Objective To investigate the effects of in-vitro monolayer culture and three-dimensional (3-D) alginate microsphere culture on the differentiation of normal human nucleus pulposus cells (NPCs), and to discuss the regulatory mechanism of restoring the phenotype of dedifferentiated NPCs by culturing resveratrol (RES) in 3-D alginate microsphere. Methods Normal human nucleus pulposus tissues were harvested for culture and identification of NPCs from 6 patients with burst lumbar vertebra fracture. NPCs at passages 1, 3, 5, and 7 in the in-vitro monolayer culture were harvested to observe the morphology, cell aging, and proteoglycan expression. The cell proliferation rates of NPCs at passage 1 in-vitro in monolayer culture and in 3-D alginate microsphere culture were detected. NPCs at passage 7 were randomly divided into 3-D alginate microsphere control group (group A), RES group (group B), silent mating type information regulation 2 homolog 1 (SIRT1)- small interfering RNA (siRNA) + RES group (group C), and negative control-siRNA + RES group (group D); and NPCs in the in-vitro monolayer culture was monolayer control group (group E). After corresponding treatment, Western blot was used for determining the protein expressions of SIRT1, Aggrecan, and collagen type II; real-time fluorescence quantitative PCR was used for detecting SIRT1 mRNA expression. Results The cultured cells were identified to be NPCs. Morphological observation, senescence-associated β-galactosidase (SA-β-gal) staining, and toluidine blue staining showed that dedifferentiation of normal NPCs tended to occur under continuous in-vitro monolayer culture, which was more obvious with increase of passage number. NPCs in 3-D alginate microsphere culture showed significantly lower proliferation rate than NPCs in the in-vitro monolayer culture (P lt; 0.05), but it could significantly improve the protein expressions of collagen type II and Aggrecan in dedifferentiated NPCs, showing significantly difference between groups E and A (P lt; 0.05). The protein expressions of SIRT1, collagen type II, and Aggrecan in group B were significantly improved when compared with that in group A (P lt; 0.05). Real-time fluorescence quantitative PCR and Western blot showed that the expressions of SIRT1 mRNA and proteins in group C were significantly inhibited after transfected with SIRT1-siRNA when compared with those in groups B and D (P lt; 0.05), and the protein expressions of collagen type II and Aggrecan in group C were significantly lower than those in groups B and D (P lt; 0.05). Conclusion Continuous in-vitro monolayer culture could efficiently cultivate numerous seeding NPCs, but it is liable to dedifferentiate. In 3-D alginate microsphere culture, RES could restore the phenotype of dedifferentiated NPCs and synthesize more extracellular matrix, which is related to the regulation of SIRT1.
Objective To observe the influence of resveratrol on superoxide dismutase (SOD), malondialdehyde (MDA), intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) of intestinal mucosal ischemia-reperfusion injury protection in rats with severe acute pancreatitis (SAP). Methods Fifty-four rats were divided into three groups randomly: sham operation group (SO group), SAP model group (SAP group) and resveratrol-treated group (Res group). SAP model was made by injecting sodium taurocholate 50 mg/kg to pancreatic bile duct and resveratrol was given intravenously at 5 min after inducing SAP model. The rats were sacrificed at 3 h, 6 h and 12 h after inducing SAP model respectively by equal number. The levels of MDA, SOD, ICAM-1 and VCAM-1 and histological changes of small intestine were measured. Results The level of MDA in small intestine tissue in SAP group was significantly higher than that in SO group (P＜0.05), while the activity of SOD was significantly lower in the relevant tissues (P＜0.05). The expressions of ICAM-1 and VCAM-1 in SAP group were higher than those of SO group (P＜0.05). The activity of SOD in small intestine tissue in Res group was significantly higher than that in SAP group (P＜0.05); while the level of MDA was significantly lower in the relevant tissues (P＜0.05). The expressions of ICAM-1 and VCAM-1 in Res group were lower than those of SAP group (P＜0.05). Conclusions Oxygen free radicals are concerned with the process of pathological changes in intestinal mucosal ischemia-reperfusion in rats with SAP. Resveratrol might increase SOD activity and decrease MDA level to attenuate lipid peroxidation in small intestine of SAP, and reduce the expressions of ICAM-1 and VCAM-1 in intestine, thus diminish the damage of the intestine in SAP. And it acts as a protective effect to small intestinal ischemia-reperfusion injury.
ObjectiveTo explore the protective mechanism and effect of the resveratrol for kidney injury of obstructive jaundice. MethodsThe rats were randomly divided into three groups: sham operation group receiving laparotomy without bile duct ligation (BDL), the obstructive jaundice group with BDL, and the obstructive jaundice + resveratrol group given resveratrol following BDL. The levels of total bilirubin (TBIL), direct bilirubin (DBIL), blood urea nitrogen (BUN), and creatinine (Cr) in the serum were tested. The superoxide dismutase (SOD) activity, malondialdehyde (MDA) content, glutathione (GSH) level in the renal tissues were detected. The expressions of the silent information regulator 1 (SIRT1) and nuclear factor-κB (NF-κB) proteins were tested by Western blot. The expression of SIRT1 mRNA was detected by RT-PCR and the renal cell apoptosis was examined by TUNEL staining. Results①Compared with the sham operation group, the levels of serum TBIL, DBIL, BUN and Cr were significantly higher (P < 0.05); the activity of SOD and level of GSH, and the expressions of SIRT1 mRNA and SIRT1 protein in the renal tissues were signi-ficantly lower (P < 0.05); the content of MDA, the expression of NF-κB protein, and the rate of cell apoptosis in the renal tissues were significantly higher (P < 0.05) in the obstructive jaundice group.②Compared with the obstructive jaundice group, the levels of serum TBIL, DBIL, BUN and Cr were significantly lower (P < 0.05); the activity of SOD and level of GSH, and the expressions of SIRT1 mRNA and SIRT1 protein in the renal tissues were significantly higher (P < 0.05); the content of MDA, the expression of NF-κB protein, and the rate of cell apoptosis in the renal tissues were significantly lower (P < 0.05) in the obstructive jaundice+resveratrol group. ConclusionThe resveratrol could alleviate renal damage and play a beneficial role to resist inflammation, oxidation, and apoptosis by activating the SIRT1 which probably inhibits the expression of NF-κB protein and promotes the activity of SOD in cholestatic kidney injury.
ObjectiveTo investigate the effects of different levels of resveratrol on oxidative stress injury in central nervous system of rats with obstructive jaundice and its protective effect and mechanism of oxidative stress injury. MethodsThirty two female SD rats of 6 weeks old were used as experimental object. The animals were randomly divided into four groups, 8 rats in each group. Sham operation group (SO group), the common bile duct were seperated without ligation; while the models of obstructive jaundice of obstructive jaundice group (OJ group), obstructive jaundice+low dose resvera-trol (L-Res)treatment group (OJ+L-Res group), and obstructive jaundice+high dose resveratrol (H-Res) treatment group (OJ+H-Res group) were established by operation. After the operation, the rats in OJ+L-Res group and OJ+H-Res group were treated with different doses of resveratrol, the rats in SO group and OJ group were given the same dose of normal saline. On the 14th day after operation, blood were tested for total bilirubin (TBIL), direct bilirubin (DBIL), ALT, and AST. And cerebral cortex specimen were collected, then malondialdehyde (MDA), total superoxidedismutase (T-SOD) activity, and HO-1 protein expression in the rats brain of the four groups were measured. ResultsThe levels rise of TBIL and DBIL after modeling suggested that obstructive jaundice model were estabilshed successfully, but there was no significant difference among the OJ group, OJ+L-Res group, and OJ+H-Res group. In the OJ group, OJ+L-Res group, and OJ+H-Res group, the levels of ALT, AST, and MDA were increased while levels of T-SOD and HO-1 protein expression were decreased when compared with SO group(P < 0.05). Among the OJ group, OJ+L-Res group, and OJ+H-Res group, levels of ALT, AST and MDA were lower in the treatment groups than in the OJ group(P < 0.05), while levels of T-SOD and HO-1 protein expression which reflects the oxidative stress were higher in the treatment group(P < 0.05). Different doses of resveratrol had different effects on T-SOD and HO-1 protein expression with statisticl significance (P < 0.05). ConclusionsResveratrol have little effect on TBIL and DBIL of obstrctive jaundice rats, but it can protect the liver function, and it has antioxidant properties of decreasing MDA and incresing SOD and HO-1 protein expression levels in the cerebral cortex cells of obstructive jaundiced rats.
ObjectiveTo investigate the effects of resveratrol on airway remodeling in mice with chronic asthma. MethodsTwenty-four female BALB/c mice were randomly divided into three groups (8 mice in each group), namely a control group, an asthma group and a resveratrol (RV) group. All mice were sensitized with ovalbumin (OVA). The sensitized mice were then challenged with OVA while the control group were challenged with phosphate-buffered saline. The mice in the RV group were intraperitoneally injected with RV 30 min before OVA challenge, while the mice in the control and the asthma group were intraperitoneally injected with equal volume of dimethylsulfoxide. Periodic acid-Schiff (PAS) staining was performed to evaluate goblet cell hyperplasia, and Masson-trichrome staining was used to evaluate the deposition of collagen matrix. In addition, immunohistochemical analysis of the α-smooth muscle actin (α-SMA) was applied to examine airway smooth muscle cell hyperplasia and hypertrophy. The positive staining with PAS, Masson, α-SMA areas (μm 2/μm) of per bronchial basement membrane perimeter was used to indicate the degree of airway remodeling. ResultsIn the asthma group and the RV group, the degree of the goblet cell hyperplasia was significantly higher than that in the control group (5.44±1.13, 4.18±0.85vs. 0.00±0.00,P<0.01), and the level of goblet cell hyperplasia in the RV group was lower than that in the asthma group (P<0.05). The Masson staining showed that the deposition of collagen in the asthma group and the RV group was significantly higher than that in the control group (9.80±2.78, 5.71±0.68vs. 1.67±0.65,P<0.01), and the collagen deposition in the RV group was further lower than that in the asthma group (P<0.01). The α-SMA immunohistochemical analysis demonstrated that the expression of α-SMA in the asthma group and the RV group was significantly higher than that in the control group (10.39±1.65, 7.57±1.98vs. 2.41±1.06,P<0.01), and the level of α-SMA in the RV group was also lower than that in the asthma group (P<0.05). ConclusionThese findings suggest that resveratrol has an inhibitory effect on the process of airway remodeling in mice with chronic asthma.
ObjectiveTo investigate the regulatory effect of resveratrol (RES) on the extracellular matrix (ECM) expression of nucleus pulposus cells (NPC), and its relative molecular mechanism.MethodsTen patients receiving discectomy were collected, of which 5 patients were young with spinal burst fracture, classified as control group; the rest 5 patients were senile with lumbar disc herniation, classified as degenerative group. The nucleus pulposus tissue of 2 groups were collected, the in situexpression of β-catenin was detected by immunohistochemistry, and the protein expressions of collagen type Ⅱ and Aggrecan were detected by Western blot. The NPC were isolated and cultured from degenerative nucleus pulposus tissues. RES treated the third-passage NPC with (group B) or without IL-1β (group C), to further determine the protein expressions of collagen type Ⅱ and Aggrecan by Western blot, the unstimulated cells were set up as blank control group (group A). Moreover, NPC treated with small interfering RNA (siRNA) targeted silent SIRT1 or β-catenin were used to determine the protein and gene expressions of β-catenin and SIRT1 by Western blot and real-time fluorescence quantitative PCR. In addition, the third-passage NPC treated with complete medium (group 1), IL-1β (group 2), RES+IL-1β (group 3), and SIRT1-siRNA+RES+IL-1β (group 4) for 24 hours were used to detect the nuclear translocation of β-catenin by cell immunofluorescence staining. Finally, the third-passage NPC treated with complete medium (group Ⅰ), IL-1β (group Ⅱ), IL-1β+β-catenin-siRNA (group Ⅲ), IL-1β+RES (group Ⅳ), and IL-1β+RES+SIRT1-siRNA (group Ⅴ) for 24 hours were used to detect the protein expressions of collagen type Ⅱ and Aggrecan by Western blot.ResultsImmunohistochemical staining and Western blot detection showed that when compared with control group, the cell proportion of expression of β-catenin were significantly increased in degenerative group (t=4.616, P=0.010); the protein expression of β-catenin was also significantly increased and the protein expressions of collagen type Ⅱ and Aggrecan were significantly decreased (P<0.05). In cytology experiments, the protein expression of β-catenin in group B was significantly higher than that in groups A and C, and the protein expressions of collagen type Ⅱ and Aggrecan in group B were significantly lower than those in groups A and C (P<0.05). After transfection of siRNA, the protein expressions of SIRT1 and β-catenin significantly decreased (P<0.05). The results of cell immunofluorescence staining further confirmed that when compared with group 3, after the SIRT1 was silenced by siRNA in group 4, the attenuated nuclear translocation of β-catenin by RES treatment was aggravated. Western blot results showed that the protein expressions of collagen type Ⅱ and Aggrecan in group Ⅱ were significantly lower than those in group Ⅰ(P<0.05); after transfection of β-catenin-siRNA in group Ⅲ, the degradation of ECM by IL-1β was obviously inhibited, the protein expressions of collagen type Ⅱ and Aggrecan were significantly increased when compared with group Ⅱ (P<0.05); after transfection of SIRT1-siRNA in group Ⅴ, the protective effect of RES on the degradation of ECM was inhibited, the protein expressions of collagen type Ⅱ and Aggrecan were significantly decreased when compared with group Ⅳ (P<0.05).ConclusionRES regulates the ECM expression of NPC via Wnt/β-catenin signaling pathway, which provide a new idea for intervertebral disc degeneration disease treatment.
ObjectiveTo improve the seawater-drowning-induced lung injury model in rats, and investigate the protective effect of resveratrol against seawater-drowning-induced lung injury and its mechanism.MethodsA total of 112 SD healthy rats were randomly assigned into 5 groups: a control group (Group C, n=8), a seawater drowning group (Group S, n=32), a resveratrol prophylactic treatment group (Group S+R, n=32), a resveratrol group (Group R, n=8), and an endotracheal intubation group (Group E, n=32). A modified endotracheal intubation model was developed, and endotracheal intubation was used instead of tracheotomy. Blood gas analysis was performed on the abdominal aorta at each time point, then the rats were sacrificed to obtain their lungs. Lung wet-to-dry ratio (W/D), malondialdehyde (MDA), superoxide dismutase (SOD), myeloperoxidase (MPO) and cysteinyl aspartate specific proteinase (Caspase-3) were measured by enzyme linked immunosorbent assay. The histological sections of rat lungs were stained with haematoxylin-eosin. Groups S+R and R were pretreated with resveratrol (50 mg/kg) through intragastric administration for 3 days; then models were established and the rats were sacrificed 24 hours after the last intragastric administration.ResultsAfter seawater perfusion, arterial oxygen pressure decreased and arterial carbon dioxide pressure increased in blood gas analysis of rats, MDA content increased, MPO and SOD activity decreased, caspase-3 content and W/D ratio increased, as well as lung tissue pathological damage. The resveratrol pretreatment group showed the same change trend, but the damage degree was relatively light.ConclusionsSeawater perfusion can induce respiratory failure, pulmonary edema and hemorrhage in rats. Lung tissue apoptosis may occur when seawater submergence causes lung injury. Resveratrol pretreatment can ameliorate hypoxia and pulmonary edema in rats.
Objective To investigate the effect of resveratrol (RES) on inflammation-induced cartilage endplate (CEP) degeneration, and its regulatory mechanism on high mobility group box-1 protein (HMGB1) signaling pathway. Methods The intervertebral CEP cells of Sprague Dawley (SD) rats aged 3 weeks were extracted and identified by toluidine blue staining and immunofluorescence staining of rabbit anti-rat collagen type Ⅱ. The cell counting kit 8 (CCK-8) method was used to screen the optimal concentration of RES on intervertebral CEP cells. Gene chip analysis was used to determine the target of RES on intervertebral CEP cells. Interleukin 1β (IL-1β) was used to construct the intervertebral CEP cell degeneration model caused by inflammation and the 7-8-week-old SD rat intervertebral disc degeneration model, and pcDNA3.1-HMGB1 (pcDNA3.1) was used as the control of RES effect. Flow cytometry and TUNEL staining were used to detect the apoptotic rate of intervertebral CEP cells and rat intervertebral disc tissue cells, respectively. ELISA kit was used to detect the content of interleukin 10 (IL-10) and tumor necrosis factor α (TNF-α) in the cell supernatant and rat serum. Western blot was used to detect the expressions of HMGB1, extracellular signal-regulated protein kinase (ERK), phosphorylated ERK (p-ERK), B cell lymphoma/leukemia 2 gene (Bcl-2), and Bcl-2-associated X protein (Bax). ResultsThe extracted cells were identified as rat intervertebral CEP cells. CCK-8 method screened out the highest activity of intervertebral CEP cells treated with 30 μmol/L RES. The gene chip analysis confirmed that the HMGB1-ERK signal was the target of RES. Both cell experiments and animal experiments showed that RES treatment can significantly down-regulate the apoptosis rate of intervertebral CEP cells, inhibit the release of TNF-α, and increase the content of IL-10; and down-regulate the expressions of HMGB1, p-ERK, and Bax, and increase Bcl-2; and pcDNA3.1 could partially reverse these effects of RES, and the differences were all significant (P<0.05). ConclusionRES can significantly inhibit the apoptosis of intervertebral CEP cells induced by inflammation, which is related to inhibiting the expression of HMGB1.
Objective To investigate the effect of solid lipid nanoparticles (SLNs) on enhancing the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in vitro by resveratrol (Res), and provide a method for the treatment of bone homeostasis disorders. MethodsRes-SLNs were prepared by high-temperature emulsification and low-temperature solidification method, and then the 2nd-3rd generation BMSCs from Sprague Dawley rat were co-cultured with different concentrations (0, 0.1, 1, 5, 10, 20 μmol/L) of Res and Res-SLNs. The effects of Res and Res-SLNs on the cell viability of BMSCs were detected by cell counting kit 8 (CCK-8) and live/dead cell staining; the effects of Res and Res-SLNs on the osteogenic differentiation of BMSCs were detected by alkaline phosphatase (ALP) staining and alizarin red S (ARS) staining after osteogenic differentiation induction, and the optimal concentration of Res-SLNs for gene detection was determined. Anti-osteocalcin (OCN) immunofluorescence staining and real-time fluorescent quantitative PCR (RT-qPCR) were used to detect the effect of Res and Res-SLNs on osteoblast-related genes (ALP and OCN) of BMSCs. ResultsLive/dead cell staining showed that there was no significant difference in the number of dead cells between Res and Res-SLNs groups; CCK-8 detection showed that the activity of BMSCs in Res group was significantly reduced at the concentration of 20 μmol/L (P<0.05), while Res-SLNs activity was not affected by Res concentration (P>0.05). After osteogenic differentiation, the staining intensity of ALP and ARS in both groups was dose-dependent. The percentage of ALP positive staining area and the percentage of mineralized nodule area in Res group and Res-SLNs group reached the maximum at the concentrations of 10 μmol/L and 1 μmol/L, respectively (P<0.05), and then decreased gradually; the most effective concentration of Res-SLNs was 1 μmol/L. The expression of OCN and the relative expression of ALP and OCN mRNA in Res-SLNs group were significantly higher than those in Res group (P<0.05). ConclusionEncapsulation of SLNs can improve the effect of Res on promoting osteogenesis, and achieve the best effect of osteogenic differentiation of BMSCs at a lower concentration, which is expected to be used in the treatment of bone homeostasis imbalance diseases.