Effects of IL-4 on cyclooxygenase-2 and platelet-derived growth factor in the lungs of COPD rats

Objective： To explore the role of interleukin 4（IL-4）, expressions of cyclooxygenase-2 （COX-2） and platelet-derived growth factor （PDGF） in the model of chronic obstructive pulmonary disease （COPD）, in the lungs of rats. Methods： Male Wistar rats were used to build up the model of COPD. Rats were randomly divided into the control group and model group, the IL-4 group and the dexamethasone group. The expressions of COX-2, PDGF-A and PDGF-B in the lung tissue were detected by western blotting and RT-PCR. Results： The expressions of COX-2, PDGF-A and PDGF-B in the model group were increased significantly. Those expressions in the IL-4 and dexamethasone group were notably decreased. Conclusion： IL-4 and dexamethasone could interfere in the establishment of COPD. The expressions of COX-2 and PDGF in the lung tissue of COPD were increased significantly and IL-4 and dexamethasone could decrease those expressions.     

Effect of Wumeiwan on Cytokines TNF-α, IL-6, IL-8, IL-10 and Expression of NF-κBp65 in Rats with Ulcerative Colitis

The effects of Wumeiwan （WMW） on TNF-α, IL-6, IL-8, IL-10 and NF-κBp65 in rats with ulcerative colitis （UC） were investigated, the curative effectiveness of WMW vs salicylazosulfapyridine （SASP） was compared, and the action mechanism was analyzed. Fifty-Six Sprague-Dawley （SD） rats were randomly divided into four groups （n=14 in each group, with equal ratio of male and female）： normal control group, model group, SASP group, and WMW group. Except normal control group, the rat UC models in the remaining three groups were established using the method of 2.4-dinitrochlorobenzene （DNCB） immunization and acetic acid local enema. The rats in model group, SASP group, and WMW group were treated with distilled water, SASP, and WMW respectively. The changes in the symptoms and signs were observed, and levels of IL-6, IL-8, TNF-α, IL-10 and the expression of NF-κBp65 in the colonic tissues were statistically analyzed. The results showed that the levels of IL-6, IL-8, and TNF-α were significantly increased （P〈0.01）, while those of IL-10 significantly reduced （P〈0.01） after establishment of rat UC models as compared with normal control group. The levels of IL-6, IL-8, and TNF-α were obviously lower, but the level of IL-10 was obviously higher in WMW and SASP groups than those in model group （P〈0.05）. The levels of IL-6, IL-8, and TNF-α were lower, while the level oflL-10 was higher in WMW group than in SASP group. NF-κBp65 was expressed negatively or weakly in normal colonic tissues. The positive expression rate of NF-κBp65 in WMW group and SASP group was obviously lower than in model group （P〈0.01）, and there was significant difference between WMW group and SASP group （P〈0.05）. It was concluded that rat UC model was established successfully. WMW could up-regulate the expression of IL-10, down-regulate the expression of TNF-α, IL-6, IL-8, and inhibit the NF-κBp65 activity to adjust immune function, indicating WMW had better curative effects on UC in rats.     

Effect of Quyu Chencuo Formula(去菀陈莝方)on Renal Fibrosis in Obstructive Nephropathy Rats

Objective: To observe the effect of Quyu Chencuo Formula(去菀陈莝方, QCF) on renal fibrosis in rats with obstructive nephropathy. Methods: Twenty-four rats were randomly divided into three groups, 4 for sham operation as the control group, 10 for unilateral ureteral obstruction(UUO) model group, and the rest 10 for QCF treating UUO model group. All rats were sacrificed under 3% pentobarbital(50 mg/kg) anesthesia on the 14 th day after surgery, then the right kidney samples of rats were harvested for hematoxylin eosin(HE) staining and Masson staining to observe the renal pathological changes. Immunohistochemistry and Western blotting were used to examine the expression of transforming growth factor β1(TGF-β1), and real-time polymerase chain reaction(RT-PCR) was employed to examine the expressions of TGF-β1, α-smooth muscle actin(α-SMA) and E-cadherin mRNA. Results: HE and Masson staining showed that the renal interstitial of the rats in the control group had no significant fibrotic lesion; in the model group, there were obvious interstitial fibrosis; for the QCF group, there were epithelial cell necrosis, infiltration of lymphocytes and mononuclear cells, aggravated interstitial fibrosis in varied degrees, but the pathological changes were less in the QCF group than in the model group. The immunohistochemistry and Western blotting results showed that the TGF-β1 expression was increased significantly in the model group, while decreased significantly in the QCF group(P0.05); RT-PCR showed that the mRNA expression of α-SMA and TGF-β1 increased significantly in the model group, while both were significantly decreased in the QCF group compared with the model group(P0.05). The mRNA expression of E-cadherin was decreased significantly in the model group, and it was significantly increased in the QCF group as compared with the model group(P0.05). Conclusion: QCF may improve renal fibrosis by regulating the expressions of TGF-β1, α-SMA and E-cadherin, and prevent the progress of kidney fibrosis.     

Effect of ecdysterone on heteroptopic heart transplantation in rats

Objective:To investigate the protective effects of ecdysterone(EDS)on the allograft heart transplant model of rats.Methods:Fifty healthy Sprayue-Dawley(SD)rats were divided into donors and recipients randomly.Hearts were harvested and placed heterotopically into allogenic recipients.All animals were divided into three groups:sham-operation group(only opening and closing the abdomen, n=10),EDS group(injected intraperitoneally with 20 mg/(kg·day)of EDS,n=10),and control group(injected intraperitoneally with normal saline,n=10).The pathological changes of myocardial tissue were analyzed by light microscopy and transmission electron microscopy and the levels of myocardial enzymes(GOT,LDH,CPK),SOD,ET-1,NO,MDA in serum were measured.Tissue samples underwent the detection of apoptotic cell death by in situ terminal deoxyribonucleotide transferase-mediated dUTP nick end labeling for apoptotic index(AI)and also by immunohistochemistry method to study the expressions of Bcl-2 and Bax.Results:Pathological examination and transmission electron microscope observation showed greater myocardium damage in the control group.EDS group showed a decrease in the amount of myocardial enzymes,MDA,ET-1 and an increase in the levels of SOD,NO,compared to the control group.The AI of EDS group became lower than that of control group,meanwhile the EDS group increased the expression of Bcl-2 and decreased the expression of Bax.Conclusion:EDS has protective effects on heteroptopic heart transplantation,which provides a new idea for myocardial protection in heart transplantation.However,the mechanism of its protective effect needs further research.     

Immunoregulative Mechanism of the Leydig Cell in the Infection by Expression1 of IL-1,IL-6,TGF-β, Fas and FasL mRNA

Objective To investigate the immune regulative mechanism of Leydig cells in the local infection of rat＇s testis. Methods Ureaplasma Urealyticum（UU） was injected into rat＇s bladder, which mimicked an ascending infectious way, and at the same time culture medium was injected into rat＇s bladder as the control. The rats were sacrificed at week 1, 2 and 3 after injection respectively. Then pathological changes in testis were analyzed by histological examination. At the same time Leydig cells were separated from rat＇s testis. The comparasion of levels of IL-1,IL-6, TGF-β, Fas and FasL mRNA expression among the three groups was made by RT-PCR. Results As compared with control group, the levels of IL-1, IL-6, TGF-fl mRNA expression for UU supernatant and living UU groups increased; and levels of Fas and FasL mRNA expression decreased and increased respectively after UU infection. Conclusion During anti-infective immunity, rat＇s Leydig cells may regulate immune function of the testis by changing the levels of IL-1, IL-6, TGF-β, Fas and FasL mRNA expression and may contribute to maintain immune privilege of the testis.     

Inhibition of rejection in murine islet xenografts by CTLA4Ig and CD40LIg gene transfer

Background Costimulatory signals play a vital role in T cell activation. Blockade of costimulatory pathway by CTLA4Ig or CD40LIg have enhanced graft survival in experimental transplantation models yet mechanisms remain undetermined.We investigated the effects of CTLA4Ig and CD40LIg gene transfer on islet xenografts rejection in rats.Methods Human islets were infected with recombinant adenoviruses containing CTLA4Ig and CD40LIg genes and implanted beneath the kidney capsule of diabetic rats. Levels of blood sugar, morphological changes, and survival of grafts were recorded. Expressions of CTLA4Ig, CD40LIg and insulin were detected by immunohistochemical staining and cytokines levels were quantified by enzyme-linked immunosorbent assay （ELISA）.Results Blood glucose levels in transplant rats decreased to normal level on the 2nd day post transplantation. The mean blood glucose in the control group, CTLA4Ig transfected group, CD40LIg transfected group and CTLA4Ig ＋CD40LIg cotransfected group increased on days 8, 24, 21, 68, post transplantation respectively. The grafts in control group, CTLA4Ig transfected group, CD40LIg transfected group and CTLA4Ig ＋ CD40LIg cotransfected group survived for （8±1）, （29±4）, （27±3）, and （74±10） days, respectively. Survival in CTLA4Ig ＋ CD40LIg cotransfected group was significantly longer. Survivals of CTLA4Ig transfected group and CD40LIg transfected group were significantly longer than control group. In controJ animals, serum interleukin-2 and tumor necrosis factor a concentration significantly increased within seven days post transplantation. Haematoxylin eosin staining of grafts showed live islets in situ of transplant rats without inflammatory cell infiltration. Immunohistochemical staining confirmed the expression of insulin at islets in all experimental groups.Conclusions Transfer of CTLA4Ig and CD40Llg genes, especially the cotransfer of both, inhibits rejection of murine islet xenografts. Downregulated expressions of Th1 cells related cytokines might be related to the beneficial effects.     

Expression of fractalkine and its receptor in acute cardiac allografts rejection

Objective To investigate the expression of fractalkine(FKN) and its receptor CX3CR1 in cardiac allografts and the effect of Cyclosporin A(CsA). Methods Three groups of rats underwent heterotopic cardiac transplantation, 45 cases in each group and 5 cases in control group:SD to SD regarded as isograft group (group A), Wistear to SD divided into CsA untreated allograft group(group B) and CsA treated allograft group (group C), normal SD rats as control group. The FKN mRNA expression was detected by one-step RT-PCR method and the expression of FKN and CX3CR1 protein was detected by standard ABC immunohistochemical technique. Results The expressin of FKN mRNA and protein was weak in both isografts and normal heart specimens. The changes of FKN mRNA expression were correkted with the process of acute allograft rejection. The peak of FKN mRNA expression(0.8 ±0.26) appeared on the seventh day after transplantation, which could be downregulated by CsA significantly ( t = 2.390, P < 0.05). FKN protein was locate     

Local Expression of Vaginal Th1 and Th2 Cytokines in Murine Vaginal Candidiasis under Different Immunity Conditions

To investigate the expression of vaginal Th1 and Th2 cytokines in rats with experimental vaginal candidiasis under different immune conditions, ICR murine vaginal candidiasis model was established and immno-suppressed murine models of vaginal cadidiasis were established in estrogen-treated mice. Non-estrogen-treated mice were used as controls. The mRNA level of Th1 （IL-2）/Th2 （IL-4, IL-10, TGF-β1） cytokines in murine vaginal tissues was determined by RT-PCR. The cykotine in local tissues was increased to different extent under normal immune condition. IL-2 mRNA was increased during early stage of infection, while IL-10 was increased transiently during late stage of infection. TGF-β1 production was found to be increased persistently. At same time, the expression of IL-2 mRNA was suppressed in immno-suppressed group, and the level of IL-4, IL-10, and TGF-β1 were higher than the normal immunity group to different degree during infection. The high level of IL-2 mRNA during early stage of infection was associated with clearance of mucosal Candidia albicans （C. albicans）, and its expression suppressed leading to decreased clearance of mucosal C. albican in immuno-suppression. The over-expression of IL-4 and IL-10 could significantly enhance the susceptibility to C. albicans infection in mice.     

Effect of Coiquhoumia Root on the Expression of Transforming Growth Factor-β in Mesangiai Proliferation Giomerulonephritis Model

Summary： To study the efficacy and the mechanism of Colquhoumia root （ Tripterygium hypoglaucure （Le,vL） Hutch） in the treatment of mesangial proliferation glomerulonephritis （MsPGN）, SD rats were injected with anti-thymoeyte serum （ATS） to make MsPGN model （anti-Thyl model）. The rats were then divided into 3 groups： normal control group, anti-Thyl model group and treatment group. Histopathologieal （HE, PAS）, immunohistoehemieal, RT-PCR technique and computer imaging analysis system were used to evaluate mesangial matrix production, the expression of TGF-β protein and mRNA in the tissues of kidney. Our result showed that proteinuria and the ratio of extraeellular matrix/glomerular capillaries area （ECM/CA） were increased significantly in model group. The expression of both TGF-β protein and mRNA in glomeruli was much higher in model group than in control group （P〈0.01）. After the treatment with Colquhoumia root, proteinuria, ECM/CA and the expression of both TGF-β1 protein and mRNA in glomeruli were significantly decreased in treatment group as compared with those in model group. It is concluded that Colquhoumia root is effective in reducing proteinuria and mesangial matrix proliferation in MsPGN and it may achieve these effects by inhibiting the expressions of TGF-β1 protein and mRNA of mesangial cells.     

Effects of angiotensin Ⅱ receptor antagonist on expression of collagen Ⅲ, collagen Ⅴ, and transforming growth factor β1 in the airway walls of sensitized rats

Background Repeated attacks of bronchial asthma lead to different degrees of airway remodeling, the mechanism of which is not yet clear. Some evidences indicate that it is related to the excessive expression of some growth promotion factors. Angiotensin Ⅱ is a polypeptide that may be involved in airway remodeling. To evaluate its role in airway remodeling in asthma, we observed the effects of an angiotensin Ⅱ type 1 receptor antagonist (valsartan) on the expression of collagen Ⅲ, collagen Ⅴ, and transforming growth factor β1 (TGF-β1) mRNA and protein in the airway walls of sensitized rats.Methods Forty Wistar rats were randomly divided into 5 groups: control group, sensitized group, and valsartan groups 1, 2, and 3. The rats in the sensitized group and in valsartan groups 1, 2, and 3 were sensitized and challenged with ovalbumin. Rats in control group were sensitized and challenged with 0.9% NaCl. Rats from valsartan groups 1, 2, and 3 were drenched with valsartan (10 μg, 20 μg, or 30 μg, respectively) at the time of the ovalbumin challenges. The expression of collagen Ⅲ, collagen Ⅴ, and TGF-β1 protein were detected using immunohistochemical method in combination with image analysis methods. The expression of TGF-β1 mRNA was detected by in situ hybridization. Results The expression in the airways of collagen Ⅲ and collagen Ⅴ was significantly higher in rats from the sensitized group (7.73±0.81, 1.34±0.28) and from valsartan groups 1, 2, and 3 (5.73±0.64, 1.13±0.15; 4.96±0.51, 0.98±0.08; 4.43±0.35, 0.93±0.06, respectively) than those in the control group (2.65±0.38, 0.67±0.08, P&lt;0.05). In addition, collagen levels were significantly lower in valsartan groups 1, 2, and 3 than those from the sensitized group (P&lt;0.05). The expression of TGF-β1 mRNA and protein in the airways was significantly higher in rats from the sensitized group (20.49%±3.46%, 29.73%±3.25%) and from valsartan groups 1, 2, and 3 (16.47%±1.94%, 19.41%±1.87%; 14.38%±1.58%, 18.29%±1.43%; 12.96%±1.73%, 18.63%±1.11%, respectively) than that from the control group (7.84%±1.61%, 5.63%±1.07%, P&lt;0.05). TGF-β1 mRNA and protein levels were significantly lower in valsartan groups 1, 2, and 3 than that in the sensitized group (P&lt;0.05). Conclusions Angiotensin Ⅱ receptor antagonist valsartan can suppress synthesis of collagen Ⅲ and collagen Ⅴ by downregulating TGF-β1 mRNA and protein expression. Valsartan can decrease airway remodeling and could play a role in asthma therapy.     

Plasma Levels of the Anti-inflammatory Cytokine IL-10 and Inflammatory Cytokine IL-6 in Patients with Unstable Angina

The plasma levels of inflammatory cytokine interleukin-6 （IL-6） and anti-inflammatory cytokine interleukin-10 （IL-10） in the patients with unstable angina or stable angina were determined and compared. In 30 patients with unstable angina and 22 patients with stable angina, plasma levels of IL-10 and IL-6 were detected by ELISA and plasma lipid parameters by lipid research clinical methods respectively. The results showed plasma levels of IL-10 were significantly lower in unstable angina group than in stable angina group （P=0. 005）, while those of IL-6 were significantly increased in unstable angina group as compared with those in stable angina group （P= 0. 039）. There was a significantly negative correlation between IL-10 and IL-6 in patients with unstable angina （r=-0.41, P=0. 003）. In the unstable angina group, IL-6 levels were obviously positively correlated with TC （r=0. 314, P=0. 023）, but not with TG and HDL. There were no significant correlations between IL-10 and plasma lipid parameters. It was suggested that the decreased IL-10 and increased IL-6 might be associated with the atheromatous plaque stability and progression of coronary heart diseases. IL-10 may play an important role in preventing coronary vascular lesions.     

Changes of CD4^＋CD25^＋ Regulatory T Cells in Patients with Acute Coronary Syndrome and the Effects of Atorvastatin

The function of CD4＋CD25＋ regulatory T lymphocytes （Treg） in patients with acute coronary syndrome （ACS） and the effects of atorvastatin were investigated. Forty-eight patients with ACS were randomly divided into two groups： group C receiving conventional therapy （n=24）, and group C＋A receiving conventional therapy＋atorvastatin （10 mg/day, n=24）. T lymphocytes from ACS patients （before and 2 weeks after the treatment） or 18 healthy subjects were separated and the flow cytometry was used to measure the percentage of Treg. The inhibitory ability of Treg on effector T cells was determined by mixed lymphocyte reaction （MLR）. ELISA was used to measure the serum levels of cytokines （IL-10, TGF-β1 and IFN-γ） before and after treatment. The results showed that as compared with normal control group, Treg percentage was decreased significantly （P〈0.01）, the inhibitory ability of Treg on the T lymphocytes proliferation was reduced （P〈0.01）, IFN-γ levels were increased and IL-10 and TGF-β1 levels were lowered in ACS patients. After treatment with atorvastatin, Treg percentage and the inhibitory ability of Treg on T lymphocytes proliferation were significantly increased in ACS patients. Serum IFN-γ was decreased significantly, while IL-10 and TGF-β1 were elevated significantly as compared with the non-atorvastatin group. The number of Treg was positively correlated with serum TGF-β1, but negatively with serum IFN-γ and CRP. It was concluded that ACS was associated with decreased number and defected function of Treg, which may play an important role in initiating immune-inflammatory response in ACS. The inhibitory effects of atorvastatin on inflammation in ACS may be due to its beneficial effects on Treg and restoration of immune homeostasis.     

Effects of Jinmaitong Capsule (筋脉通胶囊) on Ciliary Neurotrophic Factor in Sciatic Nerves of Diabetes Mellitus Rats

Objective:To study the effects of the Chinese medicine Jinmaitong Capsule(筋脉通胶囊,JMT) on the pathomorphology of sciatic nerves,ciliary neurotrophic factor(CNTF),and the mRNA expressions of CNTF in rats with streptozotocin-induced diabetes mellitus(STZ-DM).Methods:The animal model was established by one time intraperitoneal injection of streptozotocin.The rats were simply divided by random into 5 groups including model group,low-dose JMT group(JL),medium-dose JMT group(JM),high-dose JMT group(JH) and neurotropin group.For each of the above 5 groups,a group of 10 normal Wistar rats matched in body weight,age and gender were set as normal group.Intragastric administrations were started after the animal model established.The JL group were administered with five times the JMT dose recommended for a human adult;the JM group were administered with ten times the JMT dose recommended for a human adult;the JH group were administered with twenty times the JMT dose recommended for a human adult.The neurotropin group was administered with ten times the neurotropin dose recommended for a human adult.All rats were given intragastric administration for 16 weeks and then killed.In the 4th,8th,12th,16th week,body weight and blood glucose level were detected before and after the intervention.The morphologic changes of the sciatic nerves were observed by optical microscope and transmission electron microscope.The CNTFmRNA expressions were detected by real-time fluorescent quantitative polymerase chain protein,and the CNTF protein expressions were detected by immunohistochemical method.Results:The blood glucose levels of the STZ-DM rats were much higher than normal group(P<0.01),and there was no apparent difference between any treatment groups and the model group(P>0.05).Before and after the intervention in the 4th, 8th,12th,16th week,there were no significant differences in the body weight among all the groups(P>0.05). The sciatic nerves of STZ-DM rats might have pathomorphological changes in axons,myelin sheaths,and interstitium.The levels of CNTF and CNTF-mRNA expressions in the STZ-DM rats were both significantly decreased(P<0.01).The sciatic nerves of STZ-DM rats might have pathomorphological changes in axons, myelin sheaths,and interstitium.Conclusion:JMT could improve the pathomorphology of sciatic nerves by increasing CNTF’s and CNTF-mRNA expressions in sciatic nerve tissues,and promote the repair and regeneration of damaged nerve fibers.     

Expression of cyclooxygenase-2 and its pathogenic effects in nonalcoholic fatty liver disease

Objective:To investigate the expression of cyclooxygenase-2 and its pathological effect in the experimental nonalcoholic fatty liver of rats,and to explore its possible mechanism.Methods:The rat NAFLD model was established by giving a fat-enriched diet.The blood samples were obtained form abdominal aorta and the levels of serum ALT,AST and IL-1,changes in the hepatic tissue 6-k-PGF1α TXB2 were measured.The expression level of COX-2 in rats livers were assayed by immunohistochemistry,RT-PCR and Western-blot.Results:Light microscope analysis revealed that hepatocytes were injured in the model group and slightly in the treatment group.The levels of serum TXB2 and IL-1 in the fatty liver rats were increased.Compared with the model group,the IL-1 and TXB2 increased significantly(P < 0.05),on the contrary,compared with the normal group,the hepatic tissue 6-Keto-prostagland decreased significantly in the model group(P < 0.05),the treatment group also increased but P > 0.05.There was no positive expression of COX-2 in hepatic tissue of normal rats.In the model group,there was positive expression of COX-2 antigen and the number of COX positive cells progressively increased at 4,8,12 wks.The intensity of expression of COX-2 had significantly increased(P < 0.05)and the intensity of COX-2 expression in the treated group decreased remarkably compared with the model group(P < 0.05).The expression of COX-2 mRNA and the level of COX-2 protein were significantly stronger in the liver of model rats compared with normal rats,and significantly weaker in treated rats,than in 8W and 12W model rats(P < 0.05).Conclusion:The increase of COX-2 expression in NAFLD is closely associated with the severity of liver inflammation and damage.COX-2 may play an important role in the progression of rat NAFLD,and the expression of COX-2 mRNA is downregulated by cyclooxygenase-2 inhibitor,which can depress the oxidative stress and control inflammatory response efficiently.