[Objective]To investigate the effects of ghrelin on inflammatory signaling protein kinase B(Akt),nuclear factor-κB(NF-κB)and inducible nitric oxide synthase(iNOS)in alveolar macrophage(AM).[Methods]24 Male SD rats were randomly divided into Sham,CLP,CLP+ghrelin,and Sham+ghrelin groups. Cecal ligation and puncture(CLP)was used to induce sepsis. Ghrelin(20 nmol/kg)was administered by intraperitoneal injection at 3 h and 15 h post-operation. Histopathological changes of lungs were observed and scored.AM were extracted from bronchoalveolar lavage fluid(BALF). Interleukin-1β(IL-1β),tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)in BALF were detected by ELISA. IL-1β,TNF-α,and IL-6 mRNA in AM were detected by qPCR.NF-κB p65,IκBα,p-IκBα,Akt,p-Akt and iNOS in AM were detected by immunofluorescence(IF)and Western blotting.[Results]The histologic score(6.7±0.8),BALF IL-1β[(146±12)pg/mL]and IL-6[(182±10)pg/mL]from CLP+ghrelin group were respectively 35.4%,44.5% and 46.42% lower than those from CLP group[(10.3±0.7),(263±17)pg/mL,and(273±5)pg/mL],P<0.05.No significant difference was found in BALF TNF-α between CLP group and CLP+ghrelin group.The IL-1β,TNF-α and IL-6 mRNA in AM from CLP+ghrelin group were respectively 54.38%,53.6% and 46.42% lower than those from CLP group,P<0.05. The nuclear NF-κB p65 and cytoplasmic p-IκBα,p-Akt and iNOS from CLP+ghrelin group were respectively 32.58%,45.42%,27.6% and 48.33% lower than those from CLP group,P<0.05. There was no significant difference in all data between Sham group and Sham+ghrelin group.[Conclusion]Ghrelin can decrease the activity of inflammatory signaling proteins Akt,NF-κB and iNOS in AM,therefore restricts AM expressing pro-inflammatory cytokines IL-1β,TNF-α,and IL-6,thus alleviates sep-sis-induced acute lung injury(ALI).

[Objective]To determine whether activated hepatic stellate cell(HSC)become quiescent phenotype if cultured in a soft extracellular matrix(ECM).[Methods]HSC-T6 cells which stably expressed myofibroblast(MFB) phenotype,were cultured in the 2D and 3D in-vitro matrix culture models that were constructed by 1 mg/mL fibrin gel. The proliferation activity of HSC-T6 was determined by cell counting kit-8(CCK8)assay at different time points(24 h, 48 h,72 h,96 h). Immunofluorescence and gelatin zymography analysis were performed to detect the expression of α-SMA,MMP-2,and MMP-9. Moreover,the cellular surface morphology and deformability was detected by AFM.[Results]After cultured in the fibrin gel,the cellular proliferation activity,expression of α-SMA,MMP-2 and MMP-9,and cellular deformability of activated HSC-T6 was obviously down-regulated in contrast to the control group (P<0.05),and there were also differences in the inhibition of cellular proliferation activity,the assembly of α-SMA stress fiber and the effect of cellular deformability between 2D and 3D fibrin gel cultured(P<0.05).[Conclusions]Soft fibrin gel inhibited the transdifferentiation of activated HSC-T6,but could not make it revert to α-SMA negative quies-cent phenotype.

[Objective]To investigate the safety and efficacy of phosphorylcholine oligomer grafted graphene oxide as a drug carrier for transcatheter arterial chemoembolization in the treatment of liver cancer.[Methods]Doxorubicin loaded folic acid labeled phosphorylcholine oligomer grafted graphene oxide(DOX@GO-PCn-FA)was prepared. Graphene ox-ide(GO)and DOX@GO-PCn-FA were injected intravenously via marginal ear vein in New Zealand white rabbits respec-tively to assess their safety and biodistribution for intravenous administration.Ten male New Zealand rabbits were used to establishe the VX2 liver cancer model and the tumor characteristics were confirmed by dynamic contrast enhanced CT scan.Catheter was inserted via femoral artery and advanced into hepatic lobar or segmental artery.Digital subtraction angi-ography(DSA)was performed to validate the tumor feeding vessels.DOX@GO-PCn-FA was injected through the cathe-ter to carry out selective transcatheter arterial chemoembolization(TACE). Dynamic enhanced CT scan and pathological examinations of major tissues and organs were implemented 7 days post TACE to evaluate the efficacy of embolization effect of DOX@GO-PCn-FA against liver tumor as well as the biodistribution and safety.[Results]Intravenous injection of GO resulted in significant thrombosis and pulmonary embolism whereas DOX@GO-PCn-FA of same dosage did not. DOX@GO-PCn-FA was capable of effectively diminishing the blood supply of liver tumors when applied in TACE. Pathologic exploration revealed that DOX@ GO-PCn-FA mainly deposited in the tumor,and no obvious complications were observed.[Conclusions]GO-PCn presented superior biocompatibility and exerted effective chemoembolization against liver cancer.

[Objective]To investigate the expression changes of miR-133b in methamphetamine(MA)-induced neuro-nal injury in PC12 cells and its regulative effects on cellular apoptosis.[Methods]PC12 cells were cultured and divided into control group and MA treated group.In MA treated group,PC12 cells were insulted with 800μmol/L MA in culture medium. The cellular injury of PC12 cells was observed under microscope. The cellular apoptosis was detected by Hoechst33342/PI double staining,and the expression level of miR-133b was examined by real-time quantitative PCR(RT-PCR). Further-more,miR-133b mimic and inhibitors were transfected into PC12 cells to analyze miR-133b's function in MA-induced cell apoptosis.[Results]The data showed that 800 μmol/L MA could induce obvious cellular injury,cause neurite shortened and increase the cell apoptosis. The RT-PCR data showed that the expression of miR-133b of PC12 cells treated with MA de-creased significantly.The apoptosis rate of PC12 cells decreased after transfection of miR-133b mimic,while increased after transfection of miR-133b inhibitors.[Conclusions]High concentration of MA causes neuron damage and induces neuronal apoptosis,and also decreases the levels of miR-133b expression. Whereas,overexpression of miR-133b can reduce the apoptosis of cultured PC12 cells.Thus,miR-133b plays a crucial role in MA mediated neurotoxicity.This study provides a theoretical basis for elucidating the mechanism of MA-induced neurotoxicity and may provide a new strategy for treating MA addiction.

[Objective]To investigate the effect of KIF23 gene expression on the proliferation,migration and invasion of human hepatocellular carcinoma HepG2 cells in vitro,and to explore the possible mechanism.[Methods]The KIF23 siRNA was transfected into HepG2 cells by lipofectamine 3000.The expression of KIF23 mRNA and protein in HepG2 cells was de-tected by qRT-PCR and Western blot.The effect of silencing KIF23 on the proliferation of HepG2 cells was studied by CCK-8 assay and plate clone formation assay.The tumor cell abilities of migration and invasion after transfection were measured by scratch assay and Transwell assay.The expression of protein kinase B(PKB/Akt)and phosphorylated Akt(p-Akt)protein in HepG2 cells transfected with KIF23-siRNA2 was detected by Western blot.[Results]KIF23-siRNA could effectively si-lence the expression of KIF23 mRNA and protein in HepG2 cells(P<0.01).The results of CCK-8 assay,plate clone forma-tion assay,scratch assay and Transwell assay demonstrated that the cell proliferation,migration and invasion ability of the KIF23-siRNA2 interference group were significantly inhibited,compared to the negative control group and the blank control group(P<0.05).The expression level of total Akt protein in HepG2 cells was not changed,but the expression level of phos-phorylated Akt protein was down-regulated(P<0.05).[Conclusions]KIF23 may promote the proliferation,migration and in-vasion of human hepatocellular carcinoma cells by activating Akt signal transduction pathway.KIF23 is expected to be a new target for gene therapy of hepatocellular carcinoma.

[Objective]To evaluate the effects of anthocyanin cyanidin-3-o-β-glucoside(Cy-3-g)on monocyte migra-tion mediated by platelet derived factors in vitro.[Methods]Thrombin-activated human gel-filtered platelets were incubated with different concentrations(0,0.5,5 or 50 μmol/L)of Cy-3-g.The level of TGF-β1,β-TG,CCL5 in platelet superna-tant was determined by ELISA.The peripheral venous blood from healthy volunteers were incubated with different concentra-tions of Cy-3-g. The expression of CCR5 on leukocytes was detected by flow cytometer. Calcein AM labeled THP-1 cells were incubated with the releasates of Cy-3-g-treated gel-filtered platelets for 120 min,and then the number of the THP-1 cells were counted by a microscope.Moreover,THP-1 cells were pre-incubated with different concentrations of Cy-3-g with or without CCR5 inhibitor maraviroc(200 μmol/L)for 60 min,then treated with recombinant CCL5(100 nmol/L)for 120 min.The number of the THP-1 cells were counted as well.[Results]Cy-3-g significantly inhibited platelet TGF-β1,β-TG,CCL5 secretion and decreased CCR5 expression of leukocytes compared with the control(P<0.05).Moreover,the sig-nificant inhibitory effects of Cy-3-g on THP-1 cell migration toward the releasates of Cy-3-g-treated platelets were also ob-served(P<0.05). In addition,THP-1 cell migration toward recombinant CCL5 was significantly suppressed by Cy-3-g.[Conclusions]Anthocyanin Cy-3-g inhibited inflammation of atherosclerosis by platelet TGF-β1,β-TG,CCL5 secretion and CCL5-mediated monocyte migration.Our results provided new ideas for prevention and treatment of atherosclerosis.

[Objective]To explore the effect of alpha2-macroglobulin(α2M)on superoxide anion(.O2-)content, superoxide dismutase(SOD)activity and the process of cell-to-myofibroblast transformation in human skin fibroblasts (HSF)after X-ray irradiation.[Methods]HSF cells were irradiated with 0,5,10,15 and 20 Gy X-ray.The change of. O2- content and SOD activity in the supernatant of cell culture medium were measured on the first day after irradiation. The protein expression of alpha-smooth muscle actin(α-SMA)was detected by Western blot on the fifth day after irradia-tion.The most sensitive radiation dose is selected.HSF cells were irradiated with the above sensitive dose.Respectively, 1h before irradiation,1 h after irradiation,the experimental group cell culture medium was added to a final concentration of 0.25 mg/mL,0.5 mg/mL of α2M.The change of.O2-content,SOD activity and the protein expression of α-SMA were detected.[Results]HSF cells were irradiated with 5~20 Gy doses of X-ray..O2- content increased,SOD activity de-creased and α-SMA protein expression increased gradually(P<0.05).The addition of α2M at 1 h after 10Gy X-ray irradi-ation reduced the.O2- content,increased the SOD activity and downregulated the protein expression of α-SMA in HSF cells(P<0.05). There was no significant change in the administration at 1 h before irradiation.[Conclusion]HSF cells increased.O2-content significantly,while SOD activity decreased,and the tendency to transform myofibroblasts after X-ray irradiation.α2M can reduce the.O2-content,increase the SOD activity in HSF cells and inhibit the transformation of fibroblasts into myofibroblasts after irradiation.Indicating that α2M can play a role in radiation protection by anti-oxida-tion and anti-fibrosis.

[Objective]To investigate the effects of continuous long-term sleep deprivation on rat's behavior,memory and neurotransmitters in hippocampus,as well as determine the effects of prevention of electro-acupuncture.[Method]We used multi-platform water environment to make the rat model of insomnia.The experiment was divided into two sections.Sec-tion one:thirty rats were divided into 5 groups(six rats each group):tank control group(TC),sleep-deprived for continu-ous 7 days group(SD group),three days of recovery after SD group(3d after SD group),six days of recovery after SD group (6d after SD group),eleven days of recovery after SD group(11d after SD group).The term of recovery refers to normal feed-ing and sleeping when rats return to cages after sleep-deprivation.The open-field test was performed following the end time of each group experiment and then the rats were perfused and fixed.Section two:Eighteen rats were divided into three groups (six rats each group):tank control group(TC),sleep-deprived for continuous 7 days group(SD group),sleep-deprived rats treated with electro-acupuncture group(SD+EA group).Rats of SD group and SD+EA group were deprived sleep for con-tinuous 7 days. During the period of sleep deprivation,the rats of SD+EA group were treated with electro-acupuncture 20 minutes every morning.At the end time of experiment,rats were tested by the Morris water maze and then perfused and get specimens.[Results]Section one:the statistical analysis of outcome of open-field test showed:as compared to the TC group,the scores of rat's horizontal movement and vertical movement were significantly fewer in the SD group(P<0.05).Af-ter 3,6,or 11 days recovery of sleep,the scores increased significantly as compared with SD group(P<0.05). Although, the outcome of open-field test in the 3d after SD and 6d after SD groups had the significant difference with TC group(P<0.05),there was no different between the 11d after SD group and TC group(P>0.05).The quantitative analysis of gamma aminobutyric acid(GABA)expression in the hippocampus showed:the SD group shows lower level of GABA when com-pared to the TC group(P<0.05).After 3 days or 6 days of recovery,the expression of GABA is higher than the SD group(P<0.05),and similar to the TC group(P>0.05).In the 11days after SD group,the expression of GABA declined as compared to the TC group and the 6d after SD group(P<0.05).Section two:In the Morris water maze experiment,when compared to the TC group and the SD+EA group,the escape latency period was significantly prolonged and the times of across the plat-form were significantly reduced in the SD group(P<0.05).On the contrary,there were no statistical differences in the escape latency period or the across platform times between the SD+EA group and the TC group(P>0.05).The results of serotonin(5-HT)and GABA in the hippocampus showed:the 5-HT level in the SD group was no significant difference as compared to the TC group and the SD group on 5-HT level,while the 5-HT level in the SD+EA group was reduced as compared to the TC group(P<0.05). In the opposite,the GABA level in the SD group was decreased as compared to the TC group(P<0.05),while the SD+EA group and the TC group appear similarly level(P>0.05).[Conclusions]The effect of 7 days'contin-uous long-term sleep-deprivation may lead to the reduction in the ability of learning and memory behavior of rats.Our results is different with 4 days'continuously sleep-deprivation which was reported on the rats'expression of GABA and 5-HT. Electro-acupuncture stimulating the Sishengcong Acupoints seems preventing the harming of long-term sleep-deprivation on the ability of learning and memory,which probably is through adjusting the disorder of central neurotransmitter expression caused by sleep deprivation.

[Objective]To construct recombinant expression vector PET28a-TAT-RabGEF1,express,purify fusion pr-otein effectively in E.coli Rosetta(DE3),and investigate its transmembrane effect in vitro on P815 cells.[Methods]With cDNA in rats′tissues as the template,two primers containing the TAT sequence and two designed enzyme restriction cutting sites were designed. TAT-RabGEF1 fragment was amplified by PCR,and its product was inserted into PET-28a vector to construct recombinant plasmid PET28a-TAT-RabGEF1 prokaryotic expression vector. The recombinant vector was trans-formed into E.coli Rosetta(DE3)and express fusion proteins.The protein products were purified by affinity chromatography. The efficiency of the transduction of fusion protein into P815 cells were detected by immunofluorescence and analyzed by fluo-rescence spectro-photometer,with using methods of CCK-8 to analyze the cells viability after transduction of different con-centrations of fusion protein.[Results]The recombinant vector PET28a-TAT-RabGEF1 was constructed and the fusion pro-tein TAT-RabGEF1 was successfully expressed in E.coli Rosetta(DE3). By western blotting and SDS-PAGE we can see that the protein products′ relative molecular mass was about 57 ku,which was consistent with the target one,TAT-Rab-GEF1.The immunofluorescence results suggested that the fusion protein had the ability to transduct into P815 cells,and sat-uration of fusion proteins to be transducted into cells was 1 μmol/L.[Conclusion]Constructed recombinant vector PET28a-TAT-RabGEF1 and expressed fusion protein,TAT-RabGEF1,which verified the TAT′s ability of transduction. And it would build a solid technical foundation for the following research on the effect of RabGEF1′s on the activation of mast cells.

[Objective]To study the resistance reversion of rapamycin on ovarian cancer cell line SKOV3/DDP,and ex-plore its underlying molecular mechanisms.[Methods]MTT method was used to detect the cell toxicity,drug-resistant multi-ple and reversing multiple of cisplatin-resistant ovarian cancer cell line SKOV3/DDP;Western blot was used to detect the changes of Akt/mTOR Pathway induced by rapamycin.[Results]① MTT detected that when rapamycin concentration was 25,50,100,500 and 1 000 μg/L,its inhibition rates on cisplatin-resistant ovarian cancer cell line SKOV3/DDP were 4.48%,25.30%,35.86%,67.82%,81.43%.The concentration of 25 μg/L was selected to be the reversal concentration,be-cause its maximum rate was less than 5%.②The resistant index(RI)of cisplatin-resistant ovarian cancer cell line SKOV3/DDP was 2.21. ③ The reversal fold of 25 μg/L rapamycin on cisplatin-resistant ovarian cancer cell line SKOV3/DDP was 1.63.④Western blot results:After the addition of rapamycin,expression of p-mTOR and its downstream protein p-p70s6k in SKOV3 and SKOV3/DDP was significantly reduced. Meanwhile,there was a feedback increase in p-Akt.[Conclusions]Rapamycin has a reversal effect on cisplatin-resistant ovarian cancer cell line SKOV3/DDP. Its reversal mechanism may be inhibiting the cell proliferation and promoting cell apoptosis by depressing the expression of p-mTOR and its downstream pro-tein p-p70s6k in Akt/mTOR Pathway.