/ml) were used. In order to divide embryonic origin between mesenchymal stem cells C3H10T1/2 of in vitro cultured mice, and the effects of in vitro proliferation efficiencies of C3H10T1/2 cells of different concentrations of OSM, the C3H10T1/2 cells were divided into four groups: (1) Basal nutrient solution group (negative control); (2) Osteogenesis induced liquid group (positive control); (3) OSM (20 ng/ml) group; (4) Experimental group (osteogenesis induced liquid + OSM (20 ng/ml)). The expressions levels of relevant osteogenesis and mineralization genes were detected. RESULTS: OSM had several effects on promoting the proliferation of embryonic origin mesenchymal stem cells C3H10T1/2 with respect to time of exposure as well as concentrations. In the present study, it has been shown that when the concentration of OSM is 20 ng/ml, the effects of promoting proliferation are most obvious. OSM can induce osteogenic differentiation of C3H10T1/2, make the process of osteogenic differentiation in advance, and promote the formation of end-stage calcium deposits and mineralized nodule, and osteogenic differentiation of C3H10T1/2 is finally achieved. CONCLUSION: OSM can promote the proliferation of C3H10T1/2, and induce its osteogenic differentiation and end-stage mineralization.

Leptin improves osteoblast differentiation of human bone marrow stroma stem cells.

OBJECTIVE: To study the effects of leptin (LEP) on the osteogenic differentiation of human bone marrow stromal cells (hBMSCs) and to explore the mechanism controlling the process. MATERIALS AND METHODS: Respectively cultivated the third-generation hBMSCs with 100 ng/ml bone morphogenetic protein (BMP) culture media containing 320, 160, 80 and 40 ng/mL LEP, and regular medium. We administered alkaline phosphatase (ALP) dye (on the 7th day) and mineralized nodules alizarin red (on the 21st day) and tested the ALP activity as well as osteocalcin (OCN) level on 7th, 14th, 21st day in each group to establish the best inducing concentration of LEP. 7 days later, we tested bone differentiation related genes expression in the control, 160 ng/mL LEP and 100 ng/mL BMP groups using RT-PCR. RESULTS: The activity of ALP and OCN in the 160 ng/mL LEP group after 7, 14 and 21 days was lower than that of the BMP group but higher than that of other groups. However, LEP significantly promoted the expression of bone differentiation related genes, namely, Cbfal, ALP, COL-I and OCN. CONCLUSIONS: LEP promoted the bone differentiation in hBMSCs by promoting the expression of genes related to bone differentiation.

Mercury and cadmium contamination of irrigation water, sediment, soil and shallow groundwater in a wastewater-irrigated field in Tianjin, China.

We investigated the concentrations of Hg, Cd, Pb and As in samples of irrigation water, sediment, soil and groundwater from a field in Tianjin that was irrigated with wastewater. The results showed that the concentrations (Hg, 0.82 microg/L; Cd, 0.18 microg/L; Pb, 1.5 microg/L; As, 8.02 microg/L) in the irrigation water did not exceed the China Surface Water Quality Standard or the maximum concentrations in irrigation water recommended by the FAO. The concentrations of metals in the groundwater of wells (Hg, 0.016 microg/L; Cd, 0.128 microg/L; Pb, 0.25 microg/L; As, 4.65 microg/L) were lower than China Groundwater Quality Standard and the WHO guideline values for drinking water. The groundwater had not yet been contaminated through vertical infiltration-induced leaching. However, a substantial buildup of Hg and Cd in river sediments (I(geo) for Hg and Cd; 5.24 and 3.04, respectively) and wastewater-irrigated soils (I(geo) for Hg and Cd; 2.50 and 3.09, respectively) was observed. Taken together, these results indicated that irrigation with wastewater damaged the soil quality over the long term and that metals more easily accumulated in vegetable fields than rice fields.

Isolation of a Xanthomonas campestris strain with elevated beta-galactosidase activity for direct use of lactose in xanthan gum production.

AIMS: To isolate a Xanthomonas campestris strain that can use lactose directly for xanthan gum production. METHODS AND RESULTS: The presence of indigenous beta-galactosidase gene in the wild-type Xc17 was detected by PCR and Southern hybridization. Treatment of Xc17 with nitrous acid resulted in the isolation of Xc17L with a 3.5-fold elevation of beta-galactosidase activity capable of growing in lactose-based medium. Xc17L is stable for at least 100 generations in terms of beta-galactosidase expression. The amounts of xanthan produced by Xc17L in lactose-based medium are comparable to those in glucose-based medium. CONCLUSIONS: Xc17L is potentially useful for xanthan production from whey, a waste containing lactose. SIGNIFICANCE AND IMPACT OF THE STUDY: A lactose-utilizing strain of X. campestris strain can be constructed without incorporation of any exotic DNA or antibiotic resistance gene and therefore concern of a gene-modified organism and fear of a spread of an antibiotic-resistant gene are avoided.