Objective:To investigate impacts of lycopene on myocardial damage in preeclampsia(PE)rats by regulating IL-6/signal transducer and activator of transcription 3(STAT3)/endothelin-1(ET-1)signaling pathway.Methods:Pregnant rats were divided into model group,experimental-L group,experimental-M group,experimental-H group,combination group and control group,with 15 rats in each group.PE rat model was established by intraperitoneal injection of 300 mg/kg N-nitro-L-arginine methyl ester(L-NAME)hydrochloride.Experimental-L,experimental-M,experimental-H groups were intraperitoneally injected with 2,4 and 8 mg/kg lycopene,combination group was intraperitoneally injected with 8 mg/kg lycopene and 1 mg/kg STAT3 activator Colivelin,control group and model group were injected with same amount of normal saline intraperitoneally,once a day for 5 consecutive days.Myocardial troponin Ⅰ(cTnⅠ),creatine kinase isozyme(CK-MB)and myoglobin(Mb)levels were detected by ELISA 24 h after the last administration.Western blot was applied to detect IL-6,p-STAT3/STAT3 and ET-1 proteins expressions.Results:Comparison between model group and control group,experimental-L,M,H groups and model group,combined group and high dose experimental group,cTnⅠ,CK-MB,Mb,IL-6 protein,p-STAT3/STAT3 protein,ET-1 protein showed statistically significant differences(all P<0.05).Conclusion:Lycopene alleviates myocardial damage in PE rats by inhibiting IL-6/STAT3/ET-1 signaling pathway.

Objective:To investigate impacts of lycopene on myocardial damage in preeclampsia(PE)rats by regulating IL-6/signal transducer and activator of transcription 3(STAT3)/endothelin-1(ET-1)signaling pathway.Methods:Pregnant rats were divided into model group,experimental-L group,experimental-M group,experimental-H group,combination group and control group,with 15 rats in each group.PE rat model was established by intraperitoneal injection of 300 mg/kg N-nitro-L-arginine methyl ester(L-NAME)hydrochloride.Experimental-L,experimental-M,experimental-H groups were intraperitoneally injected with 2,4 and 8 mg/kg lycopene,combination group was intraperitoneally injected with 8 mg/kg lycopene and 1 mg/kg STAT3 activator Colivelin,control group and model group were injected with same amount of normal saline intraperitoneally,once a day for 5 consecutive days.Myocardial troponin Ⅰ(cTnⅠ),creatine kinase isozyme(CK-MB)and myoglobin(Mb)levels were detected by ELISA 24 h after the last administration.Western blot was applied to detect IL-6,p-STAT3/STAT3 and ET-1 proteins expressions.Results:Comparison between model group and control group,experimental-L,M,H groups and model group,combined group and high dose experimental group,cTnⅠ,CK-MB,Mb,IL-6 protein,p-STAT3/STAT3 protein,ET-1 protein showed statistically significant differences(all P<0.05).Conclusion:Lycopene alleviates myocardial damage in PE rats by inhibiting IL-6/STAT3/ET-1 signaling pathway.

Objective To study the immunoadjuvant effects of chitosan oligosaccharide(COS),including the immune activation and the triggering of lysosomal escape,and to explore whether COS can be used as an adjuvant for attenuated live bacteria vector vaccines.Methods 1)Mouse macrophages RAW264.7 cells were cultured with COS at 0 mg/mL(the control group)and 0.1-4 mg/mL for 24 h and the effect on cell viability was measured by CCK8 assay.Mouse macrophages RAW264.7 were treated with COS at 0(the control group),1,2,and 4 mg/mL for 24 h.Then,the mRNA expression levels of the cytokines,including IFN-γ,IL-10,TGF-β,and TLR4,were determined by RT-qPCR assay.2)RAW264.7 cells were treated with 1 mL of PBS containing different components,including calcein at 50 μg/mL,COS at 2 mg/mL,and bafilomycin A1,an inhibitor,at 1 μmol/mL,for culturing.The cells were divided into the Calcein group,Calcein+COS group,and Calcein+COS+Bafilomycin A1 group accordingly.Laser scanning confocal microscopy was used to observe the phagocytosis and the intracellular fluorescence distribution of calcein,a fluorescent dye,in RAW264.7 cells in the presence or absence of COS intervention to determine whether COS was able to trigger lysosomal escape.3)LM?E6E7 and LI?E6E7,the attenuated Listeria vector candidate therapeutic vaccines for cervical cancer,were encapsulated with COS at the mass concentrations of 0.5 mg/mL,1 mg/mL,2 mg/mL,4 mg/mL,and 8 mg/mL.Then,the changes in zeta potential were measured to select the concentration of COS that successfully encapsulated the bacteria.Phagocytosis of the vaccine strains by RAW264.7 cells was measured before and after LM?E6E7 and LI?E6E7 were coated with COS at 2 mg/mL.Results 1)CCK8 assays showed that,compared with the findings for the control group,the intervention of RAW264.7 cells with COS at different concentrations for 24 h was not toxic to the cells and promoted cell proliferation,with the difference being statistically significant(P＜0.05).According to the RT-qPCR results,compared with those of the control group,the COS intervention up-regulated the mRNA levels of TLR4 and IFN-γ in RAW264.7 cells,while it inhibited the mRNA expression levels of TGF-β and IL-10,with the most prominent effect being observed in the 4 mg/mL COS group(P＜0.05).2)Laser scanning confocal microscopy revealed that the amount of fluorescent dye released from lysosomes into the cells was greater in the Calcein+COS group than that in the Calcein group.In other words,a greater amount of fluorescent dye was released from lysosomes into the cells under COS intervention.Furthermore,this process could be blocked by bafilomycin A1.3)The zeta potential results showed that COS could successfully encapsulate the surface of bacteria when its mass concentration reached 2 mg/mL.Before and after the vaccine strain was encapsulated by COS,the phagocytosis of LM?E6E7 by RAW264.7 cells was 5.70%and 22.00%,respectively,showing statistically significant differences(P＜0.05);the phagocytosis of LI?E6E7 by RAW264.7 cells was 1.55%and 6.12%,respectively,showing statistically significant differences(P＜0.05).Conclusion COS has the effect of activating the immune response of macrophages and triggering lysosomal escape.The candidates strains of coated live attenuated bacterial vector vaccines can promote the phagocytosis of bacteria by macrophages.Further research is warranted to develop COS into an adjuvant for bacterial vector vaccine.

Objective To construct Listeria monocytogenes(LM)and Listeria ivanovii(LI)balanced lethal systems expressing cervical cancer antigens,to study their basic biological characteristics,and to provide reference data for the immunotherapy of cervical cancer.Methods Through seamless cloning via in vitro ligation kit,the HPV16 E6E7 fusion protein antigen gene constructed in our lab was spliced to the complement plasmid pCWgfp-LM dal-Amp that contained the nutritional gene dal.Then,we replaced the ampicillin(Amp)resistance gene of the complement plasmid with the asd nutrition gene.The ligation reaction mixture was transformed into Escherichia coli(E.coli)recipient bacteria DH5аΔasd and the complement plasmid pCWgfp-E6E7-LM dal-Ampfree,which expressed cervical cancer antigens and had no Amp resistance,was obtained by nutrition screening from the E.coli DH5аΔasd.The plasmid pCWgfp-E6E7-LM dal-Ampfree was complemented into LMΔdd and LIΔdd,the attenuated nutrition-deficient Listeria strains with the virulence genes actA and plcB and nutrition genes dal and dat deleted by electroporation,thereby obtaining LM and LI balanced lethal systems expressing cervical cancer antigen genes.The in vitro growth of the strains was observed.Western blot was performed to examine the status of antigen protein expression.PCR was performed to measure the in vitro passage stability of complement plasmid pCWgfp-E6E7-LM dal-Ampfree.Their basic biological characteristics were examined by biochemical reaction tests and hemolysis assay.Results Two Listeria balanced lethal systems expressing cervical cancer antigen were successfully constructed.The HPV16 type E6E7 fusion protein was successfully expressed in the two Listeria balanced lethal systems.pCWgfp-E6E7-LM dal-Ampfree,the positive plasmid expressing cervical cancer antigen,maintained stable existence in the two Listeria balanced lethal systems.The two Listeria balanced lethal systems expressing cervical cancer antigen showed significantly better recovery growth in comparison with Listeria nutrition deficiency strains.The results of biochemical reaction tests showed that most of the biochemical reaction of the two Listeria balanced lethal systems expressing cervical cancer antigen were consistent with those of Listeria attenuated strains.The two Listeria balanced lethal systems expressing cervical cancer antigen still maintained the hemolytic ability,although their hemolytic ability was slightly inferior to that of the Listeria balanced lethal systems not expressing cervical cancer antigen and the Listeria attenuated strains.Conclusion The two Listeria balanced lethal systems expressing cervical cancer antigen genes are constructed successfully.They display normal in vitro growth.The complement plasmid pCWgfp-E6E7-LM dal-Ampfree can maintain stable existence in vitro,showing little change in its biochemical characteristics and hemolytic ability.Further research should be conducted to investigate the potential of these two recombinant strains to be used as candidate strains for cervical cancer therapeutic vaccine.