Interventional Effect And Preliminary Mechanism Of NaNO₃ On ApoE^-/- Mouse Aortic Atherosclerosis With Aging

Senescent cells can drive the body aging and promote the occurrence of many diseases.At present,there still need new treatment for alleviating cellular senescence and related diseases.In this study,different aging models were established in vivo and in vitro to study the aging associated with aortic atherosclerosis in mice,and to explore the anti-aging effect of Na NO₃in aortic atherosclerosis and its potential mechanism.【Objective】To explore the effect of Na NO₃on anti-Apo E^-/-mouse aortic atherosclerosis associated with senescence and its preliminary mechanism.【Methods】Atherosclerosis model was established with Apo E^-/-mice.Apo E^-/-mice were randomly divided into normal group（normal diet with normal drinking water）,high-fat group（high-fat dietwith normal drinking water）and high-fat+Na NO₃group（high-fat diet,drinking water with concentration of 1g/L Na NO₃）.During the feeding process,the body weight of the mice was recorded,and the serum concentration of total cholesterol（CHO）,triglyceride（TG）,low density lipoprotein（LDL）and high density lipoprotein（HDL）were detected.At the end of the experiment（the 12th week）,the mouse aorta was taken and the formation of aortic plaque was observed by oil red O staining,the changes of aortic SA-β-Gal activity were detected by gross SA-β-Gal staining and the senescence of aortic tissue was detected by P16immunohistochemical staining.The expression of cell cycle regulation related genes（P53 and P16）,inflammatory factors（IL-1β、TNF-αand IFN-γ）),mitochondrial function related genes（AMPK and PTPMT1）and mi R-34a in aorta were detected by fluorescence quantitative PCR（QPCR）.The expression level of inflammatory factors（IL-6,TNF-α,IL-1βand IFN-γ）in serum of mice were detected by ELISA.Senescence model of human aortic endothelial cells（HAo ECs）were established by using H₂O_2.To determine the appropriate concentrations of H₂O_2,we detected the effect of different concentrations of H₂O₂on the proliferation of HAo ECs through CCK-8 and on the senescence of HAo ECs by using SA-β-Gal staining.Then,QPCR was used to detecte the expression level of cell cycle regulation related genes（RB,P53 and P21）and inflammatory factors（IL-6,IL-8 and IL-1β）,and Western Blot was used to detect the expression level of senescence related proteins such as RB,P53 and P16 of HAo ECs induced by H₂O₂and treated by Na NO₃.【Results】Compared with the control group,the body weight of Apo E^-/-mice in the high-fat group did not change significantly.The concentration of CHO,TG and LDL in serum increased significantly,while the concentration of HDL decreased slightly.The oil red O positive area,the SA-β-Gal positive area and P16 positive staining of aortic sections were increased in high-fat group.The expression level of cell cycle regulation related genes（P53 and P16）,inflammatory factors（IL-1β、TNF-αand IFN-γ）and mi R-34a in aorta increased markedly.But the expression level of mitochondrial function-related genes（AMPK and PTPMT1）was significantly down-regulated.It indicates that the model of atherosclerosis with senescence has been successfully established.Compared with the high-fat group,there were nearly no change in the body weight of Apo E^-/-mice in the Na NO₃group.The serum concentration of CHO and TG was decreased,while the concentration of LDL and HDL had no significant difference in Na NO₃group when compared with high-fat group.The oil red O positive area,the SA-β-Gal positive area and P16 positive staining of aortic sections were decreased after Na NO₃treatment.And the expression of P16 in aorta was markedly down-regulated.P53 expression level decreased,but the difference was no statistical significance.The expression level of inflammatory factor TNF-α,IFN-γand IL-1βand mi R-34a were decreased,and increased expression of mitochondrial function-related genes AMPK and PTPMT1.With the increase of H₂O₂concentration,the relative absorbance value of HAo ECs decreased,and the SA-β-Gal positive area increased.Compared to the control group,the expression level of P21 in HAo ECs was increased after H₂O₂induction.Though the increased expression of P53 and RB genes in H₂O₂group had no statistical significance with the control group,the expression level of RB,P53 and P16 protein increased significantly.Inflammatory factors IL-6,IL-8 and IL-1βwere increased after H₂O₂treated.Compared with the H₂O₂group,the expression level of P53 and P21 genes after Na NO₃treatment decreased significantly,while the expression level of the RB gene had no difference.The expression levels of RB,P53 and P16 proteins,inflammatory factors IL-6,IL-8and IL-1βwere significantly decreased.【Conclusion】Na NO₃can reduce the aortic atherosclerotic plaque,alleviate cell aging,restore mitochondrial dysfunction and down-regulate the expression of mi R-34a of Apo E^-/-mice induced by high-fat diet.Na NO₃can alleviate H₂O₂-induce cell senescence of HAo ECs.It is suggested that Na NO₃may inhibit cell senescence and alleviate aortic atherosclerosis through reducing the expression of mi R-34a and regulating the downstream target gene.Spinal cord injury（SCI）is one of the most complex and destructive diseases of the nervous system,which can lead to permanent loss of tactile perception.But existing treatment methods have limited effects.In this study,a spinal cord hitting injury model was established to explore the therapeutic effect of OIC gene modified dental pulp stem cells on spinal cord injury.【Objective】 To explore the therapeutic effect of OIC gene modified dental pulp stem cells（DPSCs）on spinal cord injury（SCI）.【Methods】OPN,IGF-1 and CNTF genes were connected to p UC57 vector plasmid to construct recombinant plasmid p UC57-syn.And the recombinant plasmid p KAd5f11p-Syn was constructed by recombining p UC57-syn with p KAd5f11 p ES-Pmel.Then recombinant plasmid p KAd5f11 p ES-Pmel was transfected into 293 cells for preparation of adenovirus（Ad-OIC）carrying OPN,IGF-1 and CNTF genes.In vitro experiments,DMEM high glucose medium,Ad-Null modified DPSC conditioned medium（DPSC-Null-CM）and Ad-OIC modified DPSC conditioned medium（DPSC-OIC-CM）were used to co-culture with mouse hippocampal neurons（HT-22）.The effects of three media on the proliferation of HT-22 cells were detected by using CCK-8 kit.Fluorescence quantitative PCR（QPCR）was used to detect the changes in the expression level of neuron marker Tubb3 and nerve function-related gene Syn1.After β3-Tublin immunofluorescence staining,laser confocal microscopy was used to detect the effect of three media on the axon length of HT-22 cells.We also established a H2O2 induced apoptosis model of HT-22 cell,and flow cytometry was applied to detect the effect of three media on HT-22 cell apoptosis.In vivo experiment,the spinal cord injury model of mice was established by hitting the local spinal cord of T10.After hitting,DPSC which carried luciferase was injected locally,and bioluminescent imaging system was used to detect the survival time of transplanted cells.PBS,DPSC-Null and DPSC-OIC were injected into the injuried area respectively,and the secondary injury after SCI was observed by magnetic resonance imaging（MRI）;The Basso Mouse Motor Rating Scale（BMS）was used for evaluating motor function of the hind limbs of the experimental mice.We evaluated the recovery of spontaneous urination through measuring the bladder diameter of mice.Immunohistochemical staining was used to detect the expression level of apoptosis-related gene cleaved caspase3 and cell proliferation marker Ki-67.Iimmunofluorescence staining was used to test the expression of nestin in the lesion area,western blot was used to detect the expression of Sox2 and fibronectin,and dot blot hybridization was used to detect the expression of NG2.【Results】The recombinant plasmid pKAd5f11p-Syn was successfully constructed and the recombinant adenovirus Ad-OIC was obtained.When cocultureed with HT22,compared with the control group,DPSC-Null-CM and DPSC-OIC-CM could promote the proliferation of HT-22 cells,up-regulate the expression level of Tubb3 and Syn1 genes,increase the length of HT-22 axon,and reduce the proportion of apoptotic HT-22 cells under the condition of H2O2 induction.DPSC-OIC-CM perfomed better than DPSC-Null-CM.The up-regulation of Tubb3 and Syn1 gene expression and the increase of axon length were statistically significant differences between DPSC-OIC-CM and DPSC-Null-CM.The effect of promoting the proliferation of HT-22 cells and protecting the apoptosis induced by H2O2 of DPSC-OIC-CM better than that of DPSC-Null-CM,but there had no statistically significance.We established SCI model successfully and verified that the transplanted human DPSC could survive in the spinal cord of mice for about two weeks.Compared with PBS group,DPSC-Null and DPSC-OIC treatment could improve the spinal cord kyphosis,reduce the bleeding and edema,make the tissue structure more complete,and improve the BMI score of SCI mice.DPSC-Null and DPSC-OIC could also reduce the bladder diameter of SCI mice to a certain extent,down-regulate the expression of cleaved caspase-3 in spinal cord tissue,and up-regulate the expression of Ki-67.The expression level of nestin,Sox2 and fibronectin in the lesion area were increased,while the expression level of NG2 was decreased in DPSC-Null and DPSC-OIC group.Compared with DPSC-Null,DPSC-OIC could markedly improve the spinal cord kyphosis and improve the BMS score of SCI mice,and the difference between the two groups had statistical significance.The better effect of down-regulating the expression of cleaved caspase3 and up-regulating the expression of nestin and Sox2 was seen in DPSC-OIC group than that of DPSC-Null group,but there was no significant difference two groups.The effects of DPSC-Null and DPSC-OIC treatment on reducing the bladder diameter of SCI mice,up-regulating the expression of Ki-67 and fibronectin in the lesion,and down-regulating the expression of NG2 were similar.【Conclusion】DPSC-OIC could promote HT-22 cell proliferation,up-regulate the expression of Tubb3 and Syn1 genes,increase the length of HT-22 axon,and protect HT-22 cell apoptosis induced by H2O2.Transplantation of DPSC-OIC in situ could promote the recovery of motor function and spinal structure,enhance cell proliferation and regeneration-promotive substance production,and reduce cell apoptosis and prohibitive factor secretion.Therefore,DPSC-OIC could be a potential therapy for SCI treatment.