ABSTRACT
AIM: To investigate the protective effect of "DuZhong-DangGui" (DZ-DG) on the knee tissue of rats with osteoarthritis (OA) and its regulation role on NLPR3 inflammasome. METHODS: Twenty-four SPF male SD rats, eighteen rats were randomly selected to establish OA model by anteri- or cruciate ligament amputation (ACLT) method, and rats were divided into control group, OA model group, DZ-DG group and positive drug group, 6 in each group, treatment for 8 weeks. The peripheral blood were collected, ELISA was used to detect the levels of IL-1β, IL-6, IL-18, and TNF-α; cartilage tissue of knee joint were collected, HE staining was used to observe pathology changes, OARSI staining was used to observe cartilage calcification and preform quantitative OARSI scoring; immunohistochemistry and TUNEL staining were used to detect the contents of Caspase1 and Collagen II and the number of apoptosis in the tissue, respectively, and western blot was used to detect the protein expressions of matrix metalloproteinase-13 (MMP-13), tissue inhibitor of metalloproteinase-1 (TIMP-1), p53, p21, NLPR3, apoptosis-associated speck-like protein containing a CARD (ASC) and Pro-Caspase-1. RESULTS: Compared to control group, OA model group rats serum levels of IL-1β, IL-6, IL-18, and TNF - α significantly increased (P<0.01), OARSI scores significantly increased (P<0.01), chondrocyte apoptosis was increased (P<0.01), Caspase-1 content and MMP-13, p53, p21, NLPR3, ASC, Pro-Caspase-1 protein expressions significantly increased (P <0.01), while Collagen II content and TIMP-1 protein expression significantly decreased (P<0.01). Compared with the OA model group, DZ-DG group and positive drug group rats serum levels of IL-1β, IL-6, IL -18 and TNF - α significantly decreased (P< 0.05), chondrocyte apoptosis were significantly decreased (P<0.01), Caspase1 content, MMP-13, p53, NLPR3, Pro-Caspase-1 significantly decreased (P< 0.05), Collagen Ⅱ content and TIMP- 1 protein expression (P<0.01); DZ-DG group rats protein expression of p21 and ASC were decreased (P<0.01). CONCLUSION: The DZ-DG have protection role on cartilage of OA rats, its effect may related to mediation of NLPR3/ASC/Pro-Caspase-1 pathway, to decrease of IL-1β, and inhibition of cell apoptosis.
ABSTRACT
The purpose of this study was to investigate how Zuogui pills from the Kidney-tonifying and Nourishing Yin formula, in combination with the gonadotrophin-releasing hormone antagonist cetrorelix, affected the ovarian local oxidative stress response in decreasing ovarian reserve (DOR) mice. All animal experiments were carried out in accordance with the guidelines and standards established by Jinan University's Experimental Animal Management Committee. Cyclophosphamide (CTX)-treated DOR mice were given Zuogui pills, cetrorelix, or a combination of the two drugs intragastrically. After treatment, there were changes in the estrous cycle, serum sex hormone levels, oxidative stress-related indexes, growth biochemical factor levels, and SIRT1/P53/P21 expression. In comparison to the model group, the Zuogui pills and the cetrorelix+Zuogui pills group had significantly prolonged estrous periods and shortened interestrous periods, and the cetrorelix+Zuogui pills group had a significantly shortened cycle length. Follicle-stimulating hormone (FSH) decreased and estradiol (E2) increased in all treatment groups compared to the model group, oxidative stress indexes nitric oxide synthase (NOS), nitric oxide (NO), and reactive oxygen species (ROS) decreased, growth biochemical factors brain derived neurotrophic factor (BDNF) and growth differentiation factor 9 (GDF-9) concentrations increased significantly, and leukemia inhibitory factor (LIF) showed no significant change. SIRT1/P53/P21 immunohistochemical results revealed that, when compared to the model group, the expression of SIRT1 increased while the expression of P53 and P21 proteins decreased in all treatment groups, with the cetrorelix+Zuogui pills group having the largest decrease, with significant differences in all indicators. We conclude that cetrorelix combined with Zuogui pills for kidney nourishing and Yin recipe improved the oxidative stress response in the follicle by regulating the SIRT1/P53/P21 pathway, reducing peroxide product production, protecting ovarian function, and regulating ovarian hormone secretion, and its efficacy is superior to that of cetrorelix or Zuogui pills alone.
ABSTRACT
OBJECTIVE@#To investigate the mechanism by which epigallocatechin gallate (EGCG) induces gene demethylation and promotes the apoptosis of acute myeloid leukemia KG-1 and THP-1 cell lines.@*METHODS@#KG-1 and THP-1 cells treated with 25, 50, 75, 100 or 150 μg/mL EGCG for 48 h were examined for gene methylation using MSP and for cell proliferation using MTT assay. The changes in cell cycle and apoptosis of the two cell lines after treatment with EGCG for 48 h were detected using flow cytometry. The mRNA and protein expressions of DNMT1, CHD5, p19, p53 and p21 in the cells were detected using RT-quantitative PCR and Western blot.@*RESULTS@#EGCG dose-dependently reversed hypermethylation of gene and reduced the cell viability in both KG-1 and THP-1 cells ( < 0.05). EGCG treatment caused obvious cell cycle arrest in G1 phase, significantly increased cell apoptosis, downregulated the expression of DNMT1 and upregulated the expressions of CHD5, p19, p53 and p21 in KG-1 and THP-1 cells ( < 0.05).@*CONCLUSIONS@#EGCG reduces hypermethylation of gene in KG-1 and THP-1 cells by downregulating DNMT1 to restore its expression, which results in upregulated expressions of p19, p53 and p21 and induces cell apoptosis.
ABSTRACT
Objective: To study the protective effect of Wuzi Yanzong Prescription (WYP) on DNA damage in testis of natural ageing rats based on P53/P21 signaling pathway and base excision repair (BER). Methods: SPF grade 16-month-old male SD rats were randomly divided into three groups with eight rats in each group: ageing model group, low and high dose of WYP groups (1 and 4 g/kg). In addition, 2-month-old SD male rats were used as adult control group. The ageing model group and the adult control group were fed with normal diet for four months. Rats in the WYP groups were given the medicated feed for four months. After fasting for 12 h, the rats were sacrificed. Then, the testes were immediately removed from rats. The expression and localization of DNA damage-related protein γH2AX and BER-related proteins OGG1, APE1, and XRCC1 were detected by immunofluorescence, and the content of 8-OHdG in testis was detected by ELISA. Also, the protein expression levels of p-P53 and P21 were detected by Western blotting. Results: Compared with the ageing model group, immunofluorescence results showed WYP significantly decreased the expression levels of DNA damage-related protein γH2AX and BER-related proteins OGG1, APE1, and XRCC1 in testis of natural ageing rats. ELISA results showed that WYP significantly downregulated 8-OHdG levels, compared with the ageing model group. Moreover, Western blotting results showed that WYP significantly decreased the protein expression levels of p-P53 and P21 of the testis when compared with the ageing model group. Conclusion: WYP reduced the DNA damage of testes in ageing related rats via P53/P21 and BER pathways.
ABSTRACT
Objective To explore the mechanism of the rat hyperplasia suppressor gene (rHSG) inhibited proliferation in C6 rat glioma cells. Methods C6 cells were cultured in vitro and transduced with the adenovirus vector which carried rHSG gene (Adv-rHSG-GFP). The transduction efficiency of adenovirus vector was measured by inverted microscope and flow cytometry in C6 cells. Flow cytometry was used to analyze the C6 cells cycle. Western blot was adopted to test the change of rHSG protein expression, the protein of cancer suppressor gene P53, cell cycle control protein of P21cip1, phosphorylation and non-phosphorylation retinoblastoma proteins (p-Rb, Rb). Results The adenovirus can insert the target gene into the genome of C6 target cell efficiently. The expression level of rHSG protein of Adv-rHSG-GFP group is obviously higher than that of PBS Group and Adv-GFP group. Meanwhile, the over-expressed C6 cells of rHSG that arrest in G0/G1 phase are largely increased (P < 0.01). Besides, there is a large increase in the protein expression of P53 and P21cip1 (P < 0.01), decrease in the expression of p-Rb (P < 0.01) and no significant change in the expression of Rb (P < 0.05). Conclusion rHSG might inhibit the proliferation of C6 rat glioma cells through P53-P21cip1 pathway.
ABSTRACT
Objective: To observe anti-aging effect and mechanism of concentrated solution of aqueous extract from Penthorum chinense. Methods: Aging mice models were caused by D-galactose, learning and memory functions were investigated by avoid dark experiment, the spleen index, activity of MAO in liver, activities of SOD and MDA in serum were recorded, and the expression of p21 and p53 protein was detected by Western blotting. Results: Concentrated solution of P. chinense by aqueous extraction increased the ability of learning and memory, improved spleen index and activity of SOD in serum, but it decreased the activity of MDA in serum and the activity of MAO in liver, and inhibited p21 and p53 protein expression in a dose-dependent manner. Conclusion: Concentrated solution of aqueous extract from P. chinense could significantly exhibit anti-aging effects, its mechanism may be related to down-regulating the p53/p21 protein expression in aging signaling pathway, enhancing immune function, and removing free radicals.
ABSTRACT
Objective To observe the intervention effect of Ginkgo biloba extract EGb761 on tert-butyl hydroperoxide ( t-BHP)-induced injury in human umbilical cord-derived mesenchymal stem cells ( hUC-MSCs) . Methods Proliferation of hUC-MSCs after primary culture was detected by methyl thiazolyl tetrazolium (MTT) assay, and then the optimal concentrations of t-BHP and EGb761 for oxidative stress injured MSC model were screened. Cell apoptosis was assessed by flow cytometry after Annexin V-fluorescein isothiocyanatel propidium iodide ( Annexin V-FITC/PI) staining. Content of malondialdehyde ( MDA) and superoxide dismutase (SOD) activity in hUC-MSCs were evaluated, and the expression levels of p53 and p21Cip1/Waf1 were analyzed by real-time fluorescence PCR. Results Pretreatment with 10~200 mg/L of EGb761 for 3 hours reduced the sensitivity of hUC-MSCs to t-BHP ( 100 μmol/L) induced proliferation inhibition, while EGb761 over 100 mg/L had no significant effect on enhancing the protection of hUC-MSCs . EGb761 at 100 mg/L prohibited hUC-MSCs apoptosis and MDA accumulation in hUC-MSCs induced by 100μmol/L of t-BHP acting for 6 hours, maintained the enzymatic activity of SOD, and decreased the expression of p53 and p21Cip1/Waf1 in hUC-MSCs with t-BHP-induced injury. Conclusion EGb761 is capable of protecting hUC-MSCs against oxidative stress injury, and its mechanism is probably related with the modulation of p53/p21 signal pathway.
ABSTRACT
This study explored the molecular mechanisms underlying the time-dependent autophagy and apoptosis induced by nutrient depletion in human multiple myeloma cell line RPMI8226 cells.RT-PCR and qRT-PCR were used to evaluate the transcriptional levels of Deptor,JNK1,JNK2,JNK3,Raf-1,p53,p21 and NFκB1 at 0,6,12,18,24 and 48 h after nutrient depletion in RPMI8226 cells.We found that transcriptional levels of Deptor were increased time-dependently at 0,6,12 and 18 h,and then decreased.Its alternation was consistent with autophagy.Transcriptional levels of Raf-1,JNK1,JNK2,p53 and p21 were increased time-dependently at 0,6,12,18,24 and 48 h accompanying with the increase of apoptosis.Transcriptional levels of NFκB 1 at 6,12,18,24 and 48 h were decreased as compared with 0 h.It was suggested that all the studied signaling molecules were involved in cellular response to nutrient depletion in RPMI8226 cells.Deptor contributed to autophagy in this process.Raf-1/JNK/p53/p21 pathway may be involved in apoptosis,and NFκB1 may play a possible role in inhibiting apoptosis.It remained to be studied whether Deptor was involved in both autophagy and apoptosis.
ABSTRACT
BACKGROUND: Aberrations of cell cycle-related genes have been reported to contribute to the formation and development of various human tumors. To investigate the gastric carcinogenesis, the expression of cell cycle-related genes (p53, p21wafl/cipl, cyclin D1 and Rb protein) compared to the morphological changes of gastric epithelial lesions were studied. METHODS: The expression of p53, p21wafl/cipl, cyclin D1 and Rb protein was immunohistochemically studied in a series of surgical specimens including the 36 normal/regenerating lesions and the 127 gastric epithelial proliferative lesions (GEPLs). The gastric epithelial proliferative lesions consisted of 25 regenerating epithelia with atypias (REAs), 27 low grade gastric dysplasias (LGDs), 17 high grade dysplasias (HGDs), 24 early gastrc carcinomas (EGCs), and 34 advanced gastric carcinomas (AGCs). RESULTS:The frequency of p53 protein overexpression was significantly associated with histologic grades of GEPLs (p=0.031); occurring in 4% of REAs, in 14.8% of LGDs, in 23.5% of HGDs, in 41.7% of EGCs and 58.9% of AGCs. The p21 wafl/cipl immunohistochemical reaction showed superficial eccentric positivity, representing an inverse correlation with histologic grades of GEPLs (p=0.04); occurring in 83.4% of normal/regenerating lesions, in 80% of REAs, in 74.1% of LGDs, in 29.4% of HGDs, 20.8% of EGCs and 8.8% of AGCs. Although Cyclin D1 and Rb proteins were expressed highly in the GEPLs, the frequency of both proteins were insignificantly associated with histologic grades of GEPLs (p=0.092). However, cases with both the Rb and cyclin D1 positivity were increased with statistical significance along histologic grades of GEPLs (p=0.044). CONCLUSIONS: The altered expression of p53, p21, Rb, and cyclin D1 was considered to be related to dysplastic progression and advancement of malignancy in GEPLs. Therefore, immunohistochemical studies of cell cycle related proteins and a combined analysis may be useful for estimating and following up cases of GEPLs.
Subject(s)
Humans , Carcinogenesis , Cell Cycle , Cyclin D1 , Cyclins , Retinoblastoma ProteinABSTRACT
Objective To investigate the role of apoptosis and apoptotic proteins p53,p21 and Bax in mechanisms of brain ischemic tolerance in rats.Methods In our study the models of focal-focal type brain ischemic preconditioning were maded by occlusion middle cerebral artery using an intraluminal filament method. Infarct sizes were measured by image analysis system.Neuronal apoptosis was identified by TUNEL staining and expressions of p53, p21 and Bax were analyzed by immunohistochemistry.Results Compared with the lethal ischemic group, the volume of infarct was greatly reduced when MCAO 20 minutes was performed as ischemic preconditioning( P