Objective:To investigate the effect of chrysophanol(CHR)on cartilage injury in rats with osteoarthritis and its mechanism of regulating SIRT1/HMGB1/NF-κB signal pathway.Methods:Rat models of osteoarthritis were established and divided into negative control group,chrysophanol low(CHR-L,10 mg/kg),middle(CHR-M,20 mg/kg),high dose group(CHR-H,40 mg/kg),SIRT1 inhibitor(sirtinol 5 mg/kg)+chrysophanol high dose group(sirtinol+CHR-H),and normal healthy control group was set up.The degree of joint swelling was measured,and the inflammatory index was evaluated;the pain threshold(tenderness and heat pain)was measured;HE staining and safranine O staining were applied to detect the pathological changes of rat articular cartilage;the levels of serum inflammatory factors(IL-6,IL-1β,TNF-α)were detected by ELISA method;oxidative stress indexes(MDA,SOD,GSH-PX)were detected by micro method;TUNEL staining was used to detect apoptosis;Western blot was used to detect the protein expressions of SIRT1,HMGB1,NF-κB p65,p-NF-κB p65,MMP-13 and C-Caspase-3.Results:Compared with the normal healthy control group,the rats in negative control group had obvious pathological injury,such as destruction of articular cartilage structure,necrosis and reduction of chondrocytes,the joint swelling degree,arthritis index,levels of IL-6,IL-1β,TNF-α,content of MDA,chondrocyte apoptosis rate,expressions of apoptotic protein C-Caspase-3,HMGB1,NF-κB p65/p-NF-κB p65,MMP-13 proteins increased obviously,the tenderness threshold,heat pain threshold,activities of SOD,GSH-PX,and the expression of SIRT1 protein decreased obviously(P<0.05);compared with negative control group,the pathological injury of articular cartilage in CHR group improved obviously with the increase of dosage,the joint swelling degree,arthritis index,levels of IL-6,IL-1β,TNF-α,content of MDA,chondrocyte apoptosis rate,expression of apoptotic protein C-Caspase-3,HMGB1,NF-κB p65/p-NF-κB p65,MMP-13 proteins decreased obviously,the tenderness threshold,heat pain threshold,activities of SOD,GSH-PX,and the expression of SIRT1 protein increased obviously(P<0.05);compared with CHR-H group,sirtinol+CHR-H group was able to reverse the protective effect of CHR on cartilage injury to a certain extent.Conclusion:CHR can reduce the inflammation of articular cartilage,inhibit the apoptosis of chondrocytes and play a protective role in the cartilage injury of osteoarthritis rats by up-regulating the expression of SIRT1 and down-regulating the expressions of HMGB1 and NF-κB p65/p-NF-κB p65.

Objective:To investigate the effect of chrysophanol(CHR)on cartilage injury in rats with osteoarthritis and its mechanism of regulating SIRT1/HMGB1/NF-κB signal pathway.Methods:Rat models of osteoarthritis were established and divided into negative control group,chrysophanol low(CHR-L,10 mg/kg),middle(CHR-M,20 mg/kg),high dose group(CHR-H,40 mg/kg),SIRT1 inhibitor(sirtinol 5 mg/kg)+chrysophanol high dose group(sirtinol+CHR-H),and normal healthy control group was set up.The degree of joint swelling was measured,and the inflammatory index was evaluated;the pain threshold(tenderness and heat pain)was measured;HE staining and safranine O staining were applied to detect the pathological changes of rat articular cartilage;the levels of serum inflammatory factors(IL-6,IL-1β,TNF-α)were detected by ELISA method;oxidative stress indexes(MDA,SOD,GSH-PX)were detected by micro method;TUNEL staining was used to detect apoptosis;Western blot was used to detect the protein expressions of SIRT1,HMGB1,NF-κB p65,p-NF-κB p65,MMP-13 and C-Caspase-3.Results:Compared with the normal healthy control group,the rats in negative control group had obvious pathological injury,such as destruction of articular cartilage structure,necrosis and reduction of chondrocytes,the joint swelling degree,arthritis index,levels of IL-6,IL-1β,TNF-α,content of MDA,chondrocyte apoptosis rate,expressions of apoptotic protein C-Caspase-3,HMGB1,NF-κB p65/p-NF-κB p65,MMP-13 proteins increased obviously,the tenderness threshold,heat pain threshold,activities of SOD,GSH-PX,and the expression of SIRT1 protein decreased obviously(P<0.05);compared with negative control group,the pathological injury of articular cartilage in CHR group improved obviously with the increase of dosage,the joint swelling degree,arthritis index,levels of IL-6,IL-1β,TNF-α,content of MDA,chondrocyte apoptosis rate,expression of apoptotic protein C-Caspase-3,HMGB1,NF-κB p65/p-NF-κB p65,MMP-13 proteins decreased obviously,the tenderness threshold,heat pain threshold,activities of SOD,GSH-PX,and the expression of SIRT1 protein increased obviously(P<0.05);compared with CHR-H group,sirtinol+CHR-H group was able to reverse the protective effect of CHR on cartilage injury to a certain extent.Conclusion:CHR can reduce the inflammation of articular cartilage,inhibit the apoptosis of chondrocytes and play a protective role in the cartilage injury of osteoarthritis rats by up-regulating the expression of SIRT1 and down-regulating the expressions of HMGB1 and NF-κB p65/p-NF-κB p65.

Objective:To evaluate the role of Jumonji domain-containing 3 (JMJD3) in cisplatin-induced renal fibrosis following acute kidney injury in mice.Methods:Forty-eight healthy C57BL/6 male mice, aged 8-10 weeks, weighing 20-30 g, were divided into 4 groups ( n=12 each) using a random number table method: control group (group CON), control plus JMJD3 inhibitor group (group CON-A), cisplatin group (group CIS), and cisplatin plus JMJD3 inhibitor group (group CIS-A). In group CIS and group CIS-A, cisplatin was intraperitoneally administered on 1st and 14th days, respectively, to develop a renal fibrosis model in mice with acute kidney injury, and the JMJD3 inhibitor GSKJ4 10 mg/kg and equal volume of PBS were intraperitoneally injected on 4th day, respectively, once every 3 days, 6 injections in total.The equal volume of PBS and GSKJ4 10 mg/kg were intraperitoneally injected at the corresponding time points in group CON and group CON-A, respectively.Six mice in each group were selected, and orbital blood samples were collected on 3rd day after the first injection of cisplatin to determine the concentrations of serum creatinine (Cr) and blood urea nitrogen (BUN), then the animals were sacrificed, and kidney tissues were obtained for microscopic examination of pathological changes after HE and PAS staining (with a light microscope), and the damage to kidneys was assessed and scored.Six mice were sacrificed on 28th day after the first injection of cisplatin, and kidney tissues were removed for determination of the area of renal fibrosis ( via Sirius red and Masson staining), expression of fibronectin (Fn), collagen type Ⅰ (Col Ⅰ) and α-smooth muscle actin (α-SMA) (by immunofluorescence), F4/80 + cell and CD3 + cell count (using immunohistochemical method), and expression of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), CXC chemokine ligand 16 (CXCL16), and monocyte chemoattractant protein1 (MCP-1) mRNA (by real-time polymerase chain reaction). Results:Compared with group CON, the serum BUN and Cr concentrations, renal injury scores, and area of renal fibrosis were significantly increased, the expression of Fn, Col Ⅰ and α-SMA was up-regulated, the F4/80 + cell and CD3 + cell count was increased, and the expression of IL-6, CXCL16, TNF-α and MCP-1 mRNA was up-regulated in group CIS ( P<0.05), and no significant change was found in the parameters mentioned above in group CON-A ( P>0.05). Compared with group CON-A, the serum BUN and Cr concentrations, renal injury scores, and area of renal fibrosis were significantly increased, the expression of Fn, Col Ⅰ and α-SMA was up-regulated, the F4/80 + cell and CD3 + cell count was increased, and the expression of IL-6, CXCL16, TNF-α and MCP-1 mRNA was up-regulated in group CIS-A ( P<0.05). Compared with group CIS, the serum BUN and Cr concentrations, renal injury scores, and area of renal fibrosis were significantly decreased, the expression of Fn, Col Ⅰ and α-SMA was down-regulated, the F4/80 + cell and CD3 + cell count was decreased, and the expression of IL-6, CXCL16, TNF-α and MCP-1 mRNA was down-regulated in group CIS-A ( P<0.05). Conclusions:JMJD3 is involved in the process of renal fibrosis following acute kidney injury in mice, and the mechanism may be related to promotion of inflammatory responses.

Objective:To evaluate the role of CXC chemokine receptor 6 (CXCR6)-mediated activation of natural killer T (NKT) cells in renal fibrosis following acute kidney injury (AKI) in mice.Methods:Eighteen male wild-type C57BL/6 mice and 18 CXCR6 knockout C57BL/6 mice, aged 8-10 weeks, weighing 20-30 g, were divided into 6 groups ( n=6 each) using a random number table method: wild-type mouse control group (group WT-CON), CXCR6 knockout mouse control group (group CXCR6 -/--CON), wild-type mouse with AKI group (group WT-AKI), CXCR6 knockout mouse with AKI group (group CXCR6 -/--AKI), wild-type mouse with AKI + NKT cell adoptive transfer group (group WT-AKI-NKT) and CXCR6 knockout mouse with AKI + NKT cell adoptive transfer group (group CXCR6 -/--AKI-NKT). Folic acid 250 mg/kg was intraperitoneally injected to establish the model of renal fibrosis in mice with AKI.NKT cellsuspension 250 μl(1×10 6 cells) was injected through the tail vein on the 4th and 9th days after folic acid injection in group WT-AKI-NKT and group CXCR6 -/--AKI-NKT, respectively.Blood samples were taken from orbital at day 14 after folic acid injection for determination of the concentrations of serum blood urea nitrogen (BUN) and creatinine (Cr). The animals were sacrificed, and renal tissues were obtained for observation of the area of renal fibrosis (by Sirius red staining) and renal injury (using H&E staining) which was scored and for determination of the proportion of CD1d Tetramer+ cells (by flow cytometry), the number of CD206 and α-smooth muscle actin (α-SMA) double positive (CD206 + -α-SMA + ) cells (by immunofluorescence) and expression of interleukin (IL)-4 and IL-13 mRNA (by real-time polymerase chain reaction). Results:Compared with group WT-CON, the BUN and Cr levels, renal injury scores, area of renal fibrosis, proportion of CD1d Tetramer + cells and CD206 + -α-SMA + cell count were significantly increased, and the expression of IL-4 and IL-13 mRNA was up-regulated in group WT-AKI and WT-AKI-NKT ( P<0.05). Compared with group WT-AKI, the BUN and Cr levels, renal injury scores, area of renal fibrosis, proportion of CD1d Tetramer + cells and CD206 + -α-SMA + cell count were significantly increased, and the expression of IL-4 and IL-13 mRNA was up-regulated in group WT-AKI-NKT ( P<0.05), and the BUN and Cr levels, renal injury scores, area of renal fibrosis, proportion of CD1d Tetramer + cells and CD206 + -α-SMA + cell count were significantly decreased, and the expression of IL-4 and IL-13 mRNA was down-regulated in group CXCR6 -/--AKI ( P<0.05). Compared with group CXCR6 -/--CON, the BUN and Cr levels, renal injury scores, area of renal fibrosis, proportion of CD1d Tetramer + cells and CD206 + -α-SMA + cell count were significantly increased in group CXCR6 -/--AKI and group CXCR6 -/--AKI-NKT ( P<0.05). Compared with group CXCR6 -/--AKI, the BUN and Cr levels, renal injury scores, area of renal fibrosis, proportion of CD1d Tetramer + cells and CD206 + -α-SMA + cell count were significantly increased, and the expression of IL-4 and IL-13 mRNA was up-regulated in group CXCR6 -/--AKI-NKT ( P<0.05). Conclusion:CXCR6-mediated activation of NKT cells is involved in renal fibrosis following AKI in mice, and the mechanism may be related to promoting Th2 cytokine-mediated M2 macrophage-myofibroblast transformation.

Objective:To evaluate the role of natural killer T cells in renal fibrosis in mice with acute kidney injury (AKI).Methods:Twenty-four clean-grade healthy male C57BL/6 mice, aged 8-10 weeks, weighing 20-30 g, were divided into 4 groups ( n=6 each) using a random number table method: control group (group C), AKI group (group A), control plus CD1d antibody group (group C-MA), and AKI plus CD1d antibody group (group A-MA). The model of renal fibrosis in mice with AKI was established by intraperitoneal injection of folic acid 250 mg/kg.In group C, homotypic control antibody 20 mg/kg was injected via the tail vein.In group AKI, homotypic control antibody 20 mg/kg was injected via the tail vein at 24 h before establishing the model. In group C-MA, anti-CD1d monoclonal antibody 20 mg/kg was injected via the tail vein.In group A-MA, anti-CD1d monoclonal antibody 20 mg/kg was injected via the tail vein at 24 h before establishing the model.On the 14th day after folic acid injection, blood samples were taken from eyeballs to determine the concentrations of blood urea nitrogen (BUN) and creatinine (Cr) in serum.Then the mice were sacrificed, and the renal tissues were taken for Sirius red staining and HE staining to determine the area of renal fibrosis, and renal injury was scored.The expression of fibronectin (FN), type I collagen (Col-Ⅰ) and alpha-smooth muscle actin (α-SMA) in renal tissues was detected by immunofluorescence method.The expression of interleukin (IL)-4, IL-13, arginase-1 (Arg-1) and found in inflammatory zone 1 (FIZZ1) mRNA in renal tissues was detected by real-time polymerase chain reaction. Results:Compared with group C, the concentrations of BUN and Cr in serum, renal injury score, and area of renal fibrosis were significantly increased, the expression of FN, Col-Ⅰ and α-SMA and IL-4, IL-13, Arg-1 and FIZZ1 mRNA was up-regulated in A and A-MA groups ( P<0.05), and no significant change was found in the above indexes in group C-MA ( P>0.05). Compared with group A, the concentrations of BUN and Cr in serum, renal injury score, and area of renal fibrosis were significantly decreased, the expression of FN, Col-Ⅰ and α-SMA and IL-4, IL-13, Arg-1 and FIZZ1 mRNA was down-regulated in group A-MA ( P<0.05). Conclusion:Activation of natural killer T cells is involved in the process of renal fibrosis in mice with AKI, and the mechanism may be related to promoting the release of Th2 cytokines and M2 polarization of macrophages.

Objective:To evaluate the relationship between chemokine CXC-ligand 16 (CXCL16) and natural killer T cells during renal fibrosis in mice with acute kidney injury (AKI).Methods:Twenty-four healthy male C57BL/6 mice, aged 8-10 weeks, weighing 20-30 g, were divided into 4 groups ( n=6 each) using a random number table method: control group (group C), AKI group, control+ rCXCL16 group (group C-rCXCL16) and AKI+ rCXCL16 group.In AKI-rCXCL16 and AKI groups, folic acid 250 mg/kg was intraperitoneally injected to induce AKI in anesthetized mice, and rCXCL16 0.1 mg/kg and the equal volume of solution were intraperitoneally injected, respectively, at 3, 6, 9 and 12 days after injection of folic acid.The equal volume of solution and rCXCL16 were intraperitoneally injected at the corresponding time points in group C and group C-rCXCL16, respectively.The orbital blood samples were taken on day 14 after injection of folic acid for determination of the serum blood urea nitrogen (BUN) and creatinine (Cr) concentrations.The renal tissues were obtained for measurement of the renal fibrosis size (using Sirius red staining and Masson staining), for determination of the expression of fibronectin (FN), collagen-Ⅲ (Col-Ⅲ) and α-smooth muscle actin (α-SMA) (by immunofluorescence) and expression of interleukin-4 (IL-4), mannose receptor (CD206) and arginase 1 (Arg-1) mRNA (by real-time polymerase chain reaction), and for evaluation of the ratio of CD1d Tetramer + -IL-4 + cells (by flow cytometry). Results:Compared with group C, the serum BUN and Cr concentrations were significantly increased, the renal fibrosis size was increased, the expression of IL-4, CD206, Arg-1 mRNA, FN, Col-Ⅲ and α-SMA was up-regulated, and the ratio of CD1d Tetramer + -IL-4 + cells was increased in AKI and AKI-rCXCL16 groups ( P<0.05), and no significant change was found in the parameters mentioned above in group C-rCXCL16 ( P>0.05). Compared with group AKI, the serum BUN and Cr concentrations were significantly increased, the renal fibrosis size was increased, the expression of IL-4, CD206, Arg-1 mRNA, FN, Col-Ⅲ and α-SMA was up-regulated, and the ratio of CD1d Tetramer + -IL-4 + cells was increased in group AKI-rCXCL16 ( P<0.05). Conclusion:The mechanism by which CXCL16 is involved in the process of renal fibrosis is related to the recruitment of natural killer T cells secreting IL-4 which regulates macrophage M2 polarization in mice with AKI.

Objective:To evaluate the role of interleukin-4 (IL-4) in renal fibrosis induced by acute kidney injury (AKI) in mice.Methods:Twenty-four male C57BL/6 mice, aged 8-10 weeks, weighing 20-30 g, were divided into 4 groups ( n =6 each) using a random number table method: control group (group CON), AKI group, control plus anti-IL-4 antibody group (group CON-A), and AKI plus anti-IL-4 antibody group (group AKI-A). In AKI-A and AKI groups, folic acid was intraperitoneally injected to induce AKI in anesthetized mice, and the anti-IL-4 antibody 40 mg/kg and the equal volume of PBS were intraperitoneally injected, respectively, at 3, 6, 9 and 12 days after injection. The equal volume of PBS and anti-IL-4 antibody was given at the corresponding time point in CON and CON-A groups, respectively.The orbital blood samples were taken on day 14 after injecting folic acid for determination of the serum blood urea nitrogen (BUN) and creatinine (Cr) concentrations.The animals were then sacrificed, and renal tissues were obtained for examination of the pathological changes (using Sirius red staining and Masson staining) and for determination of the expression of fibronectin(FN), collagen-Ⅰ(Col-Ⅰ) and α-smooth muscle actin (α-SMA) (by Western blot) and expression of CD206, Arg-1 and FIZZ1 mRNA (by real-time polymerase chain reaction). The area of renal fibrosis was measured. Results:Compared with group CON, the serum BUN and Cr concentrations were significantly increased, the area of renal fibrosis was increased, and the expression of FN, Col-Ⅰ, α-SMA and CD206, Arg-1 and FIZZ1 mRNA in renal tissues was up-regulated in group AKI ( P<0.05), and no significant change was found in the parameters mentioned above in group CON-A( P>0.05). Compared with group AKI, the serum BUN and Cr concentrations were significantly decreased, the area of renal fibrosis was reduced, and the expression of FN, Col-Ⅰ, α-SMA and CD206, Arg-1 and FIZZ1 mRNA in renal tissues was down-regulated in group AKI-A ( P<0.05). Conclusion:IL-4 is involved in the process of renal fibrosis following AKI, and the mechanism may be associated with the regulation of macrophage M2 polarization in mice.

Objective: To evaluate the effect of dexmedetomidine on hypoxia-inducible factor-1alpha (HIF-1 α) signaling pathway during hypoxia in mice with lung cancer. Methods: Eighteen clean-grade healthy adult male BALB/c nude mice, aged 8 weeks, weighing 20-30 g, were divided into 3 groups (n=6 each) using a random number table method: lung cancer group (group L), hypoxia+ lung cancer group (group HL), and hypoxia plus lung cancer plus dexmedetomidine group (group HLD). Human lung adenocarcinoma A549 cell suspension 1×10⁶/150 μl was injected via the tail vein to establish the mouse model of lung cancer.After the model was established successfully, chronic intermittent hypoxia was performed as follows: the mice were placed in air-tight modular incubation chambers, and the atmosphere was controlled by a constant gas flow containing 10% O₂ for 3 h once a day for 3 weeks.The mice in group HL were exposed to hypoxia.After the end of hypoxia exposure, the animals in group HLD were intraperitoneally injected with dexmedetomidine 25 μg/kg 3 times a week for 3 weeks in total.The mice in group L were placed in air-tight modular incubation chambers and the atmosphere was controlled by a constant gas flow containing 21% O₂ for 3 h once a day for 3 weeks, and the equal volume of normal saline was injected intraperitoneally.The mice were sacrificed by cervical dislocation, and the lung tissues were removed for determination of lung cancer nodules count, HIF-1α expression (by immunohistochemistry), expression of HIF-1α, survivin and X chromosome linked inhibitor of apoptosis protein (XIAP) (by Western blot), and expression of matrix metalloproteinase-2 (MMP-2) and MMP-9 (by real-time polymerase chain reaction). Results: Compared with group L, the number of lung cancer nodules was significantly increased, and the expression of HIF-1α, survivin, XIAP, MMP-2 and MMP-9 was up-regulated in the other two groups (P<0.05). Compared with group HL, the number of lung cancer nodules was significantly increased, and the expression of HIF-1α, survivin, XIAP, MMP-2 and MMP-9 was up-regulated in group HLD (P<0.05). A small number of HIF-1α positive cells were found in group L, a medium number of HIF-1α positive cells in group HL, and a large number of HIF-1α positive cells in group HAD. Conclusion Dexmedetomidine can promote proliferation of tumor cells in mice with lung cancer during hypoxia, and the mechanism is related to activating HIF-1α signaling pathway.

Objective To evaluate the role of interleukin-4 receptor (IL-4R) in renal fibrosis following renal ischemia-reperfusion (I/R) injury in mice.Methods Twelve male wild type BALB/C mice and 12 IL-4Rα gene-knockout mice,aged 8-10 weeks,weighing 20-30 g,were used in the study.The mice of either type were divided into 2 groups (n =6 each) using a random number table:sham operation group (group S) and group I/R.In group I/R,renal I/R was induced by occlusion of the right renal artery for 1 h with atraumatic microclips followed by 2 weeks of reperfusion.The right renal artery was only isolated in group S.At 2 weeks of reperfusion,blood samples were taken from the orbital vein for determination of the concentrations of serum blood urea nitrogen (BUN) and creatinine (Cr).The renal tissues were obtained,and the renal fibrosis area was measured by Sirius Red staining.The expression of fibronectin (FN),collagen Ⅰ (COL-Ⅰ) and α-smooth muscle actin (α-SMA) in renal tissues was detected by immunofluorescence.The expression of signal transducer and activator of transcription 6 (STAT6) and phospho-STAT6 in renal tissues was determined by Western blot.The ratio of phoshop-STAT6 to STAT6 was calculated to reflect the phosphorylation of STAT6.Results Compared with group S of wild type mice,the serum BUN and Cr concentrations and renal fibrosis area were significantly increased,the expression of FN,COL-Ⅰ and α-SMA in renal tissues was significantly up-regulated,and the phosphorylation of STAT6 in renal tissues was significantly increased in group I/R of wild type and IL-4Rα KO mice (P＜0.05).Compared with group I/R of wild type mice,the serum BUN and Cr concentrations and renal fibrosis area were significantly decreased,the expression of FN,COL-Ⅰ and α-SMA in renal tissues was significantly down-regulated,and the phosphorylation of STAT6 in renal tissues was significantly decreased in group I/R of IL-4RαKO mice (P＜0.05).Conclusion The mechanism of renal fibrosis following renal I/R injury is partially related to IL-4R,and IL-4R results in renal fibrosis through promoting activation of STAT6 signaling pathway in mice.

Objective To discuss the neuroimaging characteristics of transcranial ultrasound in Parkinson's disease (PD) patients with or without depression.Methods Transcranial sonography (TCS) was performed in PD patients with depression (PDD +,n =50),PD patients without depression (PDD-,n =50),depression patients (D,n =50) and healthy controls (n =50),who were enrolled in the Second Affiliated Hospital of Soochow University from September 2010 to July 2016.The differences of the neuroimaging characteristics of TCS in brainstem raphe (BR) and substantia nigra (SN) in four groups were analyzed.According to the degree of depression,PDD + and D groups were divided into three subgroups:mild,moderate and severe depression,and the differences of echo characteristics in BR were analyzed among the subgroups.Results The rate of abnormal BR echogenicity was significantly higher in PDD + (78.0％,39/50) and D (82.0％,41/50) groups than that in PDD-(18.0％,9/50) and healthy control (10.0％,5/50) groups (x2 =87.80,P ＜0.01),and there was no statistically significant difference among the subgroups (PDD + group,P =0.98;D group,P =0.57).The rate of SN hyperechogenicity was significantly higher in PDD + (80.0％,40/50) and PDD-(86.0％,43/50) groups than that in D (8.0％,4/50) and healthy control (10.0％,5/50) groups (x2 =110.07,P＜ 0.01).Conclusion The echogenicity changes of BR and SN on TCS could provide some useful neuroimaging information for the diagnosis and differential diagnosis of PDD-from PDD +.