Cyclosporin A induces myocardial fibrosis and affects expression of matrix metalloproteinases and their inhibitors in rats

AIM: To investigate the changes of matrix metalloproteinases (MMP2 and MMP9) and tissue inhibitor of metalloproteinases (TIMP2 and TIMP1) in the myocardial fibrosis induced by cyclosporin A (CsA), and to explore the underlying mechanism. METHODS: Female Wistar rats (n=64, 6~8 weeks old) weighing (200±25) g were randomly divided into 4 groups[control, low dose of CsA (5 mg·kg^-1·d^-1), medium dose of CsA (12.5 mg·kg^-1·d^-1) and high dose of CsA (25 mg·kg^-1·d^-1)]. The rats were intraperitoneally injected with saline and different doses of CsA, respectively. CsA was continuously administered for 1, 2 and 3 weeks, and then the animals were killed to collect samples. HE staining was used to observe the morphological structure of myocardium. Masson staining was used to observe the deposition of myocardial collagen and the degree of myocardial fibrosis. The protein expression levels of MMP2, MMP9, TIMP2 and TIMP1 were determined by immunohistochemistry and Western blot. RESULTS: HE staining showed that CsA induced cardiomyocyte edema, eosinophilic changes in the cytoplasm, fine granular and vacuolar changes in cardiomyocytes, disappearance of myocardial striae, nuclear condensation, and myocardial focal necrosis and fibrosis. Masson staining showed that the degree of myocardial interstitial fibrosis was getting worse with the increase in the duration and doses of CsA exposure. The results of immunohistochemistry and Western blot showed that the expression of MMP2 in CsA groups was significantly increased in the process of CsA-induced myocardial fibrosis. In each CsA dose group, MMP2 was highly expressed at the first week, and the expression was gradually decreased over time. In contrast, the expression of TIMP2 was increased gradually in a time-and dose-dependent manner (P<0.05). Compared with control group, MMP9 expression level was low at the first week, increased at the second week, and decreased at the third week (P<0.05). Compared with control group, the expression of TIMP1 was dose-dependently increased (P<0.05). CONCLUSION: CsA induces myocardial injury and interstitial fibrosis in rats in a dose-and time-dependent manner. CsA-induced myocardial fibrosis is associated with changes of MMPs and TIMPs, and is affected by the imbalance of MMPs/TIMPs.     

Role of MMP2/TIMP1 in the vascular remodeling after angioplasty in rabbits

AIM: To discuss the effects of matric metalloproteinasez and tissue inhibitor 1 (MMP2/TIMP1) on the vascular remodeling after angioplasty and the regulatory effect of tongxinluo on it in rabbits. METHODS: 65 male white rabbits (2.5-3.5 kg; 6-8 months) were divided into 4 groups: control group (n=5, normal diet ), injury group (n=20, normal diet plus intimal injury), high-lipid group (n=20, high cholesterol diet plus intimal injury) and tongxinluo group (n=20, tongxinluo 0.5 mg·kg-1·d-1, 4 weeks, also high cholesterol diet plus intimal injury). Under X-ray the narrow parts of the vessel underwent angioplasty. All rabbits were killed and vessel samples were excised, respectively, for the detection of histomorphometry immunohistochemically and RT-PCR. The data were expressed as ±s. RESULTS: (1) The change of vascular morphological results showed that the intimal area in tongxinluo group was less than that in high-lipid group (P<0.05), inversely the lumen area in tongxinluo group was higher than that in high-lipid group (P<0.05). The difference between the increase in lumen area and the decrease in intimal area suggested that the vascular remodeling exist. (2) RT-PCR showed that the relative level of mRNA of MMP2 and TIMP1 in tongxinluo group was less than those in high-lipid group (P<0.05). (3) The immunohistochemical staining showed that the light absorption of MMP2 and TIMP1 in Tongxinluo group was less than that in high-lipid group (P<0.05). CONCLUSION: Tongxinluo inhibits the accumulation and secretion of extracellular matrix, also inhibits vascular remodeling in rabbits, which is related with MMP2 and TIMP1.     

Effects of bilberry anthocyanins on MMP2 and collagen I in human fetal scleral fibroblast in vitro

AIM:To investigate the effects of bilberry anthocyanins on matrix metalloproteinase 2 (MMP2) and collagen type I (collagen I) expression in human fetal scleral fibroblasts (HFSF) in vitro and to provide experimental data for the prevention and treatment of myopia by bilberry anthocyanins. METHODS:HFSF were treated with bilberry anthocyanins at different concentrations (0, 10^-6, 10^-5, 10^-4, 10^-3, 10^-2, 10^-1 and 1 g/L). The effects of bilberry anthocyanins on the viability of HFSF for different time (6 h, 8 h, 12 h, 24 h and 48 h) were measured by MTT assay. The cell cycle distribution and apoptosis of HFSF with highest viability (10^-1 g/L bilberry anthocyanins for 12 h) were analyzed by flow cytometry. The mRNA expression of MMP2, COL1A1 and COL1A2 in the HFSF was detected by RT-qPCR. The protein expression of MMP2 and collagen I was determined by Western blot. RESULTS:The cell viability was the highest after treatment with bilberry anthocyanins at a concentration of 10^-1 g/L for 12 h. Compared with blank control group, 10^-1 g/L bilberry anthocyanin group showed increased cell numbers in S and G₂ phases (P<0.05), but no significant difference of the apoptotic rate was observed. The mRNA expression of MMP2 was decreased (P<0.05), and that of COL1A1 was increased (P<0.05). No significant difference of COL1A2 expression was seen. The protein expression of MMP2 was decreased (P<0.05), and collagen I protein was increased (P<0.05). CONCLUSION:Bilberry anthocyanins inhibit the expression of MMP2 and increase the expression of collagen I in the HFSF.     

Effect of edaravone on expression of Aβ and AQP4 and activity of MMP2 and MMP9 in acute cerebral ischemia/reperfusion rats

AIM: To investigate the effect of edaravone on acute cerebral ischemia/reperfusion rats. METHODS: SD rats were randomly divided into sham operation group (saline), model group (modeling given saline), low dose group (edaravone at 6 mg/kg) and high dose group (edaravone at 10 mg/kg). The rat model was established by Zea Longa suture method. The nerve function scores were evaluated after operation, and the infarct volume was measured by TTC assay. The mRNA expression of aquaporin 4 (AQP4) and amyloid β-protein (Aβ) in the brain tissue was detected by RT-qPCR. The protein levels of APQ4 and Aβ were determined by Western blot. The activity of matrix metalloproteinase 2 (MMP2) and matrix metalloproteinase 9(MMP9) was detected by gelatin zymography. RESULTS: Compared with model group, edaravone administration markedly alleviated neurological deficits, histological damages and brain edema. The mRNA and protein levels of AQP4 and Aβ, and the activity of MMP2 and MMP9 were downregulated (P<0.05). Furthermore, the improvements in high dose group were significantly more effective than those in low dose group. CONCLUSION: Edaravone significantly reduces neurological deficits and brain edema in the rats with acute ischemic stroke, and the mechanisms may be related to the downregulation of AQP4 and Aβ, and the activation of MMP2 and MMP9.     

Coordinated expression of discoidin domain receptor 2 and matrix metalloproteinase 2 in alcoholic liver disease with hepatic sinusoidal cappillatization

AIM: To observe the expression of discoidin domain receptor 2 (DDR2) and matrix metalloproteinase 2 (MMP2) in the rat model of alcoholic liver fibrosis (ALF) with hepatic sinusoidal cappillatization and to investigate the possible effect of DDR2 on the cappillatization of the sinusoids.METHODS: The ALF rat model was established by alcoholic intragastric administration plus with olive oil diet. The liver samples were harvested at the end of 4th, 8th, 12th and 16th week respectively to observe the development of hepatic sinusoidal cappillatization by reticular fiber straining and immunohistochemical staining of collagen type I, Ⅳ and laminin (LN). The expression of DDR2 and MMP2 at mRNA and protein levels was also detected by RT-PCR and Western blotting respectively to make a dependability analysis with the index of hepatic sinusoidal cappillatization. RESULTS: The liver fatty degeneration was observed at the end of 4th week of alcoholic feeding, then aggravated to inflammation and fibrosis as the process of modeling extended. Compared to control group, the mRNA and protein levels of DDR2 in the liver increased markedly in ALF model groups, which also raised gradually as the process of modeling went on (P<0.01). The mRNA and protein levels of MMP2 increased gradually in ALF groups following the aggravation of fibrosis, in which the maximum was observed in 12th week group (P<0.05). The expression of DDR2 had an obviously positive correlation with the expression of MMP2, reticular fibers, collagen type I, Ⅳ and LN. CONCLUSION: DDR2 may participate in hepatic sinusoidal capillarization through the interaction with MMP2.     

Effect of berberine on invasion and migration of PG cells from a high metastatic human giant lung carcinoma cell line

AIM:To study the effect of berberine(Ber) on invasion and migration of PG cells from a high metastatic human giant lung carcinoma cell line and to explore its mechanism. METHODS:Agarose drop method was used to detect PG migration; transwell cabin with FN in lower chamber was adopted to detect PG chemotaxis. PG adhesion to FN and martrigel was detected by MTT; PG invasive ability was determined by transwell cabin covered with martrigel. Expression of MMP2/TIMP2 protein and mRNA were detected by quantitative immunocytochemical method and RT-PCR respectively. RESULTS:After PG was treated by Ber（10 mg/L） for 24 h: 1) migration distance of Ber-treated PG cells was markedly shorter than that of control cells (P<0.01) and the number of passed membrane cells towards FN was much fewer than that of control cells (P<0.01); 2) PG adhesion to FN and martrigel was inhibited remarkably by Ber compared with control PG; 3) the migration of PG cells through the martrigel-coated transwell was significantly inhibited by the addition of Ber; 4) MMP2 expression was reduced significantly(P<0.01), while the TIMP2 expression showed up-regulating tendency, but had no differences compared with control group(P>0.05). The MMP2/TIMP2 ratio was decreased; 5) the MMP2 mRNA/TIMP2 mRNA ratio was decreased by Ber.CONCLUSION:Inhibition of cell migration, adhesion to ECM and invasion into ECM of tumor cells and regulation of homeostasis between MMPs and TIMPs to maintain ECM integrity may be the basic mechanism of inhibitive effect of Ber on invasion and metastasis of tumors.     

Effects of spironolactone on atrial structural remodeling in rabbit model of chronic atrial fibrillation

AIM: To evaluate the effects and potential mechanism of spironolactone (SP) on atrial structural remodeling in rabbit model of chronic atrial fibrillation (AF). METHODS: The sternotomy was performed and the pacing electrodes were fixed to the left atria of New Zealand white rabbits. The animals were randomly divided into 3 groups. The rabbits were subjected to rapid atrial pacing (RAP) for 3 weeks in RAP group (intragastric administration with placebo) and RAP+SP group (intragastric administration with spironolactone at 20 mg·kg^-1·d^-1), respectively. The rabbits in sham group did not receive RAP and drugs. Before and after RAP, the structure and function of the atria were evaluated and AF inducibility was tested. After RAP, the atrial fibrosis was evaluated, and the expression levels of collagen I, collagen Ⅲ, matrix metalloproteinase (MMP)-2 and MMP-9 were determined. RESULTS: After 3 weeks of RAP, compared with sham group, obvious left atrial enlargement and dysfunction were observed in RAP group and RAP+SP group, but those had no significant differences in these 2 groups. Sustained AF was induced in 7, 5, and 0 rabbits in RAP group, RAP+SP group, and sham group, respectively. Compared with sham group, atrial interstitial fibrosis and the protein expression levels of collagen Ⅰ, collagen Ⅲ, MMP-2 and MMP-9 were all significantly increased in RAP group and RAP+SP group(P<0.05). Compared with RAP group, the the above indexes were all decreased in RAP+SP group(P<0.05). CONCLUSION: Spironolactone suppresses the atrial interstitial fibrosis and collagen expression, thus preventing atrial structural remodeling in rabbit model of chronic AF. The effect of spironolactone on reducing atrial MMP-2 and MMP-9 levels may be the potential mechanism.     

Effects of down-regulation of AEG-1 expression on cell cycle and invasion of human cervical carcinoma cells

AIM: To investigate the effects of down-regulation of astrocyte elevated gene-1 (AEG-1) expression on cell cycle and invasion of human cervical carcinoma SiHa cells.METHODS: The protein expression of AEG-1 was detected by Western blotting in normal cervical tissues, cervical squamous cell carcinoma tissues, HeLa cells, SiHa cells and CaSki cells. Control siRNA or AEG-1 siRNA was transfected into SiHa cells, and the protein expression of AEG-1 in SiHa cells was detected by Western blotting. The changes of cell cycle distribution and cell invasion were determined by flow cytometry and Boyden chamber, respectively. The protein levels of cyclin D1, cyclin-dependent kinase 2(CDK2) and matrix metalloproteinase-9 (MMP-9) were analyzed by Western blotting.RESULTS: The protein expression of AEG-1 in cervical squamous cell carcinoma tissues was significantly higher than that in normal cervical tissues (P<0.05). Meanwhile, the protein expression of AEG-1 in the 3 cervical carcinoma cell lines was obviously higher than that in normal cervical tissues, in which SiHa cells displayed the highest AEG-1 protein level (P<0.05). In addition, AEG-1 siRNA effectively down-regulated the protein expression of AEG-1 in SiHa cells, which led to increase the percentage at G0/G1 phase and reduced the invasion of SiHa cells. Furthermore, the protein levels of cyclin D1, CDK2 and MMP-9 in AEG-1 siRNA group were markedly lower than those in non-treatment group and control siRNA group (P<0.05).CONCLUSION: Over-expression of AEG-1 may be closely associated with the occurrence and development of cervical carcinoma, and the AEG-1 down-regulation-mediated cell cycle arrest and attenuation of invasion may be tightly related to the down-regulations of cyclin D1, CDK2 and MMP-9 at protein levels.     

Carnosine protects against high glucose-induced injury in H9c2 cells

AIM： To explore the protective effect of carnosine (Car) on cardiomyocytes with high glucose (HG)-induced injury. METHODS：Rat H9c2 cardiomyocytes were cultured in vitro and divided into three groups: normal control (NC) group, HG group and Car pretreatment (Car+HG) group. The survival rate of H9c2 cells was measured by MTT assay. Intracellular level of reactive oxygen species (ROS) was detected by fluorescent probe DCFH-DA. The protein expression of caspase-8, caspase-9 and caspase-3 was determined by Western blotting. RESULTS：The survival rate of H9c2 cells decreased with the increases in glucose concentration and time, while pretreatment with 20 mmol/L Car could increase the survival rate significantly (P<0.05). The intracellular level of ROS in HG group was significantly increased compared with NC group (P<0.05), while that in Car+HG group was significantly decreased compared with HG group (P<0.05). The expression of caspase-8, caspase-9 and caspase-3 proteins in HG group was significantly increased compared with NC group (P<0.05). Compared with HG group, the expression of caspase-9 and caspase-3 was significantly decreased in Car+HG group (P<0.05), but the expression of caspase-8 did not obviously change (P>0.05). CONCLUSION：Carnosine can protect H9c2 cells against the injury of oxidative stress and apoptosis induced by high glucose.     

Effect of Yiqi Huayu Huatan decoction on expression of KLF15, HMGB1, NF-κB and its downstream inflammatory factors in rats with UUO-induced renal interstitial fibrosis

AIM: To observe the effect of Yiqi Huayu Huatan decoction (YHHD) on unilaterral ureteral obstruction (UUO)-induced renal interstitial fibrosis in rats, and to investigate the possible mechanism. METHODS: Female SD rats (n=48) were randomly divided into sham group, model group, telmisartan group, and low-, middle-and high-dose YHHD groups, with 8 rats in each group. The UUO model rats was established by ligating left ureter. The rats in sham group and model group were treated with equal volume of normal saline, others were treated with the corresponding drugs daily. After 12 weeks, the rats were sacrificed. The serum samples were collected for determining the concentrations of cystatin C (Cys-C) and uric acid (UA). The morphological changes of the renal tissue were observed by PAS staining. The collagen fiber was observed by Masson staining. The mRNA expression of Krüppel-like factor 15 (KLF15), high-mo-bility group box protein 1 (HMGB1), nuclear factor-κB (NF-κB), IκB, monocyte chemotactic protein-1 (MCP-1), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), fibronectin (FN), collagen type I (Col I) and Col-Ⅳ was detected by real-time PCR. The protein expression of KLF15, HMGB1 and NF-κB was detected by Western blot. The protein expression of MCP-1 was determined by the method of immunohistochemistry. RESULTS: Compared with sham group, the deposition rate of collagen fibers and the concentration of Cys-C in model group were significantly increased (P<0.05), the mRNA and protein expression of KLF15 was significantly down-regulated (P<0.05), while the mRNA expression of HMGB1, NF-κB, IκB, MCP-1, IL-1β, TNF-α, FN, Col I and Col Ⅳ and the protein expression of HMGB1, NF-κB and MCP-1 were significantly up-regulated (P<0.05). Compared with model group, the deposition rates of collagen fibers in middle-and high-dose YHHD groups and telmisartan group were significantly decreased (P<0.05), with down-regulated protein expression of HMGB1 and NF-κB and mRNA expression of IL-1β and TNF-α (P<0.05). The protein expression of KLF15 was significantly up-regulated in high-dose YHHD group and telmisartan group (P<0.05), while the protein expression of MCP-1 and the mRNA expression of FN were significantly down-regulated (P<0.05). The mRNA expression of KLF15 was significantly up-regulated in high-dose YHHD group (P<0.05), while the mRNA expression of MCP-1, Col I and Col IV was significantly down-regulated (P<0.05). The mRNA expression of NF-κB and IκB was significantly down-regulated and the concentration of Cys-C was significantly decreased in each dose of YHHD groups and telmisartan group (P<0.05). No significant difference of UA level among the groups was observed. CONCLUSION: YHHD alleviates renal interstitial fibrosis in a dose-dependent manner, and YHHD at high dose shows the most obvious effect. The mechanism may be associated with the up-regulation of KLF15 and the down-regulation of HMGB1, NF-κB and its downstream inflammation-related factors in the renal tissue.     

Changes of histone modification levels in rats with liver fibrosis

AIM: To observe the changes of histone modifications in the liver of rats with hepatic fibrosis and its possible role in the development of hepatic fibrosis. METHODS: Male Wistar rats (n=20) were randomly divided into liver fibrosis group and normal control group. The liver fibrosis model was established by hypodermic injection of CCl₄, and the rats in normal control group were injected with vegetable oils. At the end of the 8th week, all rats were killed. Liver function indexes including alanine aminotransferase (ALT) and aspartate aminotransferase (AST), and liver fibrosis indexes including haluronic acid (HA), laminin (LN), collagen type IV (Col Ⅳ) and procollagen type Ⅲ (PCⅢ) were determined by biochemical and RIA methods. The liver index was analyzed, and the liver fibrosis degree and the morphological change of the liver were detected by HE and Masson staining. The levels of α-smooth muscle actin (α-SMA), collagen type Ⅰ (ColⅠ), H3K4me2, H3K9me2, acH3K9 and acH4K12 were detected by Western blot. RESULTS: After hypodermic injection of CCl₄ for 8 weeks, the liver index, ALT, AST, HA, LN, Col Ⅳ and PCⅢ of the rats in liver fibrosis group were higher than those in control group (P<0.05). Compared with control group, the level of acH4K12 was decreased (P<0.05), while acH3K9, H3K9me2, α-SMA and ColⅠ were increased (P<0.05), but H3K4me2 had no significant change.CONCLUSION: The levels of acH4K12, acH3K9 and H3K9me2 may play essential roles in the pathogenesis of liver fibrosis, and these histone modifications may regulate gene expression associated with extracellular matrix metabolism.     

Expression of MMP3 in subchondral bones in early experimental osteoarthritis

AIM: To demonstrate that matrix metalloproteinase 3(MMP3)plays MMP3 play an important role in the pathogenesis of osteoarthritis by studying the expression of MMP3 in subchondral bones in early experimental osteoarthritis at gene and protein levels. METHODS: The SD rats were randomly divided into 2 groups: experimental group and control group, each containing 30 rats with similar body weight. The right knee joints of the rats in experimental group underwent surgery, which involved in both medial collateral ligament (MCL) transection and medial meniscectomy, while the animals in control group were only carried out a sham operation. The rats were killed at the 1st, 2nd and 4th weeks post-surgery to obtain the right knee joints. Pathological analysis was performed to validate this early osteoarthritic rat model. The expression of MMP3 in subchondral bones at mRNA and protein levels was evaluated by real-time polymerase chain reaction and immunohistochemical staining, respectively. RESULTS: The expression of MMP3 in subchondral bones was significantly increased at both the 1st and 2nd weeks post-surgery in experimental group, with the fold changes of 8.34 (P<0.05) and 4.85 (P<0.05), respectively, as compared with control group. No differentially expression of MMP3 was observed at the 4th week post-surgery between these 2 groups. A lot of MMP3 positive cells, including small mononuclear cells and bigger polynuclear giant cells, were found in subchondral bones in experimental group at the 1st and 2nd weeks post-surgery,but not in control group. At the 4th week post-surgery, no MMP3 positive cells were found in subchondral bones of both groups. CONCLUSION: MMP3 plays an important role in the pathogenesis of early experimental osteoarthritis.     

Mechanism of miR-30c over-expression inhibiting malignant phenotypes of cervical cancer cells

AIM:To investigate the molecule mechanism of microRNA (miR)-30c over-expression inhibiting malignant phenotypes of cervical cancer cells. METHODS:Cervical cancer cell lines C33A, HeLa, SiHa and CaSki were transfected with pGenesil-1-miR-30c plasmid using Lipofectamine 2000 kit, and the expression of miR-30c was determined by TaqMan real-time PCR. The cell viability inhibition rate, colony formation ability, migration rate and apoptotic rate were measured by MTT assay, colony formation assay, Transwell experiment, and flow cytometry with Annexin V-FITC staining. The protein expression of Bax, Bcl-2, matrix metalloproteinase (MMP)-9, MMP-13 and tissue inhibitor of metalloprotei-nase-1 (TIMP-1) was detected by Western blot. RESULTS:The expression levels of miRNA-30c in the cervical cancer cell lines transfected with pGenesil-1-miR-30c plasmid were significantly higher than those in negative control groups (cell lines transfected with pGenesil-1 plasmid) (P<0.01). Significantly increased cell viability inhibition rate, and decreased colony formation ability and migration rate were found in the cervical cancer cell lines over-expressing miR-30c as compared with negative control groups (P<0.05). The apoptotic rate in the cervical cancer cell lines over-expressing miR-30c was dramatically higher than that in control groups (P<0.05). Over-expression of miR-30c in cervical cancer cells promoted the protein expression of Bax and TIMP-1, and decreased the protein expression of Bcl-2 and MMP-13 (P<0.05 or P<0.01). CONCLUSION:Over-expression of miR-30c significantly inhibits the viability and migration, and induces apoptosis of cervical cancer cells. The mechanism may be related to activating apoptosis pathway and inhibiting MMP-13 protein expression.     

Molecular mechanism of BMSC intracerebral transplantation in improving learning and memory abilities of AD mice

AIM: To investigate the effect of bone marrow mesenchymal stem cell (BMSC) transplantation on learning and memory abilities and pathological changes of Alzheimer disease (AD) mice and the molecular mechanisms. METHODS: C57/BL6 wild-type (WT) and transgenic (Tg) mice were randomly divided into 4 groups:WT/PBS group, WT/BMSCs group, Tg/PBS group and Tg/BMSCs group. The mice were administered with PBS or BMSCs via intracerebroventricular injection. Spatial learning and memory abilities of the mice were evaluated by Morris water maze test on the 3rd day after surgery. Real-time PCR was applied to detect the mRNA expression of CX3C chemokine ligand 1 (CX3CL1), CX3C chemokine receptor 1 (CX3CR1), IL-1β, TNF-α, Nurr1, YM1, insulin-degrading enzyme (IDE) and matrix metalloproteinase 9 (MMP9). The protein levels of CX3CL1 and Aβ42 were measured by ELISA. Western blot was used to detect the protein expression of postsynaptic density protein 95 (PSD95) and synaptophysin (SYP). RESULTS: The transplanted BMSCs were observed near the hippocampus of APP/PS1 mice on the 10th postoperative day. The escape latency of the mice in Tg/PBS group was significantly longer than that in the WT/PBS mice (P<0.05). Compared with Tg/PBS group, the escape latency of Tg/BMSCs group was significantly shorter (P<0.05), and the mRNA and protein levels of CX3CL1 in Tg/BMSCs group were significantly higher than those in Tg/PBS group (P<0.01). The results of immunohistofluorescence staining showed that BMSC transplantation promoted the activation of microglia in the brain of WT and Tg mice. The mRNA expression of YM1 was up-regulated in WT/BMSCs group and Tg/BMSCs group (P<0.05). Compared with WT/PBS mice, the mRNA expression of TNF-α in the cortex and hippocampus of Tg/PBS group was significantly increased (P<0.05), and the mRNA expression of Nurr1 in the cortex was significantly decreased (P<0.01). Meanwhile, the mRNA expression of TNF-α in the cortex of Tg/BMSCs mice was decreased (P<0.01) and the mRNA expression of CX3CR1 and Nurr1 was up-regulated compared with Tg/PBS group (P<0.05). The results of Western blot showed that the protein levels of PSD95, p85, p110 and p-Akt in Tg/BMSCs group were significantly higher than those in Tg/PBS group (P<0.05). Finally, BMSC transplantation reduced the protein level of Aβ42 in APP/PS1 mice (P<0.05), and increased the mRNA expression of IDE and MMP9 in the hippocampus (P<0.05). CONCLUSION: BMSC transplantation modulates neuroinflammatory responses and promotes neuroprotective factor and synaptic protein expression, thus improving the learning and memory abilities in the APP/PS1 mice, which may be achieved by up-regulating the expression of CX3CL1.     

Effect of Yangyu Tuji on content of type Ⅰ, Ⅲ collagen and the expression of MMPs and TIMP-1 in wound caused by streptozotocin in rats

AIM: To study the effects of Yangyu Tuji (YYTJ) on delayed healing wound of diabetic rats caused by streptozotocin (STZ). METHODS: SD male rats were randomly divided into control group (control), model group (model); and 3 different dose groups of YYTJ. 55 mg/kg STZ were given by intraperitoneal injection except for control group. After 30 days, a round skin of 1.6 cm diametre was excised on all dorsal back of rats. The healing time and healing rate were observed according to re-epithelization. The content of collagenⅠ and Ⅲ was observed by Picric acid-Sirius red staining , Matrix metalloproteinase-1, 13 (MMP-1, -13), tissue inhibitor of metalloproteinases-1 (TIMP-1) by immuno-histochemistry assay. All data were analyzed by IPP software. RESULTS: The healing time in each group treated with YYTJ was shorter than that in model group (P<0.01), and the healing rate was increased (P<0.01, P<0.05). Content of type I collagen, ratio of type Ⅰ and Ⅲ collagen of high and mid dose group were significantly higher than that in model group (P<0.01) at 3rd, 7th, 11th day. The expression of MMP-1, -13 of each groups were higher than that in model group at 7th day (P<0.01, P<0.05), and MMP-1 trend to equal with model group at 11th day. MMP-13 was significantly lower than that in model group at 11th day (P<0.01, P<0.05). TIMP-1 of each group of wound was higher than that in model group at 3rd, 7th, 11th day (P<0.01, P<0.05). The ratio of type Ⅰ and Ⅲ collagens in each group was lower than that in model group at 11th day (P<0.01). Ratio of MMP-13 and TIMP-1 of high dose group and mid dose group were higher than that in model group at 3rd and 7th day (P<0.01). The ratio of each group was lower than that in model group at 11th day (P<0.01). Meanwhile, ratio of MMP-13 and TIMP-1 of high dose group and mid dose group were lower than that of lower dose group (P<0.05). CONCLUSION: It is possible that YYTJ accelerates wound healing by increasing collagen content of type Ⅰ and Ⅲ, especially type Ⅰ, as well as improves collagen deposition by regulating the balance of MMP and TIMP.     

Effects of icariin on expression of transforming growth factor β1 and type Ⅳ collagen in diabetic rat testis

AIM: To determine the beneficial effects of icariin on streptozotocin (STZ)-induced diabetic testopathy in rats. METHODS: The diabetic animal model was induced in male Sprague-Dawley rats by an injection of streptozotocin (40 mg/kg, iv). The rats were randomly divided into 3 groups: control group, model group and icariin (80 mg/kg, ig) group. Twelve weeks after injected with streptozotocin, all rats were anaesthetized and killed to remove the testes from scrotum. Serum concentrations of glucose and testosterone, and the levels of succinate dehydrogenase (SDH), acid phosphatase (ACP), γ-glutamyl transpeptidase (γ-GT) and lactate dehydrogenase (LDH) in testes were measured. The morphology of the testicular tissues was observed under light microscope. Immunohistochemistry was employed to determine the protein levels of TGF-β₁ and type Ⅳ collagen. RESULTS: Compared with control group, the content of serum glucose increased while the serum level of testosterone and the activitiy of SDH, ACP, γ-GT and LDH in testis decreased in model group (P<0.01). The histopathological examination showed that the diameters of seminiferous tubules and various grades of spermatocytes in the testis were markedly decreased. Compared with control group, the expression of TGF-β₁ and collagen Ⅳ was significantly increased in model group. These alterations were significantly attenuated in icariin group (P<0.01). CONCLUSION: Icariin evidently relieves testicular damage in rats with diabetic testopathy by improving the secretion of testosterone and reducing the expression of TGF-β₁ and collagen Ⅳ at protein level.     

Effects of furin inhibitor on metastasis of human breast cancer MCF-7 cells

AIM：To investigate the mechanism underlying breast cancer metastasis and to provide theoretical data for studying the pathogenesis of breast cancer onset and development. METHODS：Human breast cancer MCF-7 cells were treated with different concentrations of furin inhibitor α1-PDX for 48 h. Wound healing assay and Transwell assay were applied to detect the migration and invasion abilities of the MCF-7 cells. The expression of cell migration-associated proteins, including membrane-type 1 matrix metalloproteinase (MT1-MMP), vascular endothelial growth factor (VEGF)-C and VEGF-D, was determined by Western blotting. The protein levels of MMP2 and MMP9 in the supernatant were measured by ELISA. RESULTS：Compared with control group, 200 nmol/L of furin inhibitor exerted significant inhibitory effects on the cell migration (P<0.05). The expression of cell migration-associated proteins MT1-MMP, VEGF-C and VEGF-D was significantly inhibited after treated with α1-PDX (P<0.05). Significant inhibitory effects of α1-PDX on the expression of MMP9 and MMP2 (P<0.05) in the supernatant were observed. CONCLUSION：Furin inhibitor suppresses the metastasis of MCF-7 cells via down-regulating the expression of MMPs and VEGFs.