Objective To investigate the effect of active vitamin D (VD) on macrophage M1 and M2 phenotype and its role in protecting podocyte impairment in diabetic nephropathy (DN).Methods Diabetes mellitus rats were established by intraperitoneal injection with streptozocin.Rats were randomly divided into four groups:normal-1 (NC-1,n=8),normal-2 (NC-2,n=8,normal rats treated with calcitriol 0.1 μg· kg-1 · d-1 by gavages),DN (n=24) and VD (n=24,DN+calcitriol 0.1 μg· kg-1 · d-1 by gavages).Blood glucose and body weight were assessed,and 24-hour urine was collected regularly.Blood and urine samples were taken for biochemical study,and kidney tissues were used for PAS staining to assess histological changes.Immunohistochemical staining was used to detect number of CD68 + macrophage.Western blotting was used to detect protein expressions of nephrin,podocin,CD68,M1 specific marker of inducible nitric oxide synthase (iNOS),TNF-α and M2 specific marker of CD163,arginase 1 (Arg-1),mannose receptor (MR).Results (1) In DN group,levels of BUN,Scr,urinary protein and glomerular mesangial matrix proliferation were significantly higher (P ＜ 0.05),and the expressions of nephrin,podocin were significantly decreased compared with NC groups (P ＜ 0.05).These above changes were significantly improved in VD group (P ＜ 0.05).(2)Number of CD68 + macrophage infiltration in DN group was increased in a time dependent manner compared with NC groups,which was significantly reduced in VD group (P ＜ 0.05).(3)To further definite M1 and M2 macrophage activation phenotype,the protein expressions of iNOS and TNF-α was increased in DN group at 8th,14th,18th weeks compared with NC groups (P ＜ 0.05),which were significantly decreased in VD group (P ＜ 0.05).Although,there were no significant difference of protein expressions of CD163,Arg-1 and MR between VD and DN group at both 8th and 14th week (P ＞ 0.05),the protein expressions of CD163,Arg-1 and MR were higher in VD group at 18th week than that in DN group (P ＜ 0.05),and the ratio of CD163/CD68 was also enhanced in VD group (P ＜0.05).(4)Moreover,the protein expression of iNOS was negatively correlated with expression of either nephrin or podocin (r =-0.707,P ＜ 0.01; r =-0.712,P ＜ 0.01),whereas the protein expression of CD163 was positively correlated with expression of either nephrin or podocin (r =0.627,P＜ 0.01; r=0.613,P ＜ 0.01).Conclusion Vitamin D can regulate macrophage phenotype,via inhibiting M 1 macrophage activation and enhancing M2 macrophage activation to protect podocyte impairment.

Objective To investigate the effect of 1,25(OH)2D3 on high glucose induced macrophage activation and its underlying signal transduction mechanism.Methods RAW 264.7 cells were used to perform cell culture,the activity of intracellular iNOS was measured.VDR siRNA and PPARγ antagonist pre-treatment with macrophages were done before using 10-8 mol/L1,25(OH)2D3 to intervene high glucose pre-incubated macrophages.M1 markers including iNOS,TNF-α,IL-12,M2 markers including MR,Arg-1,IL-10 and nuclear receptors VDR and PPARγ were separately examined.Results The iNOS activity was increased in a glucose-dose and time dependent manner.Particularly,25 mmol/L glucose at 24 h gave the maximum response.After being treated with 25 mmol/L glucose for 24 h,not only inflammatory cytokines of TNF-α,IL-12 in the supernatant were increased,but quantitative real-time PCR and Western blotting analysis showed iNOS was also up-regulated (P ＜ 0.05).However,M2 markers,i.e.MR and Arg-l were significantly decreased (P ＜ 0.05).When in the presence of 1,25(OH),D3,the trends were reversed:the markers of M1,including TNF-α,IL-12 and iNOS were obviously reduced (P ＜ 0.05),while M2 markers,IL-10,Arg-1 and MR were increased (P ＜ 0.05).In addition,VDR and PPARγ were also increased (P ＜ 0.05).However,the above effects of 1,25 (OH)2D3 were abolished when further inhibited the expression of VDR and PPARγby VDR siRNA and PPARγ antagonist.Besides,accompanied by VDR,PPARγwas also decreased upon the treatment with VDR siRNA (P ＜ 0.05).Conclusion 1,25(OH)2D3 can promote high glucose induced classically activated macrophages (M1) converting to alternatively activated macrophages (M2) and this is achieved through VDR-PPARγ pathway.

OBJECTIVE:To investigate the effects of levocarnitine on peripheral blood T cell subsets and inflammatory factors in diabetic patients underwent peritoneal dialysis. METHODS:A total of 100 diabetic patients underwent peritoneal dialysis in se-lected as research objects were divided into conventional therapy group and levocarnitine group according to random number table, with 50 cases in each group. Conventional therapy group received peritoneal dialysis and conventional therapy. Levocarnitine group was additionally given levocarnitine 1.0 g added into 0.9% Sodium chloride injection 20 mL intravenously,3 times a week,on the basis of conventional therapy group for 12 weeks. CD3+,CD4+,the proportion of Th1 and Th2,the contents of TNF-α,INF-γ, IL-10 and IL-4 in supernatant were all detected before and after treatment. The expression of T-bet and GATA-3 were compared be-tween 2 groups. RESULTS:After treatment,CD3+,CD4+,Th2 cells,GATA-3 expression and IL-10,IL-4 levels were significantly increased,the expression of Th1 cells,T-bet content and TNF-αand INF-γwere significantly reduced,and levcarnitine group was sig-nificantly better than conrentional therapy group,with statistically significant (P<0.05). CONCLUSIONS:Levocarnitine can im-prove inflammatory reaction and the expression of related transcription factors by promoting T cell subsets and Th1/Th2 cell subsets.

Objective To investigate the effects of nuclear factor κB(NF-κB) antisense oligodeoxynucleotides (AS-ODNs) on transforming growth factor β1 (TGF-β1)-induced epithelial mesenchymal transition (EMT) in human renal tubular epithelial cells. Methods NF-κB AS-ODNs were transferred into the human renal tubular epithelial cells (HK-2), and the cells were stimulated by 10 μg/L TGF-β1 for 24 hours. The expression of NF-κB mRNA and α-SMA mRNA were measured by RT-PCR. α-SMA protein expression was assessed by fluorescence spectrum.Results TGF-β1 significantly up-regulated the expression of NF-κB mRNA, which was 8 folds of blank control (P<0.01). TGF-β1-indueed epithelial mesenchymal transition was inhibited by NF-kB AS-ODN and the NF-KB mRNA expression of AS-ODNs was decreased by 75%(P<0.05).The expression of α-SMA mRNA and protein was also down-regulated obviously (P<0.05).Conclusion NF-κB AS-ODN can inhibit the expression of NF-κB and the epithelial-mesenchymal transition, which may be a new therapeutic strategy against tubulointerstitial fibrosis.

Objective To investigate the effects and underlying mechanism of calcitriol on ameliorating podocytes impairment in DN rats.Methods SD rats were randomly divided into four groups:normal control (NC) group,calcitriol treatment (VD) group:calcitriol 0.1μg· kg--1 d-1,diabetic nephropathy (DN) group:streptozocin (STZ) 58 mg/kg,DN treated with calcitriol (DN + VD) group:calcitriol 0.1 μg · kg-1 · d-1 + STZ 58 mg/kg.Rats were sacrificed at the end of 18 weeks.Results Compared with the DN group,the DN + VD group exhibited significantly lower proteinuria by 36％,improved renal histology at the end of the experiment (P ＜ 0.05),and similar levels of blood glucose,serum urea nitrogen as well as body weight (P ＞ 0.05).There were no significant differences in the serum concentrations of creatinine,calcium and phosphorus among the four groups (P ＞ 0.05).In DN group,the expressions of nephrin,podocin,VDR,PI3K-p85 and p-Akt were significantly decreased and the expression of desmin was increased compared to NC group.Calcitriol treatment could attenuate the above changes.Additionally,a positive correlation was observed between the expressions of nephrin and VDR (r=0.776,P ＜ 0.05).Likewise,the expression of nephrin was positively correlated with either PI3K -p85 or p-Akt (r=-0.736,r=0.855,all P ＜ 0.05).Conclusion Calcitriol can ameliorate podocytes injury in DN rats,which might be related with the further up-regulation of PI3K/p-Akt signaling pathway.

Objective To investigate the effect of 1,25(OH)2D3 on high glucose induced podocyte injury and its signal transduction mechanism.Methods Differentiated mouse podocytes were exposed to normal glucose,high glucose,and different concentrations of 1,25(OH)2D3 or LY294002 (a selective PI3K inhibitor) for 24 h.PCR and immunofluorescent staining were used to detect nephrin,podocin,and desmin.Western blotting was used to detect protein expression of nephrin,podocin,desmin,PI3K,Akt and p-Akt.Results Compared with high glucose group,1,25(OH)2D3 (100 nmol/L and 1000 nmol/L) significantly up-regulated the expression of podocin and nephrin in podocytes induced by high glucose (P ＜ 0.05).Meanwhile,1,25(OH)2D3 (100 nmol/L) significantly reduced the expression of desmin (P ＜ 0.05).PI3K and p-Akt were obviously reduced in high glucose group.In the presence of 1,25(OH)2D3,the trends were reversed.However the above effects of 1,25(OH)2D3 were abolished when p-Akt was blocked by the PI3K inhibitor LY294002.Conclusions 1,25 (OH)2D3 can inhibit high glucose-induced pedocyte injury through PI3K/p-Akt signaling pathway.

Objective To investigate the protective effect and mechanism of active vitamin D3 on podocyte injury in type 1 diabetic rats.Methods Animals were randomly divided into normal control group (NC group),diabetic nephropathy group (DN group),diabetes nephropathy plus active vitamin D3 group (DN + VD group).Random tail vein blood glucose was measured and 24 hours of urine was collected every 3 weeks to observe the dynamic changes of blood glucose and 24-hour urine volume and urinary albumin.Rats were sacrificed at the end of 18th week,the kidney weight to body weight ratio,serum creatinine,blood urea nitrogen,serum calcium,and serum phosphorus levels were measured.Pathological in glomeruli were observed by PAS staining.Immunohistochemistry and Western blotting were used to observe the expression of slit diaphragms proteins including Nephrin,Podocin,and vitamin D receptor protein VDR.The mRNA level of autophagy-related protein P62 was detected by realtime quantitative PCR,and expression of autophagy-related protein including LC3B/A,Beclin1,and P62 were detected by Western blotting.Ultrastructure of podocytes and autopbagosomes in podocytes were observed by electron microscopy.Results Levels of serum creatinine,blood urea nitrogen,and blood glucose in diabetic rats were higher than those in NC group (P＜0.05),but without significant difference between DN and DN+VD groups (P＞0.05).Compared with the DN group,the urinary protein and kidney weight to body weight ratio in the DN +VD group were significantly lower (P＜ 0.05).Mesangial matrix hyperplasia and basement membrane thickening were improved,and podocyte fusion and shedding were partially reversed.The expressions of Nephrin,Podocin,VDR,LC3B/A and Beclin1 were increased,and P62 mRNA and protein were down-regulated (P ＜ 0.05).The number of autophagosomes in podocytes increased.Besides,positive correlations were found between Nephrin and Beclin 1 (r =0.939 8,P＜0.05),as well as Nephrin and VDR (r=0.948 3,P＜0.05),and Beclin1 andVDR (r=0.9093,P＜0.05).Conclusion Active vitamin D3 inhibits the injury of diabetic nephropathy podocytes by up-regulating VDR expression and enhancing autophagy activity,thereby reducing proteinuria and delaying the development of diabetic nephropathy.

Objective To investigate the role and mechanism of growth differentiation factor (GDF) 15 in ischemia-reperfusion injury (IRI) during kidney transplantation. Methods Nine wild type donor mice and 9 wild type recipient mice were selected. The renal graft of 3 recipient mice were harvested at 4, 24 and 72 h after transplantation. GDF family transcriptome analysis was carried out, and the expression of GDF15 in renal tissues of each group were detected. Five wild type donor mice, 5 GDF15 knockout donor mice and 10 wild type recipient mice were selected. According to the experimental scheme, the mice were divided into wild type sham operation group, wild type transplantation group, GDF15 knockout sham operation group and GDF15 knockout transplantation group. Serum and renal tissue samples were extracted 72 h after transplantation. The renal function, renal tubular injury, inflammatory cell infiltration, inflammatory factors, Toll-like receptor 4 (TLR4) and nuclear factor (NF)-κB expression level were compared in each group. Nine wild type donor mice, 9 GDF15 knockout donor mice and 18 wild type recipient mice were selected. According to the experimental scheme, the mice were divided into wild type transplantation group and GDF15 knockout transplantation group, and the survival rate of two group after kidney transplantation was observed. Results Transcriptome sequencing of renal graft tissues indicated that GDF15 was the most up-regulated GDF family gene, which was mainly expressed in renal tubules. Compared with the sham operation group, the renal function of mice was declined in the transplantation group. Compared with the wild type transplantation group, the serum creatinine and blood urea nitrogen levels of mice were significantly up-regulated in the GDF15-knockout transplantation group (both P < 0.05). The 1-week survival rate of mice was 87.6% in the wild type transplantation group and 41.8% in the GDF15 knockout transplantation group. In the GDF15 knockout transplantation group, the expression level of kidney injury molecule (KIM)-1 was up-regulated, and the renal tubule injury score was increased. In the wild type transplantation group, the renal tubules were dissolved or necrotized, and tubular formation was seen in the extramedullary and cortex area, whereas tubular necrosis and tubular formation were more evident in the GDF15 knockout transplantation group. The expression levels of myeloperoxidase (MPO) and F4/80 were up-regulated in the transplantation group, and the inflammatory cell infiltration was aggravated in the GDF15 knockout transplantation group. Compared with the sham operation group, the expression levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6 in the transplantation group were up-regulated. Compared with the wild type transplantation group, the expression levels of TNF-α, IL-1β and IL-6 were also up-regulated in the GDF15 knockout transplantation group (all P < 0.05). In the transplantation group, the expression levels of TLR4 and NF-κB in the renal graft tissues were higher than those in the sham operation group. In the GDF15 knockout transplantation group, the expression levels of TLR4 and NF-κB in the renal graft tissues were higher compared with those in the wild type transplantation group. Conclusions GDF15 may alleviate the IRI of renal graft probably via inhibiting the TLR4-NF-κB signaling pathway.

Objective: To explore the clinical efficacy of Xiaoshuan enteric-coated capsule (XSECC) in treating cerebral infarction and its potential mechanism of action. Methods: Patients with acute ischemic stroke (AIS) of the qi deficiency and blood stasis type were randomly assigned to the control and observation groups. They were evaluated using the National Institutes of Health Stroke Scale (NIHSS), Activities of Daily Living (ADL), Hachinskilnchemic Scale (HIS), Barthel Index (BI), clinical efficacy scores, and TCM syndrome scores on days 0, 14, 30, and 90. Furthermore, VEGF and BDNF levels were measured on days 30 and 90. Finally, we analyzed the changes in each scale score and vascular neurological factor in both groups. Results: After 14 days of treatment, the difference values in NIHSS, ADL, and BI were higher, and TCM syndrome and clinical efficacy scores were increased in the observation group compared with those of the control group (all P < .05). After 30 days, the NIHSS, ADL, HIS, and TCM syndrome scores were decreased compared with those of the control group, while BI and clinical efficacy scores were increased (all P < .05). After 90 days, the difference value in ADL was higher, and TCM syndrome score was increased in the observation group compared with that of the control group (P = .047, P = .005, respectively). The levels of VEGF and BDNF were higher in the observation group than in the control group on days 14, 30, and 90 (all P < .05). VEGF and BDNF levels on day 0 were associated with prognosis of patients with AIS; therefore, they have a predictive value for the prognosis of acute cerebral infarction. Conclusions XSECC therapy can improve clinical outcomes in patients with acute and recurrent cerebral infarctions. Its mechanism of action may be associated with the secretion of VEGF and BDNF.

Objective To evaluate the effect of the rehabilitation software based on information-motivation-behavioral skills(IMB)model on meeting the rehabilitation needs, increasing the continuation of rehabilitation and the rehabilitation compliance, improving the outcome of rehabilitation among elderly patients with fracture. Methods Based on the theory of"information-motivation-behavior"and the unity of human- computer interaction as the design concept, the status quo of rehabilitation training and rehabilitation needs of elderly patients with fracture were reviewed. The rehabilitation training test was applied to 30 patients with fracture in orthopedic department of our hospital. 30 patients in the same period were randomly selected as control group. All patients underwent Self-Rating Anxiety Scale (SAS) and Self-rating Depression Scale (SDS) scores before and 1 and 3 months after operation. Differences were tested using Student's t test. Results Developed rehabilitation application software based on information module、motivation module and behavior module. The test group using this software for rehabilitation training was compared with the control group. The results showed that there was no significant difference in preoperative SAS and SDS scores between the two groups (t=-0.648,-0.284, P>0.05). At 1 and 3 months after operation, the SAS scores in the test group were (40.05 ± 6.77), (32.01 ±5.86), which were lower than (45.50 ± 11.32), (39.55 ± 5.67) in the control group (t=-4.14,-4.89, P<0.01). The SDS scores in the test group were (42.30 ± 9.86), (33.23 ± 6.56), which were lower than (46.50 ± 10.32), (38.45 ± 7.80) in the control group (t=-3.52,-3.82, P=0.001). Conclusion Rehabilitation software can influence the rehabilitation behavior of elderly fracture patients from information and motivation factors, provide professional guidance and rehabilitation intervention, and promote the implementation of active rehabilitation.