BACKGROUND:Tissue inhibitor of matrix metaloproteinase 1 (TIMP-1) is the corresponding antagonist of matrix metaloproteinase 13 (MMP-13), and their balance between expression and functional activity exerts an important role in the metabolic state of the extracelular matrix. During the development of osteoarthritis, however, TIMP-1 and MMP-13 expressions and their expression ratio show unclear changes in DH guinea pigs.
OBJECTIVE:To explore the expression levels of MMP-13 and TIMP-1 in DH guinea pigs with different ages, and to analyze the relationship between the ratio of MMP-13 to TIMP-1 and the age-dependent degenerative changes in the articular cartilage.
METHODS:Twenty-four female Dunkin Hartley guinea pigs were sacrificed at age of 2, 4, 8, 12 months separately, with six animals at each time point. The knee joints were colected and gross visual appearance of the articular cartilage was observed, then were decalcified and prepared for paraffin sections. VG staining and Mankin score were used to analyze the histological changes. Immunohistochemistry was conducted to assess the expression levels of MMP-13 and TIMP-1 in the cartilage. Integrated absorbance values were used as the quantitive analysis calculated by Image pro-Plus 6.0. Linear regression analysis was done to analysis the relationship between Mankin score and the ratio of MMP-13/TIMP-1. RESULTS AND CONCLUSION:Normal appearance in the articular cartilage was observed in 2-month-old DH guinea pigs, while degenerative changes in the articular cartilage were shown in 4-month-old animals, which became severer with age. Significant difference was found in Mankin score between any two groups (P < 0.05). The ratio of MMP-13 to TIMP-1 increased with age, and the ratio was positively correlated to the Mankin score (P < 0.05). Age-related articular cartilage degeneration occurred in Dunkin Hartley guinea pigs at 4 months of age, and devoloped with age, which is related with the imbanlance of the expression ratio of MMP-13 to TIMP-1.

Aim To explore the role of endoplasmic re-ticulum stress( ERS) in Astragaloside Ⅳ-induced car-dioprotection against ischemia/reperfusion injury in rats. Methods A model of myocardial ischemia 30 min followed by 120 min reperfusion was made by liga-ting coronary artery in male Wistar rats. Rats were di-vided randomly into 4 groups: sham group, ischemia/reperfusion group, ERS inhibitor TUDCA group, As-tragaloside Ⅳgroup. Myocardial samples were collect-ed from the risk zones during ischemia and reperfu-sion, ERS was determined by measuring levels of glu-cose regulated protein 78 ( GRP78 ) , an established marker of ERS with Western blot. Immunofluorescence study was used to test GRP78 intensity with laser scan-ning confocal microscopy, TTC method was used to measure the infarct size,hematoxylin-eosin staining was used to observe the changes of morphological changes of myocardium. Results There was no statistical difference in GRP78 expression during ischemia com-pared to the sham group, but was markedly increased upon reperfusion. Astragaloside Ⅳ could mimic TUD-CA and significantly decreased the GRP78 expression, reduced infarct size and improved the morphology of myocardial tissue with a significant statistical difference compared with the control group ( P<0. 05 ) . Conclu-sions ERS is induced upon reperfusion but not during ischemia in isolated rat hearts. Astragaloside Ⅳ pre-vents myocardial reperfusion injury presumably by the inhibition of ERS.

Aim To explore the role of endoplasmic reticulum stress(ERS) in Astragaloside Ⅳ-induced cardioprotection in H9c2 cardiac cells, and to explore the potential mitochondrial mechanism.Methods Conventional culture was performed of rat heart tissue-derived H9c2 cells.Experiment was randomly divided into the control group, the ERS inducer 2-Deoxy-D-Glucose(2-DG) group, Astragaloside Ⅳ and 2-DG combination group, Astragaloside Ⅳ group.Confocal microscopy was used to observe the changes of TMRE fluorescence intensity so as to confirm the influence of ERS on the mitochondrial potential, and further speculate on the opening of the mitochondrial permeability transition pore(mPTP).Western blot analysis was applied to detect the expressions of ERS proteins GRP 78, GRP 94 and IRE1.Transmission electron microscopy was used to observe the ultrastructure of the cells.Results Different doses of 2-DG could mimic the mPTP opener atractyloside to induce the mPTP opening with the peak at 100 μmol·L-1;Astragaloside Ⅳ significantly reduced 2-DG-induced mPTP opening, the expression of GPR 78, GRP 94 and IRE1 and reduced injury of mitochondria and endoplasmic reticulum.Conclusions Endoplasmic reticulum stress could be induced by 2-DG.Astragaloside ⅳ-induced mitochondrial cardioprotection involves inhibition of the ERS through GRP 78, GRP 94 and IRE1 by prevention of the mPTP opening.

BACKGROUND:Studies have shown that human telomerase reverse transcriptase (hTERT) has the ability to enhance cel proliferation, maintain telomere length, prolonged cel life cultured in vitro. OBJECTIVE:To observe the effect of hTERT gene-modified bone marrow mesechymal stem cel transplantation on neural function recovery of rats with cerebral infarction. METHODS:Rat models of middle cerebral artery occlusion were established and randomized into model group, cel transplantation group and hTERT-modified cel transplantation group, with 20 rats in each group. Rats in the three groups were respectively injected via tail vein with 1 mL PBS, passage 9 bone marrow mesenchymal stem cel suspension (2.5×107/L) and hTERT-modified passage 9 bone marrow mesenchymal stem cel suspension (2.5×107/L), respectively. Modified neurological severity scores were determined before and after transplantation; RT-PCR and western blot assay were used to measure hTERT expression at gene and protein levels; TUNEL method was adopted to detect cel apoptosis in the brain. RESULTS AND CONCLUSION:hTERT-modified bone marrow mesenchymal stem cels had prolonged cel cycle, and with the increase in passage number, the cels showed good growth with no changes in morphology. The expressions of hTERT mRNA and protein were superior in the hTERT-modified cel transplantation group than the cel transplantation group, and there was a significant difference (P < 0.05). Modified neurological severity scores and number of apoptotic cels were decreased significantly in the hTERT-modified cel transplantation group compared with the other two groups (P < 0.05). These findings indicate that hTERT-modified bone marrow mesenchymal stem cels can promote neural functional recovery of rats with cerebral infarction.

BACKGROUND:Numerous clinical studies have confirmed that the microenvironment at a spinal cord injury site can be obviously improved through hyperbaric oxygen therapy; however, what effect does hyperbaric oxygen have on the microenvironment of the injured brain? OBJECTIVE:To investigate the effect of hyperbaric oxygen therapy on nerve regeneration microenvironment and the recovery of rat nerve function after focal cerebral infarction. METHODS:Rat models of focal cerebral infarction were established by occlusion of the middle cerebral artery and subjected to hyperbaric oxygen therapy. Sham group and model group were established as comparison. In the sham group, rat models of focal cerebral infarction were established but did not receive any treatment. Rats in the model group were placed in a hyperbaric oxygen therapy chamber but the pressure and oxygen concentration were not administered. RESULTS AND CONCLUSION:Compared with the model group, the score of rat limb function at 16 days after treatment and the expression of growth associated protein 43 in the rat cerebral infarcted area at postoperative 14 days were significantly increased , but infarct volume at postoperative 24 hours was al significantly decreased in the hyperbaric oxygen therapy group (alP < 0.05). These results confirmed that hyperbaric oxygen therapy can improve nerve regeneration microenvironment and promote the recovery of rat nerve function after focal cerebral infarction.

OBJECTIVETo investigate the effects of the furin inhibitor α1-PDX on the growth, invasion, and tumorigenicity of cervical cancer cells and explore the mechanisms.

METHODSThe changes in the growth, migration and invasion of α1-PDX-transfected HeLa cells were observed using MTT assay, Boyden migration and invasion assay. The protein levels of furin and MT1-MMP were measured using Western blotting and furin activity was detected by enzyme activity assay in the transfected cells. HeLa cells were seeded subcutaneously in nude mice and the tumor volume changes were recorded.

RESULTSCompared with the control cells, α1-PDX-treated cells showed a significant growth inhibition by 18.4% at 24 h (P<0.01) with obviously lowered migration ability and cell invasiveness (P<0.01). Treatment with α1-PDX significantly reduced furin enzyme activity and MTI-MMP protein levels in HeLa cells. In nude mice, α1-PDX-treated HeLa cells exhibited a delayed and lowered tumorigenicity with reduced size of the tumors.

CONCLUSIONα1-PDX can inhibit the growth, metastasis and tumorigenicity of HeLa cells, the mechanism of which may involve a decreased furin activity and MTI-MMP expression.

Animals ; Female ; Furin ; antagonists & inhibitors ; HeLa Cells ; drug effects ; Humans ; Mice ; Mice, Nude ; Neoplasm Transplantation ; Transfection ; Uterine Cervical Neoplasms ; pathology ; alpha 1-Antitrypsin ; pharmacology

Objective:To investigate whether astragaloside IV (AS-IV) exerts neuroprotective effects via zinc ion (Zn 2+) modulation of mitochondria-associated endoplasmic reticulum membranes (MAMs) and to elucidate the possible mechanisms. Methods:PC12 cells (rat adrenal pheochromocytoma cells) were routinely cultured; they were divided into 7 groups: control group (routinely cultured), 2-DG group (treated with 2-DG at 50 μmol/L for 30 min), astragaloside IV+2-DG group (treated with 50 μmol/L astragaloside IV for 20 min, and then treated with 50 μmol/L 2-DG for 30 min), astragaloside IV group (treated with 50 μmol/L astragaloside IV for 20 min), N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (a zinc chelator, TPEN)+astragaloside IV+2-DG group (treated with 10 μmol/L TPEN for 10 min, 50 μmol/L astragaloside IV for 20 min, and then 50 μmol/L 2-DG for 30 min); TPEN group (treated with 10 μmol/L TPEN for 10 min), TPEN+2-DG group (treated with 10 μmol/L TPEN for 10 min and then treated with 50 μmol/L 2-DG for 30 min). The expressions of glucose-regulated protein (GRP)78, GRP94, cysteine-containing aspartate proteolytic enzyme caspases-8, B cell receptor associated protein 31 (BAP31) and mitochondrial fission protein 1 (Fis1) were detected by Western blotting. Annexin V-FITC/PI kit was used to detect the apoptosis level. Immunofluorescence was used to detect the Fis1 protein expression, and mitochondrial fluorescence probe TMRE was used to detect the opening of mitochondrial permeability transition pore (mPTP).Results:(1) Compared with the control group, the 2-DG group had significantly increased expressions of GRP78, GRP94, Caspase-8, Bap31 and Fis1, and green fluorescent intensity of Annexin V-FITC ( P<0.05); compared with the 2-DG group, the astragaloside IV+2-DG group had significantly decreased expressions of GRP78, GRP94, Caspase-8, Bap31 and Fis1, and green fluorescent intensity of Annexin V-FITC ( P<0.05); compared with the astragaloside IV+2-DG group, the TPEN+astragaloside IV+2-DG group had significantly increased expressions of GRP78, GRP94, Caspase-8, Bap31 and Fis1, and green fluorescent intensity of Annexin V-FITC ( P<0.05). (2) Compared with the control group, the 2-DG group had significantly enhanced Fis1 protein fluorescent intensity ( P<0.05); the astragaloside IV+2-DG group had significantly decreased Fis1 protein fluorescent intensity compared with 2-DG group ( P<0.05); Compared with astragaloside IV+2-DG group, TPEN+astragaloside IV+2-DG group had significantly enhanced Fis1 protein fluorescence intensity ( P<0.05); compared with the control group, the TPEN+2-DG group had significantly enhanced Fis1 protein fluorescent intensity ( P<0.05). (3) TMRE fluorescence intensity in 2-DG group was significantly decreased compared with control group ( P<0.05); TMRE fluorescence intensity in astragaloside IV+2-DG group was significantly enhanced compared with 2-DG group ( P<0.05); TMRE fluorescence intensity in TPEN+astragaloside IV+2-DG group was significantly decreased compared with astragaloside IV+2-DG group (P<0.05); TMRE fluorescence intensity in TPEN+2-DG group was significantly decreased compared with the control group ( P<0.05). Conclusion:Astragaloside IV can exert neuroprotective effects through Zn 2+ inhibiting endoplasmic reticulum stress-induced apoptosis and preventing mPTP opening in PC12 cells, whose mechanism may be related to Fis1 and Bap31 expressions.