Objective:To explore the mechanism of DEC2 deficiency promoting high incidence of post infectious cough(PIC)and the therapeutic effects of ephedrine hydrochloride(EH).Methods:C57BL/6 wild-type(WT)mice and DEC2-knockout(DEC2-KO)mice were treated with lipopolysaccharide(LPS)nose drops and smoke stimulation to establish PIC models,and some model mice were treated with EH intervention.Counting capsaicin-induced cough at 12,14,16 and 17 day of modeling;Single-cell sequenc-ing,flow cytometry,RT-qPCR and other techniques were used to detect the expressions of IL-1,IL-6,IFN-γ and other inflammatory factors and receptors in the lungs of mice before and after PIC modeling and EH intervention.The expressions of inflammatory factors and their receptors,as well as NF-κB signaling molecules,were analyzed by RT-qPCR using mouse lung CD45-primary cell culture system under LPS stimulation or EH intervention.Results:The transcription of DEC2 was increased in WT-PIC model mice,and the deficiency of DEC2 in DEC2-KO-PIC mice induced the cough frequency 2-fold higher than that in WT-PIC mice.The absence of DEC2 exacerbated LPS-induced pulmonary inflammatory response,especially increased expressions of IL-1,IL-6,IL-1Ra,IL-6Ra,IFN-γR in lung CD45-cells(P<0.01),and excessive activation of NF-κB signal(P<0.01).After EH treatment,the cough frequency in DEC2-KO-PIC mice was significantly reduced(P<0.001),the expression of IL-1,IL-6,IFN-γ in lung and IL-1Ra,IL-6Ra,IFN-γR in pulmonary CD45-cells were down-regulated(P<0.01).The expression and activation of NF-κB induced by DEC2 deletion were sig-nificantly inhibited(P<0.01).Conclusion:The deficiency of DEC2 can activate NF-κB signal and promote the high expression of in-flammatory cytokine and receptors in pulmonary CD45-cells,which is a risk factor in the high incidence of PIC.EH can inhibit the ex-cessive-activation of NF-κB signal and the inflammatory characteristics of lung local stromal cells caused by DEC2 absence,and that is the main part of pharmacological mechanism for effective treatment of EH targeting PIC.

Objective:To explore the mechanism of DEC2 deficiency promoting high incidence of post infectious cough(PIC)and the therapeutic effects of ephedrine hydrochloride(EH).Methods:C57BL/6 wild-type(WT)mice and DEC2-knockout(DEC2-KO)mice were treated with lipopolysaccharide(LPS)nose drops and smoke stimulation to establish PIC models,and some model mice were treated with EH intervention.Counting capsaicin-induced cough at 12,14,16 and 17 day of modeling;Single-cell sequenc-ing,flow cytometry,RT-qPCR and other techniques were used to detect the expressions of IL-1,IL-6,IFN-γ and other inflammatory factors and receptors in the lungs of mice before and after PIC modeling and EH intervention.The expressions of inflammatory factors and their receptors,as well as NF-κB signaling molecules,were analyzed by RT-qPCR using mouse lung CD45-primary cell culture system under LPS stimulation or EH intervention.Results:The transcription of DEC2 was increased in WT-PIC model mice,and the deficiency of DEC2 in DEC2-KO-PIC mice induced the cough frequency 2-fold higher than that in WT-PIC mice.The absence of DEC2 exacerbated LPS-induced pulmonary inflammatory response,especially increased expressions of IL-1,IL-6,IL-1Ra,IL-6Ra,IFN-γR in lung CD45-cells(P<0.01),and excessive activation of NF-κB signal(P<0.01).After EH treatment,the cough frequency in DEC2-KO-PIC mice was significantly reduced(P<0.001),the expression of IL-1,IL-6,IFN-γ in lung and IL-1Ra,IL-6Ra,IFN-γR in pulmonary CD45-cells were down-regulated(P<0.01).The expression and activation of NF-κB induced by DEC2 deletion were sig-nificantly inhibited(P<0.01).Conclusion:The deficiency of DEC2 can activate NF-κB signal and promote the high expression of in-flammatory cytokine and receptors in pulmonary CD45-cells,which is a risk factor in the high incidence of PIC.EH can inhibit the ex-cessive-activation of NF-κB signal and the inflammatory characteristics of lung local stromal cells caused by DEC2 absence,and that is the main part of pharmacological mechanism for effective treatment of EH targeting PIC.

Objective:To investigate mechanisms of IL-17D on cytotoxic function of CD93+CTL in lung tumor microenviron-ment(TME)and promotion of Platycodon grandiflorum(PG).Methods:Lung of B16 melanoma model mice and control mice were treated with PG or IL-17D,and lung tumor clone formation was observed.IL-17D expression change in lung was detected by single cell sequencing,immunofluorescence staining and flow cytometry.Single cell sequencing and flow cytometry were used to detect CD93+CTL content,cytotoxic phenotype and functional factors changes,including CD107a,perforin,granzyme B,chemokine CCL2,CXCL9 and their ligand CCR2 and CXCR3.Western blot and RT-PCR were used to detect effect of Platycodonopsis saponin D on IL-17D and its transcriptional regulator NRF2 expressions in EL4 cells.Results:Pulmonary CD93+CTL highly expressed cytotoxic effectors such as perforin and CD107a and chemokine receptors CXCR3 and CCR2 than CD93-CTL,while there was no significant difference in secretion of granzyme B.In mouse lung tumor model,pulmonary IL-17D and CD93+CTL were significantly decreased(P<0.001);in tumor-bearing mice after IL-17D backfill assay,or after 10 days of treatment with PG,proportion and absolute number of IL-17D and CD93+CTL in lungs were significantly increased(P<0.05),and tumor clones were significantly reduced;meanwhile,tumor-local expressions of cytokines CCL2,CXCL9,which are related to recruitment and function of CD93+CTL,and IL-17D were significantly upregulated.Up-regulation of IL-17D and its transcriptional regulator NRF2 by PG was verified in vitro experiments on EL4 cell line by PD.Conclusion:Traditional Chinese medicine PG and its extracts can up-regulate expression of IL-17D in lungs,improve infiltration and cytotoxic function of CD93+CTL and antagonize malignant progression of lung tumors,this is an important phar-macological mechanism of PG in improving immune TME of tumors in lung.

Objective:To investigate the key mechanism of transforming growth factor-β (TGF-β) inducing the expression of interleukin-17D (IL-17D) in lung cancer-associated fibroblast (CAF) and promoting the recruitment of myeloid-derived suppressor cells (MDSCs).Methods:C57BL/6 mice were established for B16 lung melanoma metastasis model (tumor model group), and control group was set up, 6 mice in each group. Flow cytometry (FACS) was used to detect the lung CAF and the changes of its ability to secrete IL-17D and the proportion of MDSCs in tumor mice. The changes of TGF-β level in lung tumor were examined by ELISA and quantitative real-time PCR (RT-qPCR). Lung fibroblasts were screened by FACS, and the effects of TGF-β on the secretion of IL-17D, C-C motif chemokine ligand (CCL)2 and CCL7 in fibroblasts were detected by RT-PCR. The migration of MDSCs under the condition of TGF-β stimulating fibroblasts was detected by Transwell.Results:The proportion of CAF (CD45 -CD326 -CD31 -) in the tumor model group was higher than that in the control group [(28.02±2.23)% vs. (7.35±2.14)%, t=9.956, P<0.001]. The ability of CAF to secrete IL-17D in the tumor model group was significantly higher than that in the control group [(38.27±2.93)% vs. (19.04±3.16)%, t=5.995, P=0.001]. The proportion of MDSCs in the tumor model group was significantly higher than that in the control group [(12.93±1.27)% vs. (8.21±1.40)%, t=4.804, P=0.009]. Compared with the control group, the protein and transcription levels of TGF-β in lung of the tumor model group were significantly increased [(1 685.07±135.61) ng/L vs. (1 047.98±68.50) ng/L, t=5.051, P=0.002; 2.17±0.03 vs. 1.00±0.05, t=51.237, P<0.001]. In vitro, lung fibroblasts were stimulated with different concentrations of TGF-β (0, 5 and 10 μg/L) for 24 hours, the relative expressions of IL-17D mRNA secreted by stimulated fibroblasts were 0.42±0.01, 0.67±0.01 and 0.84±0.04 respectively, the relative expressions of CCL2 mRNA in each group were 0.89±0.08, 1.08±0.04, 1.19±0.01 and CCL7 were 0.53±0.05, 0.65±0.04, 0.74±0.03 respectively. With the increase of TGF-β concentration, the expression levels of IL-17D, CCL2 and CCL7 in fibroblasts were significantly increased ( F=57.384, P<0.001; F=15.802, P=0.004; F=14.544, P=0.005). In addition, compared with the control group (0 μg/L TGF-β), fibroblasts treated with 10 μg/L TGF-β for 24 hours could promote the migration of MDSCs in spleen of tumor mice [(9.59±0.21)% vs. (2.14±0.24)%, t=6.585, P<0.001]. Conclusion:TGF-β can induce high expression of IL-17D in lung CAF, which is an important factor in promoting the expressions of CCL2 and CCL7 and the migration of MDSCs in tumor microenvironment.

OBJECTIVE: To explore the molecular etiology for a Chinese family with mitochondrial DNA depletion syndrome. METHODS: Genomic DNA was extracted from peripheral blood samples of the patient and her parents.Targeted capture and next-generation sequencing was carried out to detect potential variants. Suspected variant was validated by Sanger sequencing. RESULTS: A novel homozygous frameshift variant c.505_508delTATC was identified in the patient, for which both his mother and father were carriers. CONCLUSION The frameshift variant c.505_508delTATC probably underlies the mitochondrial DNA depletion syndrome in this patient. The result also enriched the variant spectrum of DGUOK gene.
Asian Continental Ancestry Group ; genetics ; DNA, Mitochondrial ; genetics ; Female ; Frameshift Mutation ; Humans ; Mutation ; Phosphotransferases (Alcohol Group Acceptor) ; genetics ; Syndrome

Objective To study the clinical efficacy of caffeine in premature infants receiving mechanical ventilation and related complications .Method From January 2014 to September 2016, preterm infants (28w≤GA<33w) treated with nasal continuous positive airway pressure (NCPAP) or conventional mechanical ventilation ( CMV ) in neonatal intensive care unit were studied .They were randomly assigned into the caffeine group and the control group .The caffeine group received caffeine when NCPAP ventilation was applied or adjusting to synchronized intermittent mandatory ventilation ( SIMV) mode.The control group was injected with 5%glucose without caffeine .The t test andχ2 test were used to analyze the clinical efficacy and related complications .Result A total of 96 patients receiving NCPAP ventilation were collected ( birth weight between 1300~2100 g), including 51 cases in caffeine group and 45 cases in the control group. 84 cases received CMV ventilation (birth weight between 1000~1499 g), with 43 cases in the caffeine group and 41 cases the control group.Among the NCPAP infants, the incidence of failure to withdraw ventilator (0% vs.13.3%) and the incidence of bronchopulmonary dysplasia (3.9% vs.17.8%) were lower in the caffeine group than the control group .The duration of assisted ventilation and hospital stay in the caffeine group were shorter than the control group [(6.2 ±3.1) d vs.(8.2 ±3.2) d, (16.3 ±8.7) d vs. (19.5 ±9.2) d], the differences were statistically significant (P<0.05).Among the CMV infants, the incidence of failure of A/C to SIMV mode transition and bronchopulmonary dysplasia were lower in the caffeine group than the control group and the duration of assisted ventilation and hospital stay were shorter . The differences were statistically significant ( P <0.05 ) . No differences were found in the related complications in each group ( P>0.05) . Conclusion Caffeine can help reduce the incidences of withdrawal failure, bronchopulmonary dysplasia , ventilation duration and hospital stay when using NCPAP and CMV ventilation.

Aim To investigate the protective effect of cinnamic aldehyde ( CA ) on hormone-induced osteo-clasts proliferation and bone resorption in vitro and its molecular mechanisms. Methods RAW264. 7 cells induced into osteoclast were treated with RANKL and M-CSF and then were divided into control group, dexa-methasone ( DEX ) group and different doses of CA (11. 6, 23. 2, 46. 4 μg·L-1 ) groups. OCs were ob-served after tartrate resistant acid phosphatase( TRAP) staining. The cell proliferation was determined by MTT assay at different time points. The expression levels of TRACP5 b in cell cultured supernatants were measured by ELISA. RT-PCR technique was applied to examine the transcriptional levels of RANK and NFATc1 . Re-sults In MTT assay, the proliferation of osteoclasts stimulated by dexamethasone was promoted seriously compared with negative control group ( P < 0. 05 ) . Meanwhile, DEX could strengthen the content of TRACP5 b and up-regulate the expressions of RANK and NFATc1 mRNA. After administration of CA, the proliferation was inhibited, while the enhanced expres-sion of TRAP5b was reversed,and the over-expressions of RANK and NFATc1 mRNA were obviously down-regulated in a time-and-dose-dependent manner ( P <0. 05 ) . Conclusion The results suggest that CA in-hibits proliferation and bone resorption of osteoclast in-duced by DEX, which may be mediated by down-regu-lation of RANK and NFATc1 mRNA.

Objective:To explore the characteristics of immunological reconstitution of T-cell subsets and the role of Cornus,a Chinese medicinal herb on T lymphocytes in mice after irradiation (IR). Methods:Irradiated model mice were exposed to a single dose of X-ray radiation (2. 6 Gy) with or without Cornus treatments. Bloodroutine was examined before or after irradiation. CD3+,CD4+, CD8+ T cells and Th1, Tc1, Th2, Th17, Treg from spleen or peripheral blood were determined by flow cytometry. Results: Total lymphocytes and CD3+T cells including CD4+T and CD8+T cells,were significantly reduced 3 days after irradiation (P<0. 05). The re-constitution of CD3+ T cells ( especially CD8+T cells ) started from 5 days post irradiation, CD4+T cells increased 8 days after irradiation. However,the production of IFN-γ by Th1 or Tc1 cells were evidently decreased compared with control group even 8 days post irradiation (P<0. 05). In contrast to Th1,Th2/Th17/Treg were observably increased in irradiation group (P<0. 05). But the percentage of Th1 was obviously increased, and Th2, Th17, Treg were markedly decreased in cornus treated mice compared with irradiated mice (P<0. 05). Conclusion: The immunological reconstitution of T lymphocytes started from 5 days after total irradiation with rapid recovery of CD8+ T cells but not CD4+T cells. Cornus effectively improved the imbalance of T cell subsets by promoting the proliferation of Th1 and suppressing Th17 and Treg.

Objective:To investigate the role of dendritic cells(DC) in unexplained recurrent spontaneous abortion(URSA) and to study the effect and molecular mechanism of Traditional Chinese Medicine Shou Tai Decoction ( STD ) on URSA treatment by regulating the function of DC.Methods:30 cases of normal pregnancy women and 30 cases of URSA patients were taken as control group and URSA group respectively.URSA patients were treated with Shou Tai Decoction.Peripheral blood mononuclear cells were taken from both control group and URSA group before and after STD administration.The proportion of CD11c+HLA-DR+cells,CD11c+CD80+cells and CD11c+CD86+cells in peripheral blood were measured by flow cytometry.Moreover,the mRNA expression of HLA-DR,CD80,CD86 and Indoleamine2,3-dioxygenase( IDO) in venous blood were detected by RT-PCR assay.The protein expression of IDO was detected by Western blot.Furthermore, the cytokines, including IL-12p70 and IL-6, in the blood serum were measured by ELISA.Results:Compared with normal pregnancy women,the proportion of CD11c+HLA-DR+,CD11c+CD80+,CD11c+CD86+cells and the mRNA expression of HLA-DR,CD80,CD86 of URSA patients in peripheral blood were both increased significantly(P<0.05), while the mRNA and protein expression of IDO were decreased markedly(P<0.05).Additionally,the level of IL-12p70 and IL-6 in serum of URSA women were significantly increased ( P<0.01 ) .When compared with URSA patients before STD administration, the proportion of CD11c+HLA-DR+,CD11c+CD80+,CD11c+CD86+cells and the mRNA expression of HLA-DR,CD80,CD86 decreased significantly after STD administration ( P<0.05 ) , while the mRNA and protein expression of IDO increased markedly after STD administration(P<0.05).Meanwhile,compared with before STD administration,serum protein level of IL-12p70 and IL-6 of URSA patients decreased significantly after STD treatment ( P<0.01 ) .Conclusion: The changes of proportion and function of DC were involved in URSA.The regulatory effect of STD on DC proportion and function contribute to the treatment of URSA.

Objective:To demonstrate the relationship between the Th 1/Th2/Th17/Treg paradigm and the bone loss induced by estrogen deficiency and looking for potential target for clinical treatment.Methods:30 BALB/c mice were divided randomly into the normal control group , the sham operation group , and the ovariectomy group.The serum estradiol ( E2 ) was assessed by ELISA.Bone mineral density (BMD) of thigh bone was measured with dual energy X ray absorptiometry.Meanwhile,the T-cell subsets (Th1:CD4+TNFα+, Th2: CD4+IL-4+, Th17: CD4+IL-17 A+, Treg: CD4+CD25+Foxp3+) in spleen lymphocytes were analyzed by flow cytometry.Results:Compared with the normal group and the sham operation group , both E2 and BMD in the ovariectomy group decreased significantly ( P<0.05 ).The percentage of Th 1 and Th17 subset increased while the percentage of Th 2 and Treg decreased significantly in ovariectomy mice compared with sham operation mice.Correlation analysis showed that BMD was positively related to E 2 level and the percentage of Th 2 and Treg subset;however ,BMD was negatively related to the percentage of Th 1 and Th17 subset ( P<0.05 ).Conclusion: Conclusion: T-cell paradigm was involved in the bone loss induced by estrogen deficiency.Modifying T-cell paradigm may become a potential target for reducing bone loss induced by estrogen deficiency .