ABSTRACT
Objective To explore the effect and potential mechanism of suppressors of cytokine signaling 3 (SOCS3) knockout on cell viability and apoptosis of retinal ganglion cells (RGCs) after optic nerve injury.Methods The optic nerve transection was used to construct optic nerve injury model of rats,and RGCs were isolated after optic nerve injury.The experimental animals were divided into optic nerve transection injury (ONT) group and sham-operation (Sham) group.The expression of SOCS3 in RGCs was detected by Western blot and RT-PCR in each group.Subsequently,SOCS3 siRNA was transfected into RGCs of Sham group and ONT group,and the experimental were further subdivided into blank control group,negative control group and SOCS3 silence groups.Cell viability was measured by CCK8 and MTr methods.Apoptosis was detected by Hoechst 33342 staining and flow cytometry.Furthermore,the mTOR siRNA and SOCS3 siRNA were co-transfected into RGCs,and cell viability and apoptosis were detected.Results The expression of SOCS3 was dramatically increased at 3 days after injury in the ONT group when compared with Sham group (P =0.049),and it showed an increased tendency gradually along with the extension of injury time.Compared with the blank control in the ONT group,SOCS3 silence markedly promoted cell viability [(49.47 ± 7.35) ％ vs.(73.24 ± 8.70) ％],reduced cell growth inhibition [(27.25 ±0.75)％ vs.(10.96 ± 1.07)％] and apoptosis [(23.06 ± 1.43)％ vs.(10.65 ± 1.77)％].The result of Hoechst 33342 staining indicated that SOCS3 silence ameliorated the cell apoptosis induced by ONT.In addition,SOCS3 silence significantly improved pS6 expression at 2 weeks after injury,and mTOR and SOCS3 co-silence reduced cell viability,increased cell growth inhibition and apoptosis compared with SOCS3 silence group after injury.Conclusion SOCS3 silence promotes injury-induced cell viability of RGCs and suppresses injury-induced apoptosis of RGCs via up-regulating mTOR activity in the later period of injury.
ABSTRACT
Objective To investigate the relationship among serum levels of suppressors of cytokine signaling-3(SOCS-3),insulin-like growth factor binding protein-1(IGFBP-1) and hyperglycemia in critically ill children. Methods The 64 critically ill children who suffered from sepsis,after surgical repair of congenital heart dis-ease by extracorporeal circulation, and after surgery of severe traumatic from January 2009 to January 2012 in Depart-ment of Pediatric Intensive Care Unit( PICU) of Guangzhou Women and Children′s Medical Center were selected as the research object. According to the blood glucose levels on admission, the 64 children were divided into the normal glu-cose group and the hyperglycemia group. The 15 cases of healthy children in the same period in Guangzhou Women and Children′s Medical Center were selected as the healthy control group. The levels of blood glucose,insulin,SOCS-3,IG-FBP-1 and insulin resistance index( HOMA-IR) were measured and compared among groups when they were on ad-mission. Results (1) The blood glucose of the hyperglycemia group was significantly higher than those in the normal glucose group and the healthy control group[(9. 83±2. 48) mmol/L vs (4. 82±0. 76) mmol/L,(4. 49±0. 81) mmol/L] (P0. 05). (4) The blood glucose was positively correlated with the insulin and HOMA-IR(r=0. 455,0. 773,P0. 05). Con-clusions In the critically ill children,hyperglycemia was related to the insulin resistance which can not be evaluated through changes in serum levels of SOCS-3 and IGFBP-1. In addition,it cannot be excluded that the critically ill chil-dren may have insulin resistance and pancreaticβ-cell dysfunction simultaneously.
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Objective To observe the effects of Shengqing Chinese herbs on the obesity and leptin resistance of diet-induced obesity (DIO) rats.Methods Wistar rats were produced a rat model including of 40 DIO rats and 10 DIO-R rats. The DIO rats were divided into DIO model group, sibutramine group, Jianpi Liqi group and Shengqing group, which were respectively by gavage of saline, sibutramine, Jianpi Liqi herbs (Citri reticulatae pericarpium, Aucklandiae radix, Aurantii fructus immaturus), Shengqing herbs (Bupleuri radix, Cimicifugae rhizoma, Puerariae lobatae radix), blank control group and DIO-R group were by gavage of saline. Their weight, were examined after 16 weeks gavage. Leptin and neuropeptide Y in the serum were examined. Leptin, TNF-α, and suppressors of cytokine signaling-3 (SOCS-3) in the homogenized fat were examined. Results Compared with the DIO-R group, DIO model group's body weight, NPY significantly increased (P<0.01);leptin in the serum and homogenized fat decreased (P<0.05);SOCS-3 increased (P<0.05). After drug intervention, weight of each drug group declined. Compared with the DIO model group, Jianpi Liqi group's leptin in the homogenized fat increased (P<0.05). Shengqing group's leptin in homogenized fat increased, body weight reduced, NPY and SOCS-3 levels decreased, and better than sibutramine group and Jianpi Liqi group (P<0.05,P<0.01).Conclusion Shengqing herbs can reduce obesity and inhibit leptin resistance.
ABSTRACT
Objective To study the construction of the lentiviral-mediated RNA interference(RNAi) vector targering rat suppressors of cytokine signaling 3(SOCS3) gene.Methods Three target sequences were selected by on-line designer software on Ambion according to rat SOCS3 mRNA sequence(NM053565),the complementary DNA contained both sense and antisense oligonucleotides were designed and synthesized.After annealing,these double strands DNA were cloned to pRNA-Lenti-green fluorescent protein(GFP),which contained U6 promoter and GFP.The resulting Lentiviral vector containing SOCS3 shRNA was named pRNA-Lenti-SOCS3-GFP.After the rat glioma cells(C6)were transduced with the constructed 1entiviral vectors,real-time polymerase chain reaction was used to evaluate the level of SOCS3 expression(including siRNA1 group,siRNA2 group,siRNA3 group,vacuity group and siRNA-Negative group).The pRNA-Lenti-SOCS3-GFP and Lentivector Pakaging plasmid mix were cotransfected into 293T to package Lentivirus particles.Culture supematant was harvested,then the virus titer was determined by serial dilution assay.Results The SOCS3 mRNA sequence was successfully cloned to pRNA-Lenti-GFP,which was proved by PCR and DNA sequence.Compared with control group,the SOCS3 mRNA expressions were obviously suppressed in all 3 experimental groups,especially the expression rate in siRNA1 group was reduced by 80%.The Lentiviral particle titer was determined by serial dilution assay with 1.0?1010 TU?L-1.Conclusion The lentiviral-mediated RNAi vector of rat SOCS3 gene has been constructed successfully,this may provide a potential tool for studying and treating SOCS3-related diseases.