1.Effect of Huangqin Qingre Chubi Capsules-containing Serum on CircRNA_0001543/NF-κB Expression in Co-cultured PBMCs and Human FLSs from Patients with Ankylosing Spondylitis
Yajun QI ; Jian LIU ; Qiao ZHOU ; Yuedi HU ; Xiang DING ; Chengzhi CONG ; Xu LI
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(15):87-95
ObjectiveThis study aims to explore the effects of Huangqin Qingre Chubi capsules-containing serum on the expression of CircRNA_0001543/nuclear factor-kappa B (NF-κB) in co-cultured peripheral blood mononuclear cells (PBMCs) and human fibroblast-like synoviocytes (FLSs) from patients with ankylosing spondylitis (AS). MethodsVenous blood was collected from patients with AS to isolate PBMCs. FLSs were co-cultured with AS patients' PBMCs, and FLSs were harvested after co-culture for subsequent experiments. The normal control group consisted of normal FLSs, while the model group comprised co-cultured AS PBMCs and FLSs to simulate AS pathology. The Huangqin Qingre Chubi capsules group involved adding Huangqin Qingre Chubi capsules-containing serum to the co-cultured cells(6.48 g·kg-1). To investigate the effect of HQC-containing serum on the viability of co-cultured cells, and the experiment was divided into the following groups based on the dilution concentration: blank group, 10% HQC group, 20% HQC group, and 30% HQC group.To study the influence of the optimal concentration of HQC-containing serum on cytokine and pathway indicators in each group, the experiment was divided into three groups: normal group, model group, and optimal concentration HQC-containing serum group.For the validation of the transfection efficiency of the CircRNA_0001543 interference plasmid, the experiment was divided into the following groups: blank group, si-NC group (with transfection reagent), si-circ_0001543-1 group (with transfection reagent and interference plasmid No. 1 targeting circ_0001543), si-circ_0001543-2 group (with transfection reagent and interference plasmid No. 2 targeting circ_0001543), and si-circ_0001543-3 group (with transfection reagent and interference plasmid No. 3 targeting circ_0001543).For the validation of the transfection efficiency of the CircRNA_0001543 overexpression plasmid, the experiment was divided into the following groups: blank group, OE-NC group (with transfection reagent), and OE-circ_0001543 group (with transfection reagent and overexpression plasmid targeting circ_0001543).To study the effects of CircRNA_0001543 interference/overexpression on cytokine and pathway indicators in each group, the experiment was divided into the following groups: si-NC group, si-CircRNA_0001543 group, OE-NC group, and OE-CircRNA_0001543 group. Enzyme-linked immunosorbent assay (ELISA) was used to detect levels of interleukin-1β (IL-1β), IL-10, IL-37, and tumor necrosis factor-α (TNF-α). Real-time quantitative polymerase chain reaction (Real-time PCR) was utilized to measure the expression of CircRNA_0001543, IκBα, and NF-κB p65. ResultsAfter 48 hours, 30% Huangqin Qingre Chubi Capsules-containing serum significantly inhibited the proliferation of co-cultured PBMCs and FLSs, which was determined to be the optimal experimental drug-containing serum concentration. Compared with those in the normal group, the expressions of NF-κB p65 mRNA, IκBα mRNA, IL-1β, and TNF-α in the model group were significantly increased (P<0.01), while the expressions of CircRNA_0001543 mRNA, IL-10, and IL-37 were significantly decreased (P<0.01). Compared with those in the model group, the expressions of NF-κB p65 mRNA, IκBα mRNA, IL-1β, and TNF-α in the Huangqin Qingre Chubi Capsules-containing serum group were significantly decreased (P<0.05), and the expressions of CircRNA_0001543 mRNA, IL-10, and IL-37 were significantly increased (P<0.05), with the most prominent changes in the 30% drug-containing serum group (P<0.01). Compared with that in the si-NC group, the expression of CircRNA_0001543 was significantly reduced in the si-CircRNA_0001543 group (P<0.01). Compared with that in the OE-NC group, the expression of CircRNA_0001543 was significantly increased in the OE-CircRNA_0001543 group (P<0.01), indicating that the si-CircRNA_0001543 and OE-CircRNA_0001543 plasmids were successfully transfected. Based on the optimal drug-containing serum of Huangqin Qingre Chubi Capsules, si-CircRNA_0001543 transfection led to significantly increased expressions of NF-κB p65 mRNA, IκBα mRNA, IL-1β, and TNF-α and decreased the expressions of IL-10 and IL-37 (P<0.01). In contrast, OE-CircRNA_0001543 transfection significantly decreased the expressions of NF-κB p65 mRNA, IκBα mRNA, IL-1β, and TNF-α (P<0.01) and increased the expressions of IL-10 and IL-37 (P<0.01). ConclusionHuangqin Qingre Chubi capsules-containing serum can improve immune inflammation in AS by increasing the expression of CircRNA_0001543, regulating the NF-κB pathway, suppressing pro-inflammatory cytokines, and enhancing anti-inflammatory cytokine expression.
2.Clinical efficacy of induction chemoimmunotherapy for locally advanced hypopharyngeal carcinoma: a prospective phase Ⅱ study
Hongli GONG ; Shu TIAN ; Hao DING ; Lei TAO ; Li WANG ; Jie WANG ; Tian WANG ; Ming ZHANG ; Yong SHI ; Chengzhi XU ; Chunping WU ; Shengzi WANG ; Liang ZHOU
Chinese Journal of Otorhinolaryngology Head and Neck Surgery 2024;59(4):350-356
Objective:To evaluate the objective response rate (ORR) of induction chemoimmunotherapy with camrelizumab plus TPF (docetaxel, cisplatin, and capecitabine) for locally advanced hypopharyngeal squamous cell carcinoma (LA HSCC) and potential predictive factors for ORR.Methods:A single-center, prospective, phase 2 and single-arm trial was conducted for evaluating antitumor activity of camrelizumab+TPF(docetaxel+cisplatin+capecitabine) for LA HSCC between May 21, 2021 and April 15, 2023, patients admitted to the Eye & ENT Hospital affiliated with Fudan University. The primary endpoint was ORR, and enrolled patients with LA HSCC at T3-4N0-3M0 received induction chemoimmunotherapy for three cycles: camrelizumab 200 mg day 1, docetaxel 75 mg/m 2 day 1, cisplatin 25 mg/m 2 days 1-3, and capecitabine 800 mg/m 2 days 1-14. Patients were assigned to radioimmunotherapy when they had complete response or partial response (PR)>70% (Group A), or assigned to surgery plus adjuvant radiotherapy/chemoradiotherapy when they had PR≤70% (Group B), and the responses were defined by using tumor volume evaluation system. Tumor diameter was also used to assess the treatment responses by Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1. Use SPSS 23.0 software was used to analyze the data. Results:A total of 51 patients were enrolled who underwent the induced chemoimmunotherapy for three cycles, and all were males, aged 35-69 years old. After three cycles of induction immunochemotherapy, 42 (82.4%) patients existed in Group A (complete response or PR>70%) and 9 patients (17.6%) in Group B (PR≤70%), the ORR was 82.4%. The primary endpoint achieved expected main research objectives. Compared to the patients of Group A, the patients of Group B showed the higher T stage and the larger volume of primary tumor before induced immunochemotherapy, and also had the less regression of tumor volume after induced immunochemotherapy (all P<0.05). The optimal cutoff value of pre-treatment tumor volume for predicting ORR was 39 cm 3. The T stage ( OR=12.71, 95% CI: 1.4-112.5, P=0.022) and the volume ( OR=7.1, 95% CI: 1.4-36.8, P=0.018) of primary tumor were the two main factors affecting ORR rate of induction chemoimmunotherapy. Conclusion:The induction chemoimmunotherapy with camrelizumab plus TPF shows an encouraging antitumor efficacy in LA HSCC.
3.circDDX17 targets miR-223-3p / RIP3 to regulate the proliferation and apoptosis of non-small cell lung cancer cells
Chengzhi DING ; Guolei WANG ; Gongqian JIANG ; Hongtao WANG ; Yuanyuan LIU ; Haoliang ZHANG ; Fang SUN ; Li WEI
Chinese Journal of Oncology 2024;46(3):239-248
Objective:To explore the molecular mechanism of circDDX17 regulating the proliferation and apoptosis of non-small cell lung cancer cells by targeting the miR-223-3p/RIP3 molecular axis.Methods:The expression levels of circDDX17, miR-223-3p, and RIP3 in human normal lung epithelial cell lines BEAS-2B and non-small cell lung cancer cells H1299, A549, and H446 were detected by reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR). The plasmids of pcDNA, pcDNA-circDDX17, anti-miR-con, anti-miR-223-3p, pcDNA-circDDX17 and miR-con, pcDNA-circDDX17 and miR-223-3p mimics were transfected into H1299 cells. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide (MTT) assay was used to detect the cell proliferation. Flow cytometry was used to detect the cell cycle and cell apoptosis. Plate cloning experiment was used to detect cell proliferation ability. The dual luciferase report experiment was applied to verify the targeting relationship between miR-223-3p with circDDX17 and RIP3. Western blot was used to detect the protein expression of cyclinD1, CDK2, cleaved caspase-3 and Bax.Results:The expression levels of circDDX17 and RIP3 mRNA in H1299, A549, and H446 cells were significantly reduced ( P<0.05), the expression level of miR-223-3p mRNA was significantly increased ( P<0.05) compared with BEAS-2B. The cell viability [(69.46±4.68)%], the number of cell clones (83.49±7.86), the proportion of cells in S phase [(22.52±1.41) %], the protein expression levels of cyclinD1 and CDK2 in PCDNa-CircDDX17 group were lower than those in pcDNA group [(97.54±7.72)%, 205.03±13.37, (28.69±1.49)%, respectively, P<0.05], while the percentage of G 0/G 1 phase cells [(64.45±3.56)%], apoptosis rate [(18.36±1.63)%], the protein expression levels of cleaved caspase-3 and Bax in pcDNA-circDDX17 group were higher than those of pcDNA group [(51.33±2.76) % and (5.21±0.54) %, respectively, P<0.05]. The viability [(72.64±5.44)%], the number of cell clones (78.16±8.23), the proportion of S-stage cells [(21.34±1.59) %], the protein expression levels of CyclinD1 and CDK2 in anti-miR-223-3p group were lower than those in anti-miR-con group [(103.47±6.25)%, 169.32±14.53, (28.43±1.26)%, respectively, P<0.05]. Percentage of G 0/G 1 phase cells [(62.86±3.28)%], apoptosis rate [(14.64±1.67)%], the protein expression levels of cleaved caspase-3 and Bax in the anti-miR-223-3p group were higher than those of anti-miR-con group [(51.33±2.71)% and (4.83±0.39)%, respectively, P<0.05]. MiR-223-3p has complementary sites with circDDX17 or RIP3. The viability [(135.45±9.28)%], the number of cell clones (174.64±10.68), the proportion of S-phase cells [(26.39±2.25)%], the protein expression levels of cyclinD1 and CDK2 in pcDNA-circDDX17+miR-223-3p group were higher than those in pcDNA-circDDX17+miR-con group [(101.56±6.68)%, 107.65±7.62, (21.64±1.72)%, P<0.05]. Percentage of G 0/G 1 phase cells [(56.64±2.76)%], apoptosis rate [(8.34±0.76)%], the protein expression levels of cleaved caspase-3 and Bax in pcDNA-circDDX17+miR-223-3p group were lower than those of pcDNA-circDDX17+miR-con group [(64.03±3.48)% and (15.21±1.18)%, respectively, P<0.05]. Conclusion:circDDX17 could inhibit the proliferation and induce apoptosis of non-small cell lung cancer cells via targeting the miR-223-3p / RIP3 molecular axis.
4.circDDX17 targets miR-223-3p / RIP3 to regulate the proliferation and apoptosis of non-small cell lung cancer cells
Chengzhi DING ; Guolei WANG ; Gongqian JIANG ; Hongtao WANG ; Yuanyuan LIU ; Haoliang ZHANG ; Fang SUN ; Li WEI
Chinese Journal of Oncology 2024;46(3):239-248
Objective:To explore the molecular mechanism of circDDX17 regulating the proliferation and apoptosis of non-small cell lung cancer cells by targeting the miR-223-3p/RIP3 molecular axis.Methods:The expression levels of circDDX17, miR-223-3p, and RIP3 in human normal lung epithelial cell lines BEAS-2B and non-small cell lung cancer cells H1299, A549, and H446 were detected by reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR). The plasmids of pcDNA, pcDNA-circDDX17, anti-miR-con, anti-miR-223-3p, pcDNA-circDDX17 and miR-con, pcDNA-circDDX17 and miR-223-3p mimics were transfected into H1299 cells. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide (MTT) assay was used to detect the cell proliferation. Flow cytometry was used to detect the cell cycle and cell apoptosis. Plate cloning experiment was used to detect cell proliferation ability. The dual luciferase report experiment was applied to verify the targeting relationship between miR-223-3p with circDDX17 and RIP3. Western blot was used to detect the protein expression of cyclinD1, CDK2, cleaved caspase-3 and Bax.Results:The expression levels of circDDX17 and RIP3 mRNA in H1299, A549, and H446 cells were significantly reduced ( P<0.05), the expression level of miR-223-3p mRNA was significantly increased ( P<0.05) compared with BEAS-2B. The cell viability [(69.46±4.68)%], the number of cell clones (83.49±7.86), the proportion of cells in S phase [(22.52±1.41) %], the protein expression levels of cyclinD1 and CDK2 in PCDNa-CircDDX17 group were lower than those in pcDNA group [(97.54±7.72)%, 205.03±13.37, (28.69±1.49)%, respectively, P<0.05], while the percentage of G 0/G 1 phase cells [(64.45±3.56)%], apoptosis rate [(18.36±1.63)%], the protein expression levels of cleaved caspase-3 and Bax in pcDNA-circDDX17 group were higher than those of pcDNA group [(51.33±2.76) % and (5.21±0.54) %, respectively, P<0.05]. The viability [(72.64±5.44)%], the number of cell clones (78.16±8.23), the proportion of S-stage cells [(21.34±1.59) %], the protein expression levels of CyclinD1 and CDK2 in anti-miR-223-3p group were lower than those in anti-miR-con group [(103.47±6.25)%, 169.32±14.53, (28.43±1.26)%, respectively, P<0.05]. Percentage of G 0/G 1 phase cells [(62.86±3.28)%], apoptosis rate [(14.64±1.67)%], the protein expression levels of cleaved caspase-3 and Bax in the anti-miR-223-3p group were higher than those of anti-miR-con group [(51.33±2.71)% and (4.83±0.39)%, respectively, P<0.05]. MiR-223-3p has complementary sites with circDDX17 or RIP3. The viability [(135.45±9.28)%], the number of cell clones (174.64±10.68), the proportion of S-phase cells [(26.39±2.25)%], the protein expression levels of cyclinD1 and CDK2 in pcDNA-circDDX17+miR-223-3p group were higher than those in pcDNA-circDDX17+miR-con group [(101.56±6.68)%, 107.65±7.62, (21.64±1.72)%, P<0.05]. Percentage of G 0/G 1 phase cells [(56.64±2.76)%], apoptosis rate [(8.34±0.76)%], the protein expression levels of cleaved caspase-3 and Bax in pcDNA-circDDX17+miR-223-3p group were lower than those of pcDNA-circDDX17+miR-con group [(64.03±3.48)% and (15.21±1.18)%, respectively, P<0.05]. Conclusion:circDDX17 could inhibit the proliferation and induce apoptosis of non-small cell lung cancer cells via targeting the miR-223-3p / RIP3 molecular axis.
5.Effects of health belief model in prevention of lower extremity deep vein thrombosis in patients with lung cancer after operative
Jieling LIU ; Luyu CHEN ; Chengzhi DING
Chinese Journal of Modern Nursing 2023;29(2):257-261
Objective:To explore the effect of health belief model in the prevention of lower extremity deep vein thrombosis (DVT) in patients with lung cancer after operative.Methods:From January 2019 to June 2020, 80 lung cancer patients admitted to Henan Provincial Chest Hospital were selected as research objects by convenience sampling method. The patients were randomly divided into control group and research group with 40 cases each. The routine health education was used in the control group, and the health belief model was used in the research group on the basis of the control group. Both groups intervened until discharge. We compared the DVT prevention knowledge and health belief of the two groups of patients before and after the intervention, and counted the incidence of DVT in the two groups of patients.Results:After intervention, the scores of all dimensions of the DVT Health Questionnaire and DVT Health Belief Questionnaire in the research group were higher than those in the control group, with statistically significant differences ( P<0.05) . The incidence of DVT in the research group was lower than that in the control group with a statistical difference ( P<0.05) . Conclusions:The health belief model can improve the health knowledge and health belief of lung cancer patients to prevent DVT, reduce the occurrence of DVT, and is worthy of clinical promotion and application.
6.Effect of electroacupuncture at Baihui ameliorated neurologic deficit and hemodynamic stability in rat model of post-cardiac arrest syndrome
Ruifeng ZENG ; Yuejia ZHONG ; Yanhua WU ; Peng WANG ; Chengzhi LAI ; Xiangqi LIU ; Yunbiao DUAN ; Zunjiang LI ; Tao YU ; Banghan DING
Chinese Critical Care Medicine 2022;34(12):1285-1290
Objective:To observe the results of electroacupuncture (EA) on the resuscitation of a rat model of asphyxia cardiac arrest (CA). And to explore its effect on the neurologic deficits and hemodynamic instability of post-cardiac arrest syndrome (PCAS).Methods:A total of 107 male SD rats were randomly divided into sham, CA, and EA groups. Each group received arterial catheterization and tracheal intubation. The sham group was not induced asphyxia. Asphyxial cardiac arrest was established by endotracheal tube clamping. Rats in the CA group received basic respiratory support and fluid resuscitation in return of spontaneous circulation (ROSC) and rats in the EA group received EA at Baihui based on the treatment of CA group after ROSC, with a dense-dispersed wave at frequencies of 4-20 Hz, while the current intensity was adjusted minimum to induce a twitch of the scalp, the course of treatment was 30 minutes. The baseline data, hemodynamics after ROSC, neurological deficit score (NDS), pathological changes of brain tissue, and levels of serum biomarker were recorded and compared among the three groups. The 72-hour survival of rats was analyzed by Kaplan-Meier survival curve. Hematoxylin-eosin (HE) staining was used to observe the pathological changes of necrotic neurons in the hippocampal CA1 region of rat brain. Meanwhile, Nissl staining and TdT-mediated dUTP nick-end labeling (TUNEL) were used to detect cell apoptosis and injury.Results:Compared with the CA group, the mean arterial pressure (MAP) in the EA group increased significantly at 15 minutes after ROSC [mmHg (1 mmHg≈0.133 kPa): 125.00 (94.00, 136.25) vs. 92.00 (72.00, 122.50), P < 0.05]. There was no significant difference in the NDS score between the EA group and the sham group. Still, the NDS score of the rats in the CA group at 6 hours after ROSC were significantly lower than that in the sham group (46.00±10.61 vs. 80.00±0.00, P < 0.05). Kaplan-Meier survival curve analysis showed that EA did not improve the 72-hour survival rate of rats (100% in the sham group, 25% in the CA group, and 30% in the EA group, P > 0.05). The analysis by TUNEL showed that the apoptosis rate of neurons in CA1 region of the hippocampus in EA group at 6 hours after ROSC was significantly lower than that in CA group [(62.84±2.67)% vs. (71.29±3.70)%, P < 0.05]. Compared with the CA group, the level of serum S100 calcium binding protein B (S100B) in the EA group at 6 hours after ROSC was significantly lower (ng/L: 19.30±13.87 vs. 132.28±31.67, P < 0.05), but there were no significant differences in the levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) between these two groups. Conclusions:In the present study, EA at Baihui can stabilize the hemodynamic, moreover, it has a particular neuroprotective effect on PCAS rats. Still, EA at Baihui does not reduce the systemic inflammatory response and improve the survival rate of rats, and its mechanism remains to be verified in further research.
7.MicroRNA-155 reduces inflammatory response induced by lipopolysaccharide in alveolar macrophages
Wei PENG ; Ning ZHAO ; Qin LIU ; Cheng NIE ; Cheng QING ; Qiang SHAO ; Fen LIU ; Kejian QIAN ; Chengzhi DING
Chinese Critical Care Medicine 2018;30(11):1061-1065
Objective To observe the effect of microRNA-155 (miR-155) on the inflammatory response of rat alveolar macrophages induced by lipopolysaccharide (LPS). Methods The alveolar macrophages NR8383 of rat were cultured in vitro, the macrophages in logarithmic growth phase were harvested to conduct experiment. ① The 1 mg/L LPS was used to stimulate the rat alveolar macrophages for 3, 6, 12, and 24 hours, a phosphate buffer solution (PBS) control group was also set up. Enzyme linked immunosorbent assay (ELISA) was used to detect the dynamic changes of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the supernatant, and real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR) was used to detect the dynamics expression of miR-155 in the cells, which confirmed the optimal time for LPS stimulation was 12 hours. ② Carboxyfluorescein (FAM) labeled mimic (FAM mimic) and inhibitor (FAM inhibitor) were used to transfect the alveolar macrophage, and the transfection effect was observed under inverted fluorescence microscope 6 hours later to confirm the optimal transfection concentration of mimic was 20 nmol/L, and the optimal transfection concentration of inhibitor was 100 nmol/L. miR-155 mimic and miR-155 inhibitor were transfected to alveolar macrophages respectively at the optimal transfection concentration for 24 hours, and 1 mg/L LPS was used to stimulate the cells for 12 hours. A mimic negative control + LPS group and an inhibitor negative control + LPS group were set up. The expressions of IL-1β and TNF-α in the supernatant were determined by ELISA to observe the regulation of miR-155 on inflammatory response of alveolar macrophages. Results ① After stimulation of 1 mg/L LPS on alveolar macrophages, the contents of IL-1β and TNF-α in the supernatant and the expression of miR-155 in the cells were increased gradually with time prolongation, IL-1β and TNF-α contents peaked at 12 hours, and the expression of miR-155 peaked at 24 hours [as compared with PBS control group, IL-1β (ng/L): 910.43±36.09 vs. 22.66±7.84, TNF-α (ng/L): 3 138.39±394.10 vs. 233.92±8.84, miR-155 (2-ΔΔCt): 7.82±0.30 vs. 1, all P < 0.05]. ② Under inverted fluorescence microscope, after 20 nmol/L FAM mimic or 100 nmol/L FAM inhibitor transfected alveolar macrophages for 6 hours, a large number of cells showed green fluorescence, indicating that the transfection was successful. The expression of miR-155 in the cells transfected with 20 nmol/L miR-155 mimic was up-regulated by (236.73±46.49) times as much as that in the negative control group (P < 0.05), and the levels of IL-1β and TNF-α in the supernatant of the cells stimulated by 1 mg/L LPS for 12 hours were significantly lower than those in the negative control group [IL-1β (ng/L): 324.37±36.59 vs. 799.31±39.44, TNF-α (ng/L): 1 554.01±342.48 vs. 3 020.49±418.30, both P < 0.05]. The miR-155 activity was significantly inhibited in the cells transfected with 100 nmol/L miR-155 inhibitor, and the expression of miR-155 was decreased by (4.00±3.26)% as compared with the negative control group, but the difference was not statistically significant (P > 0.05), and the levels of IL-1β and TNF-α in the supernatant of the cells stimulated by 1 mg/L LPS for 12 hours were significantly higher than those in the negative control group [IL-1β (ng/L): 1 358.98±212.04 vs. 878.68±53.42, TNF-α (ng/L): 4 225.57±281.11 vs. 2 881.32±286.08, both P < 0.05]. Conclusion In LPS induced inflammatory response of alveolar macrophages, miR-155 plays an obvious inhibitory role.
8.The inflammatory effect of exosomes derived from alveolar epithelial cells stimulated by lipopolysaccharide on the alveolar macrophages
Chengzhi DING ; Wei PENG ; Yong LI ; Yun YANG ; Qiang SHAO ; Ning ZHAO ; Jiaquan CHEN ; Kejian QIAN ; Fen LIU
Chinese Journal of Emergency Medicine 2018;27(10):1126-1131
Objective To explore the inflammatory effect of exosomes derived from alveolar epithelial cells stimulated by lipopolysaccharide (LPS) on the alveolar macrophages (NR8383). Methods The alveolar epithelial cells disposed with different treatments were co-cultured with alveolar macrophages by using a Transwell system separately. Alveolar epithelial cells (RLE-6TN) were randomly divided into 4 groups: normal group, LPS-stimulated group, exosome inhibitor group, and exosome inhibitor pretreatment + LPS stimulation group. NR8383 cultured alone was considered as a blank control. After the 12-h co-culture, the real-time PCR (qPCR) was performed to examine the mRNA relative expression of IL-6, TNF-α, and IL-1β in NR8383 cells. To further explore the role of exosomes derived from RLE-6TN on alveolar macrophages mediated inflammationary response, the experimental exosomes (exosomes derived from LPS-induced RLE-6TN) and control exosomes exosomes derived from normal RLE-6TN were extracted by gradient ultracentrifugation. Transmission electron microscopy and Western blotting analyses was performed to identify the exosomes, and qNano particle diameter analyzer was conducted to measure the particle diameter of exosomes. In vitro, NR8383 cells were divided into 3 groups which were cultured with exosomes derived from LPS-stimulated RLE-6TN at a concentration of 10 μg/mL (experimental group), exosomes derived from untreated RLE-6TN at the same concentration of 10 μg/mL (control group), and the PBS at the same volume with experimental group (PBS group), respectively for 12 h. After the treatment, the phagocytosis of NR8383 cells was observed by laser confocal microscope and the release of interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α in supernatants of NR8383 was detected by enzyme-linked immunosorbent assay ELISA Results (1)In the co-culture experiment, the mRNA relative expression of pro-inflammatory cytokine in the LPS group was significantly increased compared with the blank control group (P<0.01), however comparing the exosome inhibitor pretreatment+LPS group with the LPS group, the expression of pro-inflammatory cytokine was decreased (P<0.01). (2) The extracted exosomes were observed as circular or elliptical vesicles with a diameter of 40-100 nm under the transmission electron microscopy. Western blotting analyses showed that the extracted exosomes express the protein marker, such as CD63 and CD9; After incubation with NR8383 cells for 5 h, laser scanning confocal microscope showed that the exosomes labeled with red fluorescent were uptaken by NR8383 cells. (3)After the exosomes derived from the LPS-disposed RLE-6TN and the normal RLE-6TN cells were incubated with NR8383 cells respectively. The ELISA test showed that treated the alveolar macrophages with LPS induced alveolar epithelial secreted exosomes led to a robustly increased release of pro-inflammatory cytokine (P<0.01), but there was no significant difference between the control group and PBS group (P>0.05). Conclusions Exosomes derived from LPS-disposed alveolar epithelial cells activate the alveolar macrophage-mediated inflammatory response.
9.Simultaneous esophagectomy and off-pump coronary artery bypass grafting for esophageal cancer:a report of five cases with literature review
Hongtao WANG ; Guolei WANG ; Wenguang WANG ; Yuhang CHEN ; Zhenjiang WU ; Chengzhi DING
Chinese Journal of Thoracic and Cardiovascular Surgery 2017;33(7):394-396
Objective To explore the possibility and safety of the simultaneous esophagectomy and off-pump coronary artery bypass grafting(OPCABG) in patients suffering from esophageal cancer combined with coronary artery and summarize the clinical experiences.Methods We retrospectively analyzed the clinical dates of 5 patients performed at the Henan province Chest Hospital from May 2009 to December 2014.The related literature was reviewed.Results All patients were performed the simultaneous esophagectomy and OPCABG through the single left posterolateral thoracotomy 4cases and through median sternotomy and left thoracotomy 1 case.Instrument anastomosis under aortic arch 2 cases and above aortic arch 1 case , left neck anastomosis by hand 2 cases.Coronary artery three ressel disease 3cases, double-vessel 1 case, left main single vessel 1 case.There was no hospital death in this series.Postoperative complications included arrhythmia,anastomotic fistula and pneumonia.Only one patient was still alive, the other patients died of tumor recurrence or metastasis and median survival time was 20.6 months.Conclusion Simultaneous esophagectomy and OPCABG is a safe and feasible treatment modality in patients with severe CAD and esophageal cancer and it may be more beneficial for the patient with early esophageal cancer.
10.A clinical study of lung protective ventilation and sequential lung recruitment maneuver for treatment of severe acute pancreatitis complicated with acute respiratory distress syndrome
Zhenguo ZENG ; Fei WANG ; Jianguo ZHANG ; Liang XIA ; Chengzhi DING ; Qiang SHAO ; Cheng QING ; Fen LIU ; Kejian QIAN
Chinese Journal of Integrated Traditional and Western Medicine in Intensive and Critical Care 2017;24(5):497-501
Objective To investigate the clinical therapeutic effects of lung protective ventilation and sequential recruitment maneuver (RM) on treatment of patients with severe acute pancreatitis (SAP) complicated with acute respiratory distress syndrome (ARDS). Methods Sixty patients with SAP complicated with ARDS admitted to the Department of Critical Care Medicine of the First Affiliated Hospital of Nanchang University from April 2014 to March 2016 were enrolled. They were divided into control group and experimental group by random number table, 30 patients in each group. On the basis of comprehensive treatment, the patients in control group were treated with lung protective ventilation mode: low tidal volume ventilation (6 mL/kg) + optimal end-expiratory positive pressure (PEEP) ventilation mode, when the intra-abdominal pressure (IAP) was essentially returned to a normal level (Ⅰ grade intra-abdominal hypertension), the patients in experimental group were treated by the combination with RM therapy, and the rest treatment was the same as the control group. Under the two types of ventilation strategies, the clinical effects, respiratory mechanics, hemodynamics and arterial blood gas indexes were compared between the two groups. Results The mechanical ventilation time (days: 13.82±4.40 vs. 19.87±7.40), the length of ICU stay (days:22.67±4.40 vs. 26.43±5.39) and incidence of ventilator associated pneumonia [VAP: 16.67% (5/30) vs. 26.67% (8/30)] of the experimental group were lower than those of the control group (all P < 0.05), the mortality rate of the experimental group was slightly lower than that of the control group [26.67% (8/30) vs. 30.00% (9/30)] without statistical significance (P > 0.05). Plateau pressure (Pplat) and the peak airway pressure (PIP) at each time point were decreased after treatment in both groups, while the static lung compliance (Cst), the arterial partial pressure of oxygen (PaO2) and oxygenation index (PaO2/FiO2) were increased compared with those before treatment, especially the changes at 72 hours after recruitment in the experimental group were more significant than those in the control group [Pplat (cmH2O, 1 cmH2O = 0.098 kPa):15.6±4.0 vs. 21.2±5.6, PIP (cmH2O): 18.3±5.0 vs. 25.1±5.4, Cst (mL/cmH2O): 41.2±4.8 vs. 31.2±6.0, PaO2 (mmHg, 1 mmHg = 0.133 kPa): 90.93±6.45 vs. 80.27±4.51, PaO2/FiO2 (mmHg): 238.33±18.31 vs. 185.83±11.14]. Heart rate [HR (bpm): 110.23±7.92 vs. 98.23±8.44] and the central venous pressure [CVP (mmHg): 8.62±1.52 vs. 6.32±1.42] were significantly higher than those before treatment, the mean arterial pressure [MAP (mmHg): 86.74±7.65 vs. 94.92±10.93] and cardiac output [CO (L/min): 5.32±1.36 vs. 6.42±1.32] were significantly reduced compared with those before treatment (all P < 0.05). The values of HR, MAP, CVP, CO at 5 minutes after recruitment were (97.87±5.77) bpm, (94.54±6.87) mmHg, (6.33±1.44) mmHg, (6.32±1.41) L/min, respectively. The changes of these parameters were not significant when compared with those of the basal conditions (P > 0.05) Conclusions Based on the lung protective ventilation in the early stage, sequential RM is applied in treatment of patients with SAP complicated with ARDS, after the IAP is essentially returned to a normal, which is beneficial to improving lung compliance, promoting oxygenation, shortening the time of mechanical ventilation, reducing the length of ICU stay, and decreasing the incidence of VAP without any obvious hemodynamic influence.

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