Protective effects of ulinastatin on kidney injury in rabbits after cardiopulmonary resuscitation

AIM: To investigate the effect of ulinastatin (UTI) on inflammatory cell infiltration, expression of tumor necrosis factor-α (TNF-α) and concentration of oxidative stress product malondialdehyde (MDA) in the kidney medulla after cardiopulmonary resuscitation (CPR) in rabbits and to access its potential mechanisms in kidney injury after CPR.METHODS: Twenty-four male New Zealand adult rabbits were randomly divided into control group and UTI group after return of spontaneous circulation (ROSC) from 5 min ventricular fibrillation induced by alternating current. The UTI at the dose of 2.5×10⁴ U/kg was administered immediately after ROSC to the animals in UTI group, the same volume of saline were injected in control group. The urinary output was recorded and the serum concentration of BUN and creatinine were detected every 4 h after ROSC. The animals were sacrificed 24 h after ROSC and the kidney medullas were obtained to observe the degree of inflammatory cell infiltration, the expression of TNF-α and the concentration of MDA.RESULTS: Six rabbits in control group and 6 animals in UTI group survived to the end point of experiment. The urinary output was decreased gradually to the lowest 8 h-12 h after ROSC and then increased in both groups. The urinary output in UTI group was more than that in control group 8 h after ROSC (P<0.05). The serum concentrations of BUN and creatinine were significantly lower in UTI group than those in control group 4 h after ROSC (P<0.05). The myeloperoxidase-positive cells in control group were much higher than that in UTI group (P<0.05). The expression of TNF-α and concentration of MDA in the kidney medullas in UTI group were lower than those in control group (P<0.05, P<0.01).CONCLUSION: The standard dose of UTI (2.5×10⁴ U/kg) administered in rabbits suffered from ventricular fibrillation for 5 min may alleviate the degree of inflammatory cell infiltration, decrease the expression of TNF-α and reduce the concentration of MDA in kidney medulla. UTI has protective effects on the renal function after CPR.     

Bone marrow-derived mesenchymal stem cell-conditioned medium atte-nuates LPS-induced acute lung injury in mice

AIM:To explore the effects of bone marrow-derived mesenchymal stem cells-conditioned medium (MSCs CdM) on lipopolysaccharide (LPS)-induced acute lung injury. METHODS:Lung injury was induced in mice by intraperitoneal injection of LPS. The mice were given a tail vein injection of MSCs CdM or normal saline 1 h after LPS administration. The mice were killed by an intraperitoneal injection of pentobarbital 6 h after LPS injection for either bronchoalveolar lavage fluid (BALF) and serum collection or lung histological analysis. RESULTS:Compared with control group, the BALF levels of protein, interleukin-10 (IL-10) and keratinocyte growth factor (KGF), the serum levels of tumor necrosis factor α (TNF-α) and IL-6, and the myeloperoxidase (MOP) activity in the lung tissues were significantly higher in LPS group, and severe pathological damages in the lung tissues were also observed. Treatment with MSCs CdM significantly reduced the BALF prtein level, the seum TNF-α and IL-6　levels and the lung MPO activity, and attenuated the lung pathological damages, but further increased the levels of IL-10 and KGF in the BALF. CONCLUSION:Treatment with MSCs CdM attenuates the lung injuries induced by LPS, which may be via regulating the expression of TNF-α, IL-6, IL-10 and KGF.     

A novel mechanism of RhoA activation induced by tumor necrosis factor-α in mouse cerebral microvascular endothelial cells

AIM: To investigate the effects of tumor necrosis factor α (TNF-α) on RhoA activity in mouse cerebral microvascular endothelial cells.METHODS: The bEnd.3 cells, a mouse brain microvascular endothelial cell line, were cultured. RhoA activity was analyzed by pull-down assay 10 min, 30 min and 60 min after TNF-α treatment. Expression of RhoA protein was determined by Western blotting 1 h, 3 h, 6 h, 12 h and 24 h after TNF-α treatment. Small interfering RNA (siRNA) targeting to p115RhoGEF or control nsRNA was transfected into bEnd.3 cells. The expression of p115RhoGEF was determined by Western blotting, and RhoA activity was detected by pull-down assay 30 min after TNF-α treatment.RESULTS: RhoA activity peaked at 30 min after TNF-α treatment(P<0.01) . TNF-α significantly increased the protein expression of RhoA at 12 h and 24 h (P<0.05). Knock-down of p115RhoGEF by siRNA in bEnd.3 cells attenuated TNF-α-induced RhoA activation (P<0.05).CONCLUSION: TNF-α up-regulates RhoA activity and expression. p115RhoGEF may play a role in TNF-α-induced activation of RhoA.     

Effect of Xueshuantong injection on LPS-induced DIC in rabbits

AIM：To evaluate the effect of Xueshuantong injection on lipopolysaccharide (LPS)-induced experimental disseminated intravascular coagulation (DIC) in rabbits. METHODS：To establish the LPS-induced DIC model, LPS was continuously injected into the rabbit autricular vein for 6 h. The survival rate of the rabbits was recorded after 24 h. The plasma levels of alanine aminotransferase (ALT) and blood urine nitrogen (BUN) were detected. Activated partial thromboplastin time (APTT), prothrombin time (PT), platelet count and fibrinogen concentration were measured. The activity of protein C, antithrombin Ⅲ (ATⅢ) and the concentration of tumor necrosis factor α (TNF-α) were also determined. RESULTS：The survival rate of DIC rabbits was only 26.7%. BUN and ALT increased obviously. APTT and PT became much longer, platelet count, fibrinogen, protein C and ATⅢ decreased obviously, and plasma TNF-α increased remarkably. The intravenous administration of Xueshuantong injection increased the survival rate of DIC rabbits up to 66.7% in a dose-dependent manner. Xueshuantong injection also decreased the plasma levels of BUN, ALT, APTT, PT and TNF-α significantly, while increased the levels of fibrinogen, platelet, protein C and ATⅢ in plasma. CONCLUSION： Xueshuantong injection has therapeutic effect on LPS-induced DIC in rabbits.     

Effect of patchouli oil on lipopolysaccharide-induced fever in rabbits

AIM:To investigate the antipyretic effect of patchouli oil on lipopolysaccharide (LPS)-induced fever in rabbits. METHODS:Male rabbits (n=42) were randomly divided into 7 groups according to their body weight and basal body temperature, including control group, model group, western medical positive group, traditional Chinese medical positive group, and high, middle and low doses (2%, 1% and 0.5%) of patchouli oil groups. Subsequently, except the controls, the rabbits were injected with LPS at a dose of 1 mL/kg (2 mg/L) through marginal ear vein to establish rabbit fever model and the rabbits in control group received the same volume of NS. The rabbits in control group and model group were injected with 0.5% Tween-80 0.5 h late, and the rabbits in the other groups were treated with correspoonding drugs. The effect of patchouli oil on the body temperature was observed, and the levels of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the serum, and prostaglandin E2 (PGE2) and cyclic adenosine monophosphate (cAMP) in the hypothalamus were measured by radioimmunoassay. RESULTS:The body temperature and the levels of IL-1β, TNF-α, cAMP and PGE2 in model group were significant higher than those in control group. Patchouli oil notably inhibited the body temperature in the febrile rabbits. From 1.5 h to 5.5 h after administration, the body temperatures were increased by (1.06±1.55), (1.62±1.36), (1.38±1.22), (0.98±0.98) and (0.48±0.95) ℃ in high patchouli oil group, respectively. From 3.5 to 5.5 h after administration, the body temperatures were elevated by (1.47±0.73), (1.15±0.68) and (0.63±0.54) ℃ in middle patchouli oil group, respectively. A tendency of downregulation of the elevated body temperatures was observed at every time point after administration in low patchouli oil group. Patchouli oil significantly decreased the levels of TNF-α in the serum and cAMP content in the hypothalamus, and attenuated the elevated tendency of the IL-1β level in the serum and PGE2 level in the hypothalamus.CONCLUSION: Patchouli oil evidently has antipyretic effect on LPS-induced fever in the rabbits. The antipyretic mechanism might be related to the inhibition of TNF-α level in serum and cAMP content in the hypothalamus.     

Effects of renal denervation on inflammatory factors in a rabbit model of early atherosclerosis

AIM: To evaluate the effects of renal denervation (RDN) on the expression of tumor necrosis factor-α (TNF-α), interleukin-1α (IL-1α) and interleukin-6 (IL-6) in a rabbit model of early atherosclerosis. METHODS: New Zealand male rabbits were divided into control group, RDN+ high-fat diet (HFD) group (RDN group), sham+HFD group (sham group) and HFD group. The rabbits in later 3 groups were fed with 2% cholesterol for 8 weeks to establish an early atherosclerosis model. The blood samples were collected to test the levels of lipids, norepinephrine (NE), TNF-α, IL-1α and IL-6. The protein expression of angiotensin Ⅱ (Ang Ⅱ) was detected by the method of immunohistochemistry. The levels of nuclear factor-κB (NF-κB) and Ang II 1 type receptor (AT1R) were evaluated by Western blot. The mRNA expression of TNF-α, IL-1α and IL-6 was determined by real-time PCR. RESULTS: After 1 d of RDN procedure, the NE level was lower in RDN group than that in sham group (P<0.01). After 8 weeks, the NE level was lower in RDN group than that in sham group and HFD group (P<0.05), and triglyceride (TG) was lower in RDN group than that in HFD group (P<0.05). The protein expression of Ang II was decreased in RDN group compared with sham group and HFD group (P<0.01). The protein expression of NF-κB was lower in RDN group than that in sham group (P<0.05). The plasma levels of TNF-α and IL-1α were reduced in RDN group compared with sham group and HFD group (P<0.05). The mRNA expression of TNF-α, IL-1α and IL-6 was reduced in RDN group compared with sham group (P<0.05). CONCLUSION: RDN inhibits sympathetic activity, decreases the plasma level of TG, and alleviates inflammatory reactions in the rabbits with atherosclerosis.     

Change of Gq protein-phosphatidyl inositol signaling in brain of rats with acute respiratory distress syndrome

AIM: To study the change of Gq protein-phosphatidyl inositol signaling in the brain of rats with acute respiratory distress syndrome (ARDS). METHODS: Forty male Wistar rats were randomly divided into oleic acid groups (OA groups of 30 min, 60 min, 90 min and 120 min exposure) and control group. The ARDS model in rats was reproduced by intravenous injection of oleic acid (0.2 mL/kg in 2 min). The mean arterial blood pressure (MABP), blood gas indexes, malondialdehyde (MDA), lactate dehydrogenase (LDH) and creatine kinase (CK) in plasma and brain tissues were measured. Gαq/11 protein and phospholipase C in brain tissues were detected by Western blotting. RESULTS: Compared to control group, MABP and PaO2 in all OA groups obviously decreased (P<0.05). MDA concentration and LDH activity in plasma and brain tissues of OA rats at time points of 90 min and 120 min were significantly higher than those in the controls (P<0.05). The CK activity in plasma of all OA groups increased (P<0.05). The CK activity in the brain tissues peaked at 60 min after OA exposure (P<0.05) and then decreased at 90 min and 120 min after treated with OA (P<0.05). Compared to control group, the concentration of Gαq/11 protein in the brain tissues of OA 60 min, 90 min and 120 min groups, and PLC concentration in the brain tissues of all OA groups obviously increased (P<0.05). A negative correlation between the change of Gαq/11 protein in the brain tissues and PaO2 (r=-0.579, P<0.05), a positive correlation between the change of Gαq/11 protein and MDA in the brain tissues (r=0.538, P<0.05), and a positive correlation between the change of Gαq/11 protein and LDH in the brain tissues (r=0.624, P<0.05) were observed. CONCLUSION: Up-regulation of Gq protein-phosphatidyl inositol signaling in the brain may play a role in the brain injury during ARDS.     

Effects of ursodeoxycholic acid on proinflammatory cytokines in children with infantile hepatitis syndrome

AIM: To observe the effect of ursodeoxycholic acid (UDCA) on the treatment of infantile hepatitis syndrome (HIS) and to investigate its mechanism. METHODS: The children with infantile hepatitis syndrome were divided into conventional treatment group and the UDCA treatment group. Twenty healthy children were selected as normal control. The children in conventional therapy group were given antiviral and hepatoprotective treatments. The children in UDCA treatment group were given ursodeoxycholic acid (10 mg·kg^-1·d^-1) in addition to the conventional treatment group for 2 to 3 weeks. The levels of total bilirubin (TBIL), direct bilirubin (DBIL), alanine aminotransferase (ALT), glutamyltransferase (GGT), total bile acids (TBA) and TNF-α, IL-6 were detected before admission and 2 weeks later.RESULTS: The levels of TNF-α and IL-6 were significantly higher in the children with IHS than those in the normal control (P<0.01). The levels of TBIL, DBIL, ALT, GGT, TBA, TNF-α and IL-6 in conventional treatment group were reduced after therapy (P<0.01). All the above index in UDCA treatment group were decreased compared with conventional treatment group (P<0.01). CONCLUSION: On the basis of conventional therapy, ursodeoxycholic acid effectively alleviates the systemic inflammatory response in the children with IHS, reduces the liver damages.     

Expression of MMP-2 and NF-κB in myocardium of mice with viral myocarditis

AIM: To observe the effects of TNF-α/nuclear factor-κB(NF-κB)/matrix metalloproteinase-2(MMP-2) pathway on the expression of MMP-2 in the mice with viral myocarditis. METHODS: Six-week-old inbred male mice were randomly assigned to control and myocarditis group. The mice in myocarditis group and control group were intraperitoneally inoculated with 0.1 mL 10^-5.69 TCID50/mL coxsackievirus B3 and vehicle (PBS), respectively. Ten mice were sacrificed at the 4th and 10th days after injection. The blood and heart specimens were harvested. The serum content of TNF-α was measured by ELISA. The myocardial levels of MMP-2, NF-κB p65 and IκBα were determined by Western blot. RESULTS: Compared with control group, the protein expression of MMP-2 and NF-κB p65 in the myocardium and the serum content of TNF-α were significantly increased in myocarditis group (P<0.05). The protein expression of IκBα was lower in myocarditis group than that in control group (P<0.05).CONCLUSION: TNF-α, NF-κB p65 and MMP-2 were higher in the mice with acute viral myocarditis. The increased expression of them might be involved in the pathogenesis of viral myocarditis.     

Effects of adrenal gland on the expression of bax and bcl-2 in hippocampus after cerebral ischemia

AIM: To observe the effects of adrenal gland on the hippocampus responses to cerebral ischemia. METHODS: 36 Wistar rats were randomly divided into three groups: sham-operated control group (sham), unilateral adrenalectomy were performed in ADX and GC group, and GC group were injected with 5 mg/per rat of dexamethasone before cerebral ischemia. Fourteen days after the first operation, all animals were performed occlusion of bilateral carotid artery for 15 min, and then reperfusion. 3 rats of each group were sacrificed at 1 h, 4 h, 8 h and 24 h after reperfusion and hippocampus were dissected. The total RNA was rapidly extracted from hippocampus tissue. The expressions of c-fos, bcl-2 and bax gene were quantified with the method of semiquantitive RT-PCR. RESULTS: The expressions of c-fos and bax in three groups showed no statistical differences (P>0.05). The expression of bcl-2 in sham group was significantly higher than that in GC and ADX groups (P<0.05). However, no differences of bcl-2 expression between GC and ADX group (P>0.05) was observed. The ratio of bax to bcl-2 in sham group was significantly lower than that in GC and ADX groups (P<0.05), no significant differences of the ratio displayed between ADX and GC group. CONCLUSION: The expression of c-fos and bax in hippocampus after cerebral ischemia is not affected by adrenal gland. The excision of unilateral adrenal gland downregulates bcl-2 expression and raises the ratio of bax to bcl-2 in rat hippocampus after cerebral ischemia. Dexamethasone treatment does not alter the expression of bcl-2 in ADX and GC groups. The results indicate that the adrenal gland can counteract cell apoptosis in hippocampus tissue induced by cerebral ischemia. Adrenal steroids are not sufficient to enable the compensatory increase in bcl-2 expression in steroid-deficient animal, some other mechanism may exist.     

Hydrogen sulfide attenuates urosepsis-induced acute kidney injury by blocking TLR4/MyD88/PI3K signaling pathway

AIM:To explore the effect of hydrogen sulfide (H₂S) on urosepsis-induced acute kidney injury. METHODS:New Zealand white rabbits were randomly divided into control group, sham group, model (sepsis) group, NaHS treatment (NaHS) group, and NaHS combined with TAK-242 (a TLR4 inhibitor) treatment (NaHS+TAK-242) group. After treatment for 72 h, HE staining was used to measure the histopathological changes of rabbit kidney. The levels of blood urea nitrogen (BUN) and serum creatinine (SCr) were detected by automatic biochemical analyzer. The serum levels of neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule 1 (KIM-1), procalcitonin (PCT), interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were measured by ELISA. The TLR4/MyD88/PI3K signaling pathway-related proteins in the kidney were determined by Western blot. RESULTS:Compared with control group, obvious damage was observed in the kidneys of septic rabbits, but the kidneys were markedly improved by treatment with NaHS. The levels of BUN, SCr, NGAL, KIM-1, PCT, IL-1β, IL-6 and TNF-α in the septic rabbits were higher than those in control group, and decreased significantly in NaHS group and NaHS+TAK-242 group. The protein levels of TLR4, MyD88, p-PI3K and p-Akt in septic rabbit kidneys were higher than those in control group. However, NaHS or NaHS+TAK-242 inhibited the activation of TLR4/MyD88/PI3K signaling pathway in the kidneys of septic rabbits. CONCLUSION:H₂S play a protective effect on the rabbits with urosepsis-induced acute kidney injury by blocking TLR4/MyD88/PI3K signaling pathway to inhibit inflammatory response.     

Effect of L-arginine on arrhythmia induced by ischemia/reperfusion of right coronary artery in rabbits

AIM: To explore the effect of L-arginine (L-Arg) on the arrhythmia induced by ischemia/reperfusion (IR) of the right coronary artery in rabbits. METHODS: 48 healthy adult rabbits were divided into 4 groups (n=12 of each) randomly: IRa group (120 min reperfusion after 30 min ischemia), IRb group (120 min reperfusion after 120 min ischemia), IRa+L-Arg group and IRb+L-Arg group. I/R model was established by occluding and loosening the root of the right coronary artery in rabbits. The changes of ECG and arrhythmia were recorded and graded. RESULTS: ① The longer time of IR was, the higher the score of the arrhythmia was found. The incidence of atrial-ventricular block (AVB), sinus-atrial block (SAB), even sinus arrest were detected and aggravated gradually. ② The incidence of AVB was decreased and from Ⅲ°→Ⅱ°→Ⅰ° markedly, some of sinus and atrial arrhythmia were transformed into sinus rhythm gradually, and all of the arrhythmia scores in IRa group were decreased significantly as compared with the same time phases of IRb group (P<0.01). ③ All of the arrhythmia scores in IRa+L-Arg and IRb+L-Arg groups were decreased dramatically as compared with that in IRa and IRb groups at the same time phases (P<0.01). ④ All of the arrhythmia scores in IRa+L-Arg group were lower compared with those in IRb+L-Arg group (P<0.01). CONCLUSION: Supplying appropriate L-arginine to the tissue is beneficial for inhibiting arrhythmia during ischemia and reperfusion, and the longer the time of ischemia is, the weaker the effect of L-arginine on the arrhythmia presented during the period of reperfusion.     

Effect of safflower injection on LPS-induced disseminated intravascular coagulation in rabbits

AIM：To study the effect of safflower injection on lipopolysaccharide (LPS)-induced disseminated intravascular coagulation (DIC) in rabbits. METHODS：The rabbit model of DIC was induced by continuous infusion of LPS at a dose of 100 μg·kg-1·h-1 for 6 h. The treatment with safflower injection and heparin were started simultaneously with LPS infusion through the contralateral marginal ear vein. The activated partial thromboplastin time (APTT), prothrombin time (PT), platelet count and fibrin(ogen) concentration were measured. The plasma levels of fibrin(ogen) degradation products (FDP), alanine aminotransferase (ALT) and blood urine nitrogen (BUN) were detected. The activity of protein C and antithrombin III (AT III) and the concentration of tumor necrosis factor α (TNF-α) were also determined. RESULTS：The gradual injury of hemostatic parameters was induced by continuous infusion of LPS. APTT, PT, BUN, ALT and plasma TNF-α were increased obviously. The platelet count, fibrinogen, FDP, protein C and AT III were decreased significantly. The intravenous administration of safflower injection attenuated the increases in the levels of APTT, PT, BUN, ALT, FDP and plasma TNF-α induced by LPS infusion, and improved the plasma levels of fibrinogen, platelet, protein C and AT III. CONCLUSION: Safflower injection protects against LPS-induced DIC in rabbits．     

Influence of ischemia-reperfusion on apoptosis of sinoatrial node cells in rabbits in vivo

AIM:To study influence of ischemia-reperfusion(IR) on apoptosis and expression of apoptosis-related genes Fas-L, Bax and Bcl-2 of sinoatrial node(SAN) cells in rabbits in vivo. METHODS:Ninety healthy adult rabbits were divided randomly into control group, ischemia groups (I_{10 min}, I_{30 min}, I_{60 min} and I_{120 min}) and IR groups (I_{10 min}R_4h, I_{30 min}R_4h, I_{60 min}R_4hand I_{120 min}R_4h). IR injury model of SAN was established by occluding and loosening the start section of right coronary artery. The apoptosis of SAN cells was detected by TUNEL staining. The expression of Fas-L, Bax and Bcl-2 of SAN cells was detected by immunohistochemistry. RESULTS:①No obvious apoptosis of SAN cells was observed in control group, I_{10 min} and I_{30 min} groups. Apoptosis of different degrees in SAN cells were found in 68.3%(41/60) rabbits in I_{60 min}, I_{120 min} and 4 subgroups of IR. ②The highest expression of Fas-L and Bax was observed in I_{120 min} group and that of Bcl-2 was in I_{60 min} group. ③The highest expression of Fas-L and Bax was observed in I_{60 min}R_4h group. The peak level of Bcl-2 was observed in I_{30 min}R_4h group. ④The expression of Fas-L and Bax was significant higher in IR group than that in ischemic group at the same time point. CONCLUSION:Ischemia and IR induced apoptosis of SAN cells in rabbit in vivo. Fas-L、Bax、Bcl-2 may participate in the regulation of apoptosis and the injury during IR aggravates the apoptosis of SAN cells.     

Effects of tumor necrosis factor alpha on proliferation and apoptosis of HSCs

AIM:To explore the role of tumor necrosis factor alpha(TNF-α) in the pathogenesis of liver fibrosis.METHODS:The proliferation and apoptosis of hepatic stellate cells (HSCs) in vitro were detected with flow cytometry, electron microscopy and TUNEL.RESULTS:The flow cytometry analysis showed that the cell proliferation index (PI) in the TNF-α(0.5 μg/L, 2.0 μg/L, 8.0 μg/L) groups was evidently lower than that in the control group (P＜0.05). In the cell cycle distribution, the portion of G₀/G₁ phase in the TNF-α groups was significantly higher than that in the control group(P＜0.05), but the portion of S phase in the TNF-α groups was evidently lower than that in the control group(P＜0.05). These indicated that TNF-α interfered with HSCs entrance into S phase from G₀/G₁ phase whereupon the proliferation of HSCs was inhibited. The apoptotic rate in the TNF-α groups was evidently higher than that in the control group(P＜0.05). The gene expression of bcl-2 and bax was also detected with flow cytometry. The expression of bcl-2 in the TNF-α groups was evidently lower than that in the control group(P＜0.05), but the expression of bax in the TNF-α groups was significantly higher than that in the control group(P＜0.05). TUNEL analysis showed the apoptotic rate of HSCs in the TNF-α(2.0 μg/L) group was 18.7%±2.5% compared with 5.3%±1.2% in the control group(P＜0.05).CONCLUSIONS:TNF-α interfered with HSCs entrance into S phase from G₀/G₁ phase whereupon the proliferation of HSCs was inhibited. TNF-α down-regulated bcl-2 gene expression and up-regulated bax gene expression whereupon the apoptosis of HSCs was induced.     

Relationship between levels of IL-2, TNF-α and nitric oxide in aqueous humor after intraocular lens implantation

AIM: To investigate the relationship between the level of interleukin-2 (IL-2), tumour necrosis factor-α (TNF-α) and nitric oxide (NO) in aqueous humor after intraocular lens implantation. METHODS: New Zealand rabbits were divided randomly into three groups: (1) control group; (2) extracapsular cataract extraction group (ECCE); (3) extracapsular cataract extraction and posterior chamber intraocular lens implantation group (ECCE+IOL). The inflammation in all experimental rabbit eyes was observed via zoom-photo slit-lamp microscope on 1, 3, 7, 14 d and 30 d postoperation. Meanwhile, aqueous humor was drawn for white blood cell (WBC) counting and classifying and for determining IL-2, TNF-α and NO₂^-/NO₃^- contents. RESULTS: (1) The level of IL-2 and TNF-α and NO₂^-/ NO₃^- in aqueous humor of ECCE+IOL group were higher than that in ECCE and control at 1 to 14 days postoperation, respectively, it increased to peak value at 3 to 7 days postoperation and decreased gradually two weeks postoperation; (2) The changes in IL-2, TNF-α and NO₂^-/NO₃^- in each group were basically similer; (3) The changes of IL-2 and TNF-α level were closely related with NO content in aqueous humor (r=0.69, P<0.01 and r=0.98, P<0.01). CONCLUSION: IL-2, TNF-α and NO play an important role in intraocular inflammation intraocular lens implantation.     

Effect of TRPV1 activation on lipopolysaccharide-induced lung inflammatory injury in mice

AIM:To investigate the effects of transient receptor potential cation channel subfamily V member 1 (TRPV1) activation by capsaicin on the inflammation and its underlying mechanisms in lipopolysaccharide (LPS)-induced lung injury in mice. METHODS:A total of 108 specific pathogen-free male ICR mice were randomly divided into 6 groups: normal control group, capsaicin (CAP) control group, capsazepine (CAPZ) control group, endotoxemia group, CAP treatment group and CAPZ treatment group. LPS was intraperitoneally injected 30 min after the subcutaneous injection of CAP or CAPZ. After modeling, the levels of tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), IL-10, substance P (SP) and calcitonin gene-related peptide (CGRP) in the lung were measured by ELISA. The expression of Toll-like receptor 4 (TLR4) and nuclear factor κB (NF-κB) in the lung tissue was assessed by Western blotting. The pathological changes of the lung tissue were observed under light microscope. RESULTS:The expression of TNF-α, IL-6, IL-10 and NF-κB in the lung tissues at 3 h, 8 h and 16 h was dramatically higher in endotoxemia group than that in normal control group. Compared with endotoxemia group, the levels of TNF-α, IL-6 and nuclear NF-κB in CAP treatment group at 3 h, 8 h and 16 h were obviously decreased, but the level of IL-10 was increased. The changes of the factors mentioned above in CAPZ treatment group were absolutely adverse to those in CAP treatment group. The levels of SP and CGRP were significantly higher in endotoxemia group and CAP control group than those in normal control group, but those in CAPZ control group were lower. Compared with endotoxemia group, SP and CGRP were markedly increased in CAP treatment group and were obviously decreased in CAPZ treatment group. The level of TLR4 in endotoxemia group was distinctly higher than that in normal control group at 3 h, 8 h and 16 h. However, as compared with endotoxemia group, the expression of TLR4 in CAP treatment group and CAPZ treatment group didn’t change much. At 8 h and 16 h after modeling, the degree of lung damage was also decreased in CAP treatment group as compared with endotoxemia group, while that in CAPZ treatment group was aggravated. CONCLUSION: TRPV1 activation obviously inhibits the increase in TNF-α, IL-6 and NF-κB in the lung tissue of endotoxemia mice, and promotes the increase in the anti-inflammatory factor IL-10, as well as the levels of SP and CGRP, but has no effect on the expression of TLR4.     

CD137 signaling molecules promote proliferation of pulmonary artery endothelial cells by aerobic glycolysis

AIM:To investigate whether CD137 signaling molecules promote the proliferation of pulmonary artery endothelial cells (PAECs) by aerobic glycolysis. METHODS:The experiments of mouse PAECs were performed as follows. (1) Stimulating factors TNF-α (10 μg/L), ET-1 (10 mmol/L) and 5-HT (1 μmol/L) were used to stimulate the cells for 24 h. (2) After stimulation with TNF-α for 24 h, the cells were divided into control group, CD137 agonist group (treatment with 5 mg/L CD137L recombinant protein to activate CD137-CD137L signaling), c-Myc inhibitor group (pretreatment with 10 μmol/L c-Myc inhibitor 10074-G5, dissolved in DMSO, for 30 min, followed by treatment with 5 mg/L CD137L recombinant protein) and DMSO group (pretreated with DMSO at the same volume to c-Myc inhibitor group for 30 min followed by CD137L recombinant protein treatment). (3) After stimulated with TNF-α for 24 h, the cells were divided into control group, CD137 agonist group and 2-deoxyglucose (2-DG) group (pretreatment with 10 mmol/L glycolysis inhibitor 2-DG for 30 min followed by CD137L recombinant protein treatment).The expression of membrane protein and total protein of CD137 in the PAECs was detected by flow cytometry and Western blot, respectively. The protein levels of glycolytic enzymes such as hexokinase (HK2), 6-phosphofructo-2-kinase/fructose-2,6-diphosphatase 3 (PFKFB3) and c-Myc were measured by Western blot. The enzyme activity of HK2 and PFKFB3 was detected by HK2 kit and PFK kit, respectively. Glucose oxidase method was used to measure the glucose uptake rate, and lactate colorimetric assay was conducted for analyzing lactic acid production. CCK-8 assay and EdU staining were used to detect proliferation of the PAECs. RESULTS:Compared with control group, TNF-α, ET-1 and 5-HT significantly increased the expression of CD137 membrane protein and total protein in the PAECs (P<0.05). The protein levels and enzyme activity of HK2 and PFKFB3 protein in CD137 agonist group were significantly higher than those in control group (P<0.05). Compared with control group, the lactic acid production and glucose consumption in CD137 agonist group were significantly increased. The protein level of c-Myc was significantly higher than that in control group after stimulation with CD137L recombinant protein, while c-Myc inhibitor 10074-G5 significantly inhibited the promoting effect of CD137L recombinant protein on glycolysis (P<0.05). The results of CCK-8 assay and EdU staining showed that the cell proliferation in CD137 agonist group was significantly increased compared with control group, while glycolysis inhibitor 2-DG significantly inhibited the proliferation-enhancing effect of CD137 signaling activation on the cells (P<0.05). CONCLUSION:CD137 signaling molecules may modulate the aerobic glycolysis by up-regulating c-Myc, thus promoting the proliferation of mouse PAECs.