OBJECTIVE: To explore the mechanism that mediates the effect of soybean isoflavones (SI) against cerebral ischemia/reperfusion (I/R) injury in light of the regulation of regional cerebral blood flow (rCBF), ferroptosis, inflammatory response and blood-brain barrier (BBB) permeability. METHODS: A total of 120 male SD rats were equally randomized into sham-operated group (Sham group), cerebral I/R injury group and SI pretreatment group (SI group). Focal cerebral I/R injury was induced in the latter two groups using a modified monofilament occlusion technique, and the intraoperative changes of real-time cerebral cortex blood flow were monitored using a laser Doppler flowmeter (LDF). The postoperative changes of cerebral pathological morphology and the ultrastructure of the neurons and the BBB were observed with optical and transmission electron microscopy. The neurological deficits of the rats was assessed, and the severities of cerebral infarction, brain edema and BBB disruption were quantified. The contents of Fe²⁺, GSH, MDA and MPO in the ischemic penumbra were determined with spectrophotometric tests. Serum levels of TNF-α and IL-1βwere analyzed using ELISA, and the expressions of GPX4, MMP-9 and occludin around the lesion were detected with Western blotting and immunohistochemistry. RESULTS: The rCBF was sharply reduced in the rats in I/R group and SI group after successful insertion of the monofilament. Compared with those in Sham group, the rats in I/R group showed significantly increased neurological deficit scores, cerebral infarction volume, brain water content and Evans blue permeability (P < 0.01), decreased Fe²⁺ level, increased MDA level, decreased GSH content and GPX4 expression (P < 0.01), increased MPO content and serum levels of TNF-α and IL-1β (P < 0.01), increased MMP-9 expression and lowered occludin expression (P < 0.01). All these changes were significantly ameliorated in rats pretreated with IS prior to I/R injury (P < 0.05 or 0.01). CONCLUSION SI preconditioning reduces cerebral I/R injury in rats possibly by improving rCBF, inhibiting ferroptosis and inflammatory response and protecting the BBB.
Rats ; Male ; Animals ; Rats, Sprague-Dawley ; Matrix Metalloproteinase 9/metabolism* ; Soybeans/metabolism* ; Occludin/metabolism* ; Tumor Necrosis Factor-alpha/metabolism* ; Ferroptosis ; Blood-Brain Barrier/ultrastructure* ; Brain Ischemia/metabolism* ; Cerebral Infarction ; Reperfusion Injury/metabolism* ; Isoflavones/therapeutic use* ; Infarction, Middle Cerebral Artery

OBJECTIVE: To investigate the protective effect of astragalus polysaccharide (APS) against blood-brain barrier in a rat model of middle cerebral artery occlusion (MCAO) and the role of P2X7R channel in the protective mechanism. METHODS: In rat microglial cell models of oxygen and glucose deprivation (OGD) or ATP treatment, the formation of blood-brain barrier in vitro was assessed using the leak test, and the effect of APS on the permeability of the blood-brain barrier was determined using LC-MS. In 12 SD rats, MCAO model was established followed by treatment with intraperitoneal injection of normal saline (n= 6) or APS (45 mg/kg, n=6) for 3 consecutive days, with another 6 rats without MCAO receiving saline injections as the control group. The permeability of the blood-brain barrier of the rats was evaluated by determining Evans blue (EB) extravasation, and ATP content in the brain tissue was detected using ELISA; the expression levels of matrix metalloproteinase-9 (MMP-9) and P2X7R in the brain tissue were detected with Western blot. RESULTS: In the in vitro cell model of OGD or ATP treatment, APS treatment obviously promoted the repair of blood-brain barrier integrity. In the rat models, the EB content in the brain tissue and the blood-brain barrier permeability increased significantly in MCAO+saline group and MCAO+APS group as compared with those in the control group (P < 0.01). Compared with saline treatment, APS treatment significantly decreased EB content in the brain tissue and improved the blood-brain barrier permeability in the MCAO rats (P < 0.05). MCAO caused a significant reduction of ATP content and obviously increased the expression levels of MMP-9 and P2X7R in the brain tissue of the rats (P < 0.01), and these changes were significantly alleviated after APS treatment (P < 0.01 or 0.05). CONCLUSION APS can protect the brain tissue of MCAO rats by stabilizing the internal environment, down-regulating the expression of MMP-9 and improving the permeability of blood-brain barrier under cerebral ischemia and hypoxia, and its mechanism may involve the inhibition of P2X7R channel.
Animals ; Rats ; Rats, Sprague-Dawley ; Blood-Brain Barrier ; Infarction, Middle Cerebral Artery ; Matrix Metalloproteinase 9 ; Polysaccharides/pharmacology* ; Evans Blue ; Oxygen ; Glucose ; Adenosine Triphosphate

This study aims to observe the therapeutic effect of salidroside on cerebral ischemia-reperfusion(I/R) model rats, and to specifically explore the protection of salidroside on endothelial cell barrier after I/R and the mechanism. In the experiment, SD rats were randomized into sham group, model group, and high-, medium-, and low-dose(10, 5, and 2.5 mg·kg~(-1)) salidroside groups. The suture method was used to induce I/R in rats. The infarct area, neurobehavioral evaluation, and brain water content were used to evaluate the efficacy of salidroside. As for the experiment on the mechanism, high-dose and low-dose salidroside groups were designed. The pathological morphology was observed based on hematoxylin and eosin(HE) staining, and ultrastructure of vascular endothelial cells based on transmission electron microscopy. The content of nitric oxide(NO) in serum, four indexes of blood coagulation, and the content of von Willebrand factor(vWF) in plasma were measured. Western blot(WB) and immunofluorescence(IF) were employed to determine the expression of tight junction proteins(ZO-1, occluding, and claudin-1) and matrix metalloproteinase 9(MMP-9) in the cortex. The results showed that the model group had obvious neurological deficit, obvious infarct in the right brain tissue, and significant increase in water content in brain tissue compared with the sham group. Compared with the model group, high-dose and low-dose salidroside groups showed decrease in neurobehavioral score, and the high-, medium-, and low-dose salidroside groups demonstrated obviously small infarct area and significant decrease in water content in brain tissue. The results of HE staining and transmission electron microscopy showed that rats had necrosis of neurons, damage of original physiological structure of endothelial cells, and disintegration of the tight junction between endothelial cells after I/R compared with the sham group. Compared with the model group, the high-dose and low-dose salidroside groups showed alleviation of neuron injury and intact physiological structure of endothelial cells. The model group had significantly lower serum level of NO, significantly higher plasma levels of vWF and fibrinogen(FIB), and significantly shorter thrombin time(TT) and prothrombin time(PT) than the sham group. Compared with model group, the high-dose and low-dose salidroside groups increased the serum content of NO in serum, decreased the plasma levels of FIB and vWF, and significantly prolonged TT and PT. WB and IF results showed that the model group had significantly lower levels of ZO-1, occluding, and claudin-1 among endothelial cells and significantly higher level of MMP-9 than the sham group. Compared with the model group, high-dose and low-dose salidroside significantly increased the levels of ZO-1, occluding, and claudin-1 in the cortex. The above experimental results show that salidroside has clear therapeutic effect on I/R rats and protects the brain. To be specific, it alleviates the damage of endothelial cells by increasing NO synthesis in endothelial cells, inhibiting coagulation reaction and MMP-9 expression, up-regulating the expression of ZO-1, occludin, and claudin-1, thereby protecting the brain.
Animals ; Rats ; Matrix Metalloproteinase 9/metabolism* ; Endothelial Cells/metabolism* ; Reperfusion Injury/metabolism* ; Blood-Brain Barrier ; Claudin-1/therapeutic use* ; von Willebrand Factor/therapeutic use* ; Rats, Sprague-Dawley ; Brain Ischemia/metabolism* ; Cerebral Infarction ; Reperfusion ; Water/metabolism*

OBJECTIVE: To investigate the effects of blocking the activation of ERK pathway on the expression of matrix metalloproteinase-9 (MMP-9) and the formation of cerebral edema in SD rats after brain injury. METHODS: Ninety SD rats were randomly divided into 3 equal groups, including a sham-operated group, modified Feeney's traumatic brain injury model group, and ERK inhibition group where the ERK inhibitor SCH772984 (500 μg/kg) was injected via the femoral vein 15 min before brain trauma. At 2 h and 2 days after brain trauma, the permeability of blood-brain barrier was assessed by Evans blue method, the water content of the brain tissue was determined, and the phosphorylation level of ERK and the expression level of MMP-9 mRNA and protein were measured by RT-PCR and Western blotting. RESULTS: Compared with the sham-operated group, the rats with brain trauma exhibited significantly increased level of ERK phosphorylation at 2 h and significantly increased expression of MMP-9 mRNA and protein 2 days after the injury ( < 0.01). Treatment with the ERK inhibitor significantly decreased the phosphorylation level of ERK after the injury ( < 0.01), suppressed over-expression of MMP-9 mRNA and protein 2 days after the injury ( < 0.01). The permeability of blood-brain barrier increased significantly 2 h after brain trauma ( < 0.05) and increased further at 2 days ( < 0.01); the water content of the brain did not change significantly at 2 h ( > 0.05) but increased significantly 2 d after the injury ( < 0.01). Treatment with the ERK inhibitor significantly lowered the permeability of blood-brain barrier and brain water content after brain trauma ( < 0.01). CONCLUSIONS Blocking the activation of ERK pathway significantly reduced the over-expression of MMP-9 and alleviates the damage of blood-brain barrier and traumatic brain edema, suggesting that ERK signaling pathway plays an important role in traumatic brain edema by regulating the expression of MMP-9.
Animals ; Blood-Brain Barrier ; Brain Edema ; Brain Injuries, Traumatic ; MAP Kinase Signaling System ; Matrix Metalloproteinase 9 ; Rats ; Rats, Sprague-Dawley

To study the effect of modified Buyang Huanwu Decoction on the hemorrhagic transformation after intravenous thrombolysis of recombinant tissue type plasminogen activator(rt-PA) in patients with super early(onset time<4. 5 h) cerebral infarction. From March 2016 to July 2018,at the brain disease zone of the First Affiliated Hospital of Henan University of Traditional Chinese Medicine,212 cases of super early cerebral infarction were selected and divided into two group according to the randomized complete blocks designs: control group(106 cases) and traditional Chinese medicine group(106 cases). The control group was treated with rt-PA intravenous thrombolysis,while the traditional Chinese medicine group was treated with modified Buyang Huanwu Decoction in addition to the therapy of the control group. Both groups were treated for 14 days. Neurological deficit score,serum matrix metalloproteinase-9(MMP-9),neuron specific enolase(NSE),vascular endothelial growth factor(VEGF) and plasma cellular fibronectin(c-FN) levels,the incidence of hemorrhagic transformation,clinical efficacy and adverse drug reactions before and after treatment were compared between the two groups. According to the findings,at the 14 thday after treatment,the rank sum test of the grade data showed that the clinical efficacy of the traditional Chinese medicine group was better than that of the control group(Z =-2. 033,P = 0. 042); on the basis of χ2 test,the total efficiency of the traditional Chinese medicine group was higher than that of the control group(χ2= 4. 895,P =0. 027); the hemorrhagic transformation rate of the traditional Chinese medicine group was lower than that of the control group within14 days of treatment(χ2= 3. 962,P = 0. 047). MMP-9 levels in the traditional Chinese medicine group were lower than those in the control group at the 3 rd,5 th,7 th,10 th,14 thd after treatment(t =-2. 474,-3. 022,-5. 163,-6. 998,-9. 821; P = 0. 014,0. 003,0,0,0). The improvement of c-FN,NSE,VEGF and NIHSS scores in the traditional Chinese medicine group was superior to that of the control group after 14 days of treatment(t =-2. 343,-3. 187,-2. 129,-3. 105; P = 0. 020,0. 002,0. 034,0. 002). No obvious adverse reactions of modified Buyang Huanwu Decoction were observed during 14 days of treatment. Modified Buyang Huanwu Decoction could reduce the expressions of MMP-9,c-FN,NSE and VEGF after rt-PA intravenous thrombolysis in patients with super early cerebral infarction,and decrease the hemorrhagic transformation rate after thrombolysis,with high safety.
Cerebral Infarction ; drug therapy ; Drugs, Chinese Herbal ; therapeutic use ; Fibronectins ; blood ; Humans ; Matrix Metalloproteinase 9 ; blood ; Medicine, Chinese Traditional ; Phosphopyruvate Hydratase ; blood ; Recombinant Proteins ; therapeutic use ; Thrombolytic Therapy ; Tissue Plasminogen Activator ; therapeutic use ; Vascular Endothelial Growth Factor A ; blood

The objective of this study was to assess the angiogenic potential expressed as a quotient of vascular endothelial growth factor A (VEGF-A), as an indicator of proangiogenic activity, and the circulating receptors (soluble VEGF receptor protein R1 (sVEGFR-1) and sVEGFR-2), as indicators of the effect of angiogenic inhibition, depending on the concentrations of matrix metalloproteinase 2 (MMP-2) and MMP-9 and their tissue inhibitor 1 (TIMP-1) and TIMP-2 in the plasma of patients with lower extremity artery disease (LEAD). These blood parameters in patients with intermittent claudication (IC) and critical limb ischemia (CLI) were compared for select clinical and biochemical features. Stimulation of angiogenesis in the plasma of individuals with LEAD was evident as indicated by the significant increase in VEGF-A concentration along with reduced inhibition depending on circulating receptors sVEGFR-1 and sVEGFR-2. Critical ischemia was associated with higher VEGF-A, MMP-9, TIMP-1, and TIMP-2 concentrations than in the case of IC.
Aged ; Angiogenesis Inhibitors/pharmacology* ; Female ; Gene Expression Regulation ; Humans ; Intermittent Claudication/drug therapy* ; Ischemia/drug therapy* ; Lower Extremity/blood supply* ; Male ; Matrix Metalloproteinase 9/blood* ; Middle Aged ; Neovascularization, Pathologic ; Tissue Inhibitor of Metalloproteinase-1/blood* ; Tissue Inhibitor of Metalloproteinase-2/blood* ; Vascular Endothelial Growth Factor A/blood* ; Vascular Endothelial Growth Factor Receptor-1/blood* ; Vascular Endothelial Growth Factor Receptor-2/blood*

Salvianolic acid A (SAA) is a water-soluble component from the root of Salvia Miltiorrhiza Bge, a traditional Chinese medicine, which has been used for the treatment of cerebrovascular diseases for centuries. The present study aimed to determine the brain protective effects of SAA against cerebral ischemia reperfusion injury in rats, and to figure out whether SAA could protect the blood brain barrier (BBB) through matrix metallopeptidase 9 (MMP-9) inhibition. A focal cerebral ischemia reperfusion model was induced by middle cerebral artery occlusion (MCAO) for 1.5-h followed by 24-h reperfusion. SAA was administered intravenously at doses of 5, 10, and 20 mg·kg. SAA significantly reduced the infarct volumes and neurological deficit scores. Immunohistochemical analyses showed that SAA treatments could also improve the morphology of neurons in hippocampus CA1 and CA3 regions and increase the number of neurons. Western blotting analyses showed that SAA downregulated the levels of MMP-9 and upregulated the levels of tissue inhibitor of metalloproteinase 1 (TIMP-1) to attenuate BBB injury. SAA treatment significantly prevented MMP-9-induced degradation of ZO-1, claudin-5 and occludin proteins. SAA also prevented cerebral NF-κB p65 activation and reduced inflammation response. Our results suggested that SAA could be a promising agent to attenuate cerebral ischemia reperfusion injury through MMP-9 inhibition and anti-inflammation activities.
Animals ; Anti-Inflammatory Agents ; administration & dosage ; Blood-Brain Barrier ; drug effects ; enzymology ; immunology ; Brain ; Brain Ischemia ; drug therapy ; enzymology ; genetics ; Caffeic Acids ; administration & dosage ; Drugs, Chinese Herbal ; administration & dosage ; Humans ; Lactates ; administration & dosage ; Male ; Matrix Metalloproteinase 9 ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; enzymology ; genetics ; immunology ; prevention & control ; Salvia miltiorrhiza ; chemistry ; Tissue Inhibitor of Metalloproteinase-1 ; genetics ; metabolism ; Transcription Factor RelA ; genetics ; immunology

PURPOSE: Metalloproteinases are a key component of the pathogenesis of abdominal hernias. Obesity is considered a risk factor in herniogenesis and hernia recurrence. The aim of this study was to evaluate the serum concentrations of metalloproteinase-2 (MMP-2), MMP-9, MMP-13, and adiponectin in morbidly obese and non-overweight controls. MATERIALS AND METHODS: The participants were recruited from among patients undergoing bariatric and non-bariatric surgery and divided into two groups: I (body mass index (BMI)≥35 kg/m², n=40) and II (BMI<25 kg/m², n=30). Serum concentrations of MMP-2, MMP-9, MMP-13, and adiponectin were measured using enzyme-linked immunosorbent assay (ELISA). RESULTS: A statistically significant difference between groups was observed for MMP-2 concentration. The median MMP-9 concentration was higher in the obese group, but the difference was not statistically significant. Median MMP-13 concentrations did not differ between groups. Serum adiponectin concentration was insignificantly higher in the non-obese group. CONCLUSIONS The elevated serum MMP-2 and MMP-9 concentrations in obese individuals may be related to the higher incidence of incisional hernias in this population.
Adiponectin/blood* ; Adult ; Aged ; Bariatric Surgery ; Body Mass Index ; Female ; Humans ; Incisional Hernia/blood* ; Male ; Matrix Metalloproteinase 13/blood* ; Matrix Metalloproteinase 2/blood* ; Matrix Metalloproteinase 9/blood* ; Metalloproteases/blood* ; Middle Aged ; Obesity, Morbid/surgery* ; ROC Curve ; Risk Factors ; Wound Healing ; Young Adult

Adipose tissue-derived stem cell (ASCs) are an attractive source of stem cells with therapeutic applicability in various fields for regenerating damaged tissues because of their stemness characteristics. However, little has reported on evaluating adverse responses caused by human ASC therapy. Therefore, in the present study, a clinical assessment after human ASC transplantation into dogs was undertaken. A total of 12 healthy male dogs were selected and divided into four groups: saline infusion, saline bolus, ASC infusion, and ASC bolus groups. Physical assessment and blood analysis were performed following ASC transplantation, and the concentrations of angiogenic factors, and pro- and anti-inflammatory cytokines were measured by enzyme-linked immunosorbent assay (ELISA). There were no adverse vital sign responses among the dogs. Blood analyses revealed no remarkable complete blood count or serum chemistry results. ELISA results for angiogenic and anti-inflammatory factors including matrix metalloproteinase 9 (MMP9), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), and interleukin-10 (IL-10) were significantly higher in the two ASCs groups than in the controls. In conclusion, this study demonstrated that transplantation of human ASCs produced no adverse effects and could be used safely in dogs. In addition, human ASCs could be involved in modulating secretions of angiogenic factors including MMP9, VEGF, bFGF, and HGF and anti-inflammatory factor IL-10.
Angiogenesis Inducing Agents ; Animals ; Blood Cell Count ; Chemistry ; Cytokines ; Dogs ; Enzyme-Linked Immunosorbent Assay ; Fibroblast Growth Factor 2 ; Hepatocyte Growth Factor ; Humans ; Interleukin-10 ; Male ; Matrix Metalloproteinase 9 ; Stem Cell Transplantation ; Stem Cells ; Transplantation ; Vascular Endothelial Growth Factor A ; Vital Signs

Objective To explore the expressions of inhibitors of DNA binding-1 (Id-1) and matrix metalloproteinase-9 (MMP-9) in colorectal carcinoma tissues and its correlation with microvessel density (MVD). Methods The expressions of Id-1 and MMP-9 as well as CD34-labelled MVD in colorectal adenocarcinoma tissues (n=50) and normal adjacent tissues (n=50) were examined by immunohistochemistry. Results The positive expressions of Id-1 and MMP-9 were seen in 72.00% (36/50) and 78.00%(39/50) of colorectal adenocarcinoma tissues,which were significantly higher than those [24.00%(12/50) and 28.00% (14/50)] in normal adjacent tissues (P=0.000). The MVD value (17.22±2.08) in colorectal adenocarcinoma tissues was significantly higher than that (5.36±2.17) in normal adjacent tissues (P=0.000). The expressions of Id-1 and MMP-9 and MVD were significantly correlated with serosa invasion,TNM stage,carcinoembryonic antigen(+),lymph node metastasis,vascular invasion,and liver metastasis (all P<0.05) but not with the patient's age,gender,tumor size,and differentiation degree (all P>0.05). The MVD value with Id-1 and MMP-9 positive expression were significantly higher than those with Id-1 and MMP-9 negative expression (all P=0.000). The expression of Id-1 in colorectal adenocarcinoma tissues showed significantly positive correlation with that of MMP-9 (r=0.429,P=0.000). Cox multivariate analysis showed that Id-1 and MMP-9 expressions were independent prognostic factors for colorectal carcinoma. Conclusions The high expressions of Id-1 and MMP-9 have high correlations with the development and progression of colorectal adenocarcinoma and have positive correlation with MVD. Both of them may be involved in the microvascular generation and the invasion and hematogenous metastasis of colorectal carcinoma.
Adenocarcinoma ; blood supply ; metabolism ; Colorectal Neoplasms ; blood supply ; metabolism ; Disease Progression ; Humans ; Immunohistochemistry ; Inhibitor of Differentiation Protein 1 ; metabolism ; Liver Neoplasms ; Lymphatic Metastasis ; Matrix Metalloproteinase 9 ; metabolism ; Microcirculation ; Microvessels ; Neovascularization, Pathologic