The protective effect of niacinamide on interleukin-1β (IL-1β)-induced annulus fibrosus (AF) type II collagen degeneration in vitro and the mechanism were investigated. Chiba’s intervertebral disc (IVD) culture models in rabbits were established and 48 IVDs from 12 adult Japanese white rabbits were randomly divided into 4 groups: normal control group, niacinamide-treated group, type II collagen degneration group (IL-1β) and treatment group (niacinamide+IL-1β). After culture for one week, AFs were collected for inducible nitric oxide synthase (iNOS), cysteine containing aspartate specific protease-3 (Caspase-3) and type II collagen immunohistochemical examination, and type II collagen reverse transcription polymerase chain reaction (RT-PCR). The results showed that rate of iNOS positive staining AF cells in the 4 groups was 17.6%, 10.9%, 73.9% and 19.3% respectively. The positive rate in treatment group was significantly lower than in the type II collagen degeneration group (P<0.01). Rate of Caspase-3 positive staining AF cells in the 4 groups was 3.4%, 4.2%, 17.6% and 10.3% respectively. The positive rate in treatment group was lower than in the type II collagen degeneration group (P<0.01). Type II collagen staining demonstrated that lamellar structure and continuity of collagen in treatment group was better reversed than in the degeneration group. RT-PCR revealed that the expression of type II collagen in treatment group was significantly stronger than that in type II collagen degeneration group (P<0.01). It was concluded that niacinamide could effectively inhibit IL-1β stimulated increase of iNOS and Caspase-3 in AF, and alleviate IL-1β-caused destruction and synthesis inhibition of type II collagen. Niacinamide is of potential for clinical treatment of IVD degeneration.

By preventing mesenchymal stem cells (MSCs) from adhering to precoated agarose to create a model of MSC suspension in vitro, we investigated anoikis in MSCs and the role of caspase-3 in the anoikis. The cultured MSCs were randomly divided into 3 groups: the anoikis group, caspase-3 inhibitor group and control group. Before experiment, we coated dishes with 1.5 % agarose; in the anoikis group, MSCs were put into the precoated dishes; and in the inhibitor group, caspase-3 inhibitor and MSCs were also put into the precoated dishes; but there were not intervention in the control group. MSCs were collected at 2 h, 6 h, 12 h and 24 h. The alteration of caspase-3 activity was evaluated by caspase-3 fluorometric assay and western blot analysis. The apoptosis rates were detected by flow cytometry. MSCs were round and suspended sufficiently in the anoikis and inhibitor groups. Caspase-3 fluorometric assay showed that there were significant differences in statistics between the anoikis group and the others (P<0.05). Western blot analysis discovered that caspase-3 expression in the anoikis group was more than that in the control and inhibitor groups (P<0.05). Flow cytometry showed that the apoptosis peak appeared in all the three groups, but it increased dramatically in the anoikis group. The apoptosis rates in the inhibitor and control groups were low and stable. And there were significant differences in statistics between the anoikis group and the others (P<0.05). MSCs will undergo anoikis in suspended condition if they are separated from the extracellular matrix. Caspase-3 inhibitors can suppress caspase-3 activity and reduce the apoptosis rate significantly. Caspase-3 plays a vital part in induced MSC anoikis in vitro. MSCs suspension culture system might be set up with argorose and caspase-3 inhibitor.

In order to evaluate the neuroprotective effect of Rosiglitazone Maleate (RSG) against brain ischemic injury, the effects of Rosiglitazone Maleate on the inflammation following cerebral ischemia/reperfusion were investigated. Focal cerebral ischemia was induced by the intraluminal thread for cerebral middle artery (MCA) occlusion. Rosiglitazone Maleate at concentrations of 0.5, 2 and 5 mg/kg was infused by intragastric gavage twice immediately and 2 h after MCA occlusion, respectively. The effects of Rosiglitazone Maleate on brain swelling, myeloperoxidase and interleukin-6 mRNA level in brain tissue after MCA occlusion and reperfusion were evaluated. The results showed that as compared with the model control group, RSG (0.5 mg/kg) had no significant influence on brain swelling (P>0.05), but 2 mg/kg and 5 mg/kg RSG could significantly alleviate brain swelling (P<0.05). All different doses of RSG could obviously reduce MPO activity in brain tissue after MCA occlusion and reperfusion in a dose-dependent manner. RSG (0.5 and 2 mg/kg) could decrease the expression levels of IL-6 mRNA in brain tissue after MCA occlusion and reperfusion to varying degrees (P<0.05) with the difference being significant between them. It was concluded that RSG could effectively ameliorate brain ischemic injury after 24 h MCA occlusion and inhibit the inflammatory response after ischemia-reperfusion in this model.

To study the cartilage differentiation of mouse mesenchymal stem cells (MSCs) induced by cartilage-derived morphogenetic proteins-2 in vitro, the MSCs were isolated from mouse bone marrow and cultured in vitro. The cells in passage 3 were induced into chondrogenic differentiation with different concentrations of recombinant human cartilage-derived morphogenetic proteins-2 (0, 10, 20, 50 and 100 ng/mL). After 14 days of induction, morphology of cells was observed under phase-contrast microscope. Collagen II mRNA and protein were examined with RT-PCR, Western blotting and immunocytochemistry respectively and the sulfate glycosaminoglycan was measured by Alcian blue staining. RT-PCR showed that CDMP-2 could promote expression of collagen II mRNA in an dose-dependant manner, especially at the concentration of 50 ng/mL and 100 ng/mL. Immunocytochemistry and Western blotting revealed a similar change. Alcian blue staining exhibited deposition of typical cartilage extracellular matrix. Our results suggest that mouse bone marrow mesencymal stem cells can differentiate into chondrogenic phonotype with the induction of CDMP-2 in vitro, which provides a basis for further research on the role of CDMP-2 in chondrogenesis.

In order to explore the method to prepare hypoxia UW solution and the stability and preservation of hypoxia UW solution, UW solution was purged by argon or air for 15 min or 60 at a flow rate of 0.8 or 2 L/min, and the oxygen partial pressure of UW solution was detected. The hypoxia UW solution was exposed to the air or sealed up to preserve by using different methods, and the changes of oxygen partial pressure was tested. The results showed that oxygen partial presure of 50 mL UW solution, purged by argon for 15 min at a flow rate of 2 L/min, was declined from 242±6 mmHg to 83±10 mmHg. After exposure to the air, oxygen partial pressure of hypoxia UW solution was gradually increased to 160±7 mmHg at 48 h. After sealed up by the centrifuge tube and plastic bad filled with argon, oxygen partial pressure of hypoxia UW solution was stable, about 88±13 mmHg at 72 h. It was concluded that oxygen of UW solution could be purged by argon efficiently. Sealed up by the centrifuge tube and plastic bag filled with argon, oxygen partial pressure of UW solution could be stabilized.

The effects of cardiomyocyte grafting on left ventricular (LV) remodeling and function in rats with chronic myocardial infarction were evaluated using high-frequency ultrasound. Chronic myocardial infarction was induced in 50 Wister rats by ligating the left anterior descending artery. They were randomized into two groups: a trial group that received neonatal rat cardiomyocyte transplantation (n=25) and a control group which were given intramyocardial injection of culture medium (n=25). The left ventricular (LV) geometry and function were evaluated by high-frequency ultrasound before and 4 weeks after the cell transplantation. After the final evaluation, all rats were sacrificed for histological study. The results showed that 4 weeks after the cell transplantation, as compared with the control group, the LV end-systolic dimension, end-diastolic dimension, end-systolic volume and end-diastolic volume were significantly decreased and the LV anterior wall end-diastolic thickness, LV ejection fraction and fractional shortening were significantly increased in the trial group (P<0.01). Histological study showed that transplanted neonatal rat cardiomyocytes were found in all host hearts and identified by Brdu staining. It was suggested that transplantation of neonatal rat cardiomyocytes can reverse cardiac remodeling and improve heart function in chronic myocardial infarction rats. High-frequency ultrasound can be used as a reliable technique for the non-invasive evaluation of the effect of cardiomyocyte transplantation.