1.Effect of PFKFB3 on inflammatory activation of polymorphonuclear myeloid-derived suppressor cell in acute myocardial infarction
Zongying YU ; Yanjun WU ; Dadong LIU
Chinese Critical Care Medicine 2024;36(1):44-49
Objective:To investigate the correlation between 6-phosphofructo-2-kinase/fructose-2, 6-biphosphatase 3 (PFKFB3) and the inflammatory activation of polymorphonuclear myeloid-derived suppressor cell (PMN-MDSC) in acute myocardial infarction (AMI), and to evaluate the effect of intervention targeting PFKFB3 on the inflammatory activation of PMN-MDSC during AMI.Methods:① Clinical trial section: a observational study was conducted. The patients with acute coronary syndrome (ACS) admitted to Zhenjiang Fourth People's Hospital were enrolled, and they were divided into AMI group and non-AMI group according to clinical diagnosis. The peripheral venous blood of the two groups was collected to detect the proportion of PMN-MDSC, and the expression of PFKFB3 gene in mononuclear cells was detected by real-time quantitative polymerase chain reaction (RT-qPCR). ② Basic experiment section: a total of 30 male C57 mice (aged 6-8 weeks) were divided into normal control group ( n = 5), Sham group ( n = 5), AMI model group ( n = 10) and PFKFB3 inhibitor PKF-15 intervention group ( n = 10) according to random number table method. The AMI model of mice was reproduced by left anterior descending coronary artery (LADCA) ligation, and the mice in the Sham group did not attach the artery after thoracotomy. The PKF-15 intervention group was intraperitoneally injected with PKF-15 (20 μg/g) at the same time of LADCA ligation. Normal control mice did not receive any treatment. Peripheral venous blood and myocardial tissue of mice were collected 24 hours after modeling. Both the circulating PMN-MDSC ratio and the infiltration of PMN-MDSC in myocardial tissue were detected. After staining with hematoxylin-eosin (HE), the degree of inflammatory damage in mouse myocardial tissue was observed under light microscopy. PMN-MDSC were isolated from mice with flow cytometry, and the gene expressions of PFKFB3 and inflammatory factors were measured by RT-qPCR. Results:① Clinical trial section: the circulating PMN-MDSC ratio of patients in the AMI group ( n = 25) was significantly higher than that in the non-AMI group [ n = 20; (8.53±0.96)% vs. (1.13±0.39)%, P < 0.01], and PFKFB3 gene expression in the peripheral blood mononuclear cells was also increased (2 -ΔΔCt: 1.18±0.19 vs. 0.96±0.16, P < 0.01). Pearson correlation analysis showed that circulating PMN-MDSC ratio was positively correlated with PFKFB3 gene expression in mononuclear cells in AMI patients ( r = 0.608, P = 0.001). ② Basic experimental section: the circulating PMN-MDSC ratio and the infiltration of PMN-MDSC in myocardial tissue of AMI mice were significantly higher than those in the normal control group and Sham group. PFK-15 intervention could reduce the ratio of PMN-MDSC in the peripheral blood and myocardial tissue of AMI mice [(26.33±5.27)% vs. (75.12±5.02)% in peripheral blood, (20.87±2.97)% vs. (35.28±4.36)% in myocardial tissue, both P < 0.01]. Under light microscopy, the myocardial cells in the AMI modal group were disordered and a large number of inflammatory cells infiltrated. PFK-15 intervention could maintain a normal arrangement of cardiomyocytes and reduce the infiltration of inflammatory cells. The gene expression levels of PFKFB3 in the peripheral blood and myocardial tissue as well as the inflammatory factors in the myocardial tissue of AMI mice were significantly higher than those in the normal control group and Sham group. PKF-15 intervention could effectively reduce the gene expression levels of PFKFB3 in the peripheral blood and myocardial tissue as well as the inflammatory factors in the myocardial tissue of AMI mice [PFKFB3 mRNA (2 -ΔΔCt): 1.01±0.09 vs. 1.40±0.12 in peripheral blood, 0.95±0.09 vs. 1.47±0.10 in myocardial tissue; myocardial tissue tumor necrosis factor-α (TNF-α) mRNA (2 -ΔΔCt) was 14.55±3.99 vs. 29.66±3.90, interleukin-1β (IL-1β) mRNA (2 -ΔΔCt) was 8.72±1.35 vs. 18.53±2.43, IL-6 mRNA (2 -ΔΔCt) was 11.87±2.97 vs. 19.82±4.32, all P < 0.01]. Conclusions:The activation of PFKFB3 is closely related to the inflammatory activation of PMN-MDSC during AMI. Inhibition of PFKFB3 activity can inhibit the inflammatory activation of PMN-MDSC and reduce myocardial inflammatory injury.
2.Predictive value of neutrophil free fatty acid receptor 3 for secondary infection in patients with severe acute pancreatitis
Min XIAO ; Peng WANG ; Baiqiang LI ; Weiqin LI ; Dadong LIU
Chinese Critical Care Medicine 2024;36(10):1082-1087
Objective:To evaluate the predictive value of neutrophil free fatty acid receptor 3 (FFAR3) for secondary infection in patients with severe acute pancreatitis (SAP).Methods:① Biological information analysis: peripheral blood microarray data sets related to acute pancreatitis (GSE194331) were obtained from the Gene Expression Omnibus (GEO), including data from 32 healthy adults, 52 patients with mild acute pancreatitis, 20 patients with moderate-to-severe acute pancreatitis, and 10 patients with SAP. The original data of GSE194331 dataset were downloaded for quality control, pruning, quantification, annotation and difference analysis, and the different genes were obtained. ② Clinical study: a prospective observational study was conducted. Forty-five SAP patients admitted to the critical care medicine department of the Eastern Theater Command General Hospital of the Chinese People's Liberation Army from January to November 2022 were enrolled, and they were divided into infected group and non-infected group according to whether secondary infection occurred during intensive care unit (ICU) stay. At the same time, 10 healthy adult volunteers were enrolled as control. Peripheral blood of subjects in each group was collected, neutrophils were isolated, and FFAR3 mRNA expression was detected by real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-PCR). Spearman correlation method was used to analyze the correlation between neutrophil FFAR3 mRNA expression and secondary infection in SAP patients. Multivariate Logistic regression analysis was used to evaluate whether neutrophil FFAR3 mRNA expression was a risk factor for secondary infection in SAP patients. Receiver operator characteristic curve (ROC curve) was plotted to evaluate the predictive value of neutrophil FFAR3 mRNA expression on secondary infection in SAP patients.Results:① Results of biological information analysis: the analysis of GSE194331 dataset showed that 301 genes were differentially expressed in peripheral blood cells between healthy controls and patients with pancreatitis. By biological function analysis, 8 biological functions involved in immune response were obtained, and 44 differential expressed genes were enriched in these 8 biological functions. The results of cell distribution analysis showed that there were 21 differential expressed genes expressions on neutrophils significantly higher than other immune cells, and the gene related to lipid metabolism was FFAR3. These results indicated that FFAR3 expression was closely related to the occurrence and development of SAP. ② Clinical study results: out of the 45 SAP patients, 24 developed into secondary infection during ICU stay, 21 did not develop into secondary infection. The expression of neutrophil FFAR3 mRNA in SAP patients with secondary infection was significantly higher than that in SAP patients without secondary infection and healthy controls [2 -ΔΔCt: 3.8 (3.0, 4.2) vs. 1.4 (1.1, 2.7), 1.0 (0.8, 1.1), both P < 0.05]. Spearman correlation analysis showed that neutrophil FFAR3 mRNA expression was positively correlated with secondary infection in SAP patients ( r = 0.799, P < 0.001). Multivariate Logistic regression analysis showed that increased FFAR3 mRNA expression was an independent risk factor for secondary infection in SAP patients [odds ratio ( OR) = 17.212, 95% confidence interval (95% CI) was 3.004-98.613, P = 0.001]. ROC curve analysis showed that the area under the ROC curve (AUC) of neutrophil FFAR3 mRNA expression for predicting secondary infection in SAP patients was 0.856 (95% CI was 0.750-0.981, P < 0.001). When the optimal cut-off value was 2.37, the sensitivity was 95.83% and the specificity was 76.19%. According to the optimal cut-off value of neutrophil FFAR3 mRNA expression (2.37) for predicting secondary infection in SAP patients obtained by ROC curve analysis, 45 SAP patients were divided into two groups for subgroup analysis. It suggested that the incidence of secondary infection in SAP patients with FFAR3 mRNA expression level ≥2.37 was significantly higher than that in SAP patients with FFAR3 mRNA expression level < 2.37 [82.14% (23/28) vs. 5.88% (1/17)], and the difference was statistically significant ( P < 0.01). Conclusion:The expression of FFAR3 mRNA in neutrophils is closely related to the secondary infection in SAP patients, and monitoring its level can effectively predict the secondary infection in SAP patients.
3.Study on the mechanism of electroacupuncture relieving retinal cell apoptosis in experimental myopic guinea pigs based on mitochondrial dynamics
Zhaohui YANG ; Jiawen HAO ; Jinpeng LIU ; Bo BAO ; Longqian LIU ; Dadong GUO ; Hongsheng BI
Recent Advances in Ophthalmology 2023;43(12):940-945
Objective To investigate the mechanism of electroacupuncture in relieving the apoptosis of retinal cells in experimental myopia based on mitochondrial dynamics.Methods In the study,140 two-week-old tricolor guinea pigs were divided into the normal control(NC)group,lens-induced myopia(LIM)group,LIM+sham acupoint(LIM+SHAM)group,and LIM+electroacupuncture intervention(LIM+EA)group,with 35 guinea pigs in each group.Guinea pigs in the NC group were fed normally,and those in the LIM group,LIM+SHAM group and LIM+EA group wore a-6.0D lens on the right eye to induce myopia.Guinea pigs in the LIM+EA group were treated at Hegu and Taiyang acupoints,while those in the LIM+SHAM group were treated at sham acupoints.After 2-and 4-week myopia induction,the diopter and axial length of the guinea pigs were measured,and the messenger ribonucleic acid(mRNA)and protein levels of dynam in-related pro-tein 1(DRP1),optic atrophy 1(OPA1),Bcl-2 associated X protein(BAX)and B cell lymphoma-2(BCL-2)in the retinas of the guinea pigs in each group were detected by quantitative PCR and Western blot,respectively.Meanwhile,HE staining was taken to observe the morphological changes in the retina of the guinea pigs in each group,and TUNEL staining was used to detect apoptosis.Results After 2-and 4-week myopic induction,compared with the NC group,the differences in diopter and axial length between the right and left eyes of guinea pigs in the LIM and the LIM+SHAM groups significantly increased(all P<0.001).Compared with the LIM group,the differences in diopter and axial length between both eyes of guinea pigs in the LIM+EA group significantly decreased(all P<0.01).At 4 weeks after myopic induction,HE staining re-sults showed that the retinas of the guinea pigs in the NC group were evenly arranged,and the morphology of inner and out-er nuclear layer cells was normal.Compared with the NC group,the retinas of the guinea pigs in the LIM and the LIM+SHAM groups were significantly thinner and disorderly arranged.Compared with the LIM group,the retinal thickness of guinea pigs in the LIM+EA group slightly increased,and the arrangement was relatively regular.The TUNEL staining re-sults showed that compared with the NC group,the green fluorescence of the guinea pig retina in the LIM group and LIM+SHAM group was significantly enhanced;compared with the LIM group,the green fluorescence of the guinea pig retina in the LIM+EA group was significantly weaken.After 2-and 4-week myopic induction,the mRNA and protein expression lev-els of DRP1 and BAX in the LIM and the LIM+SHAM groups were significantly higher than those in the NC group(all P<0.05),while the mRNA and protein expression levels of OPA1 and BCL-2 were significantly lower(all P<0.05).The mR-NA and protein expression levels of DRP1 and BAX in the LIM+EA group were significantly lower than those in the LIM group(all P<0.05),while the mRNA and protein expression levels of OPA1 and BCL-2 were significantly higher(all P<0.05).Conclusion Experimental myopia can enhance retinal mitochondrial fission.Electroacupuncture intervention can alleviate retinal cell apoptosis and improve the morphological structure of the retina by inhibiting retinal mitochondrial fis-sion and increasing mitochondrial fusion,thereby delaying myopia development.
4.Peripheral 5-hydroxytryptophan aggravates lung injury in septic mice by inducing the formation of neutrophils extracellular trap
Yumeng HUANG ; Qian JI ; Yanyan ZHU ; Dadong LIU ; Shengqiao FU ; Xu WANG ; Ningzheng TAI
Chinese Critical Care Medicine 2021;33(12):1423-1427
Objective:To observe the effect of peripheral 5-hydroxytryptophan (5-HT)-induced neutrophil extracellular trap (NET) on lung injury in septic mice.Methods:Wild-type (WT type) and Tph1 knockout (KO) C57 mice (6-8 weeks) were selected and divided into WT mice sham group, WT mice sepsis group, Tph1 KO mice sham group and Tph1 KO mice sepsis group according to the random number table method. Mice in the sham group received sham surgery (only open the abdominal cavity to flip the cecum without ligation and puncture, and then close the abdominal cavity); the mice in the sepsis group received cecal ligation and puncture (CLP) to establish sepsis model. The mice were sacrificed 12 hours after the operation, and the levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in bronchialalveolar lavage fluid (BALF) were detected by enzyme linked immunoadsordent assay (ELISA); at the same time, the lung tissues were collected, and the pathological changes of lung tissues were observed under light microscope, and the production of NET in lung tissues was observed by immunofluorescence microscope. Results:The pathological results suggested that the lung tissue structure in sham groups was intact without exudation, while the alveolar structures of mice in the sepsis groups were damaged, with obvious exudation in the alveolar cavity and thickened alveolar walls accompanied by a large number of inflammatory cell infiltration, and the degree of lung injury in the sepsis group of WT mice was more severe than that of the sepsis group of Tph1 KO mice. ELISA results showed that there was no statistically significant difference in the contents of TNF-α and IL-6 in mice BALF from different strains of the sham group; while the contents of TNF-α and IL-6 in BALF of septic mice group were significantly higher than those in sham group [WT mice: TNF-α (μg/L) was 158.20±28.46 vs. 14.00±3.28, IL-6 (μg/L) was 304.98±21.78 vs. 57.70±12.30; Tph1 KO mice: TNF-α (μg/L) was 85.88±20.13 vs. 14.95±1.53, IL-6 (μg/L) was 169.50±45.61 vs. 55.05±12.68, all P < 0.01], and the above index levels in the sepsis group of WT mice were significantly higher than the sepsis group of Tph1 KO mice [TNF-α (μg/L): 158.20±28.46 vs. 85.88±20.13, IL-6 (μg/L): 304.98±21.78 vs. 169.50±45.61, both P < 0.01]. Immunofluorescence staining showed that a very small amount of NET formation was detected in the mice lungs from the sham group; a large amount of NET formation was detected in the lung tissues in the sepsis group, which were significantly higher than those in sham group [WT mice: (34.75±7.27)% vs. (1.75±0.96)%, Tph1 KO mice: (14.25±5.74)% vs. (2.50±1.29)%, both P < 0.01], and the amount of NET produced in the lung tissues of the WT mice sepsis group was significantly higher than that of the Tph1 KO mice sepsis group [(34.75±7.27)% vs. (14.25±5.74)%, P < 0.01]. Conclusions:In sepsis, the increased production of inflammatory factors in the mice lung tissues induces to lung injury. The mechanism may relate to the increased production of NET in the lung tissues mediated by peripheral 5-HT synthesized by enterochromaffin cells and released into the blood; inhibiting the production of 5-HT in the peripheral blood can effectively reduce the production of NET in the lung tissues, thereby reducing lung injury.
5.Value of neutrophil to lymphocytes and platelets ratio for predicting 28-day mortality in sepsis patients
Dadong LIU ; Zongying YU ; Dehou ZHANG ; Jianguo ZHANG ; Yafeng ZHANG ; Xu WANG
Chinese Critical Care Medicine 2021;33(1):33-37
Objective:To evaluate the value of neutrophil to lymphocyte and platelet ratio (N/LPR) for predicting 28-day mortality in sepsis patients.Methods:A retrospective analysis was conducted. The clinical data of 154 sepsis patients admitted to intensive care unit (ICU) of the Affiliated Hospital of Jiangsu University from June 2017 to June 2020 were enrolled. The time of first diagnosis of sepsis in ICU was taken as the research starting point, and the death or 28 days as the end point. The 28-day outcomes of patients were recorded. The counts of peripheral blood neutrophil (NEU), lymphocyte (LYM) and platelet (PLT) were collected from all the enrolled patients within 3 days after diagnosis of sepsis. The ratios of N/LPR and NEU/LYM (NLR) were calculated respectively. The differences of N/LPR and NLR between survival group and death group were compared. Receiver operating characteristic (ROC) curve analysis was used to analyze the value of N/LPR and NLR on predicting the 28-day mortality of sepsis patients. According to the best cut-off value of ROC curve analysis, the 28-day mortality of patients with sepsis was analyzed by subgroup analysis, and the 28-day cumulative survival of patients with sepsis was analyzed by Kaplan-Meier survival curve.Results:Of the 154 sepsis patients, the patients with age < 18 years, pregnancy, blood disease, taking aspirin or other antiplatelet drugs within 1 week, taking leucocyte drugs within 1 week, length of ICU stay < 3 days and incomplete data were excluded. Finally, 50 patients were enrolled. Among them, 30 patients survived on the 28th day and 20 died. Compared with the survival group, the levels of N/LPR and NLR in the death group were significantly increased (N/LPR: 23.85±11.99 vs. 12.41±5.25, NLR: 17.83±8.69 vs. 10.75±3.63), with statistical differences (both P < 0.01). ROC curve analysis indicated that the area under ROC curve (AUC) of N/LPR for predicting 28-day death of sepsis patients was 0.827, it was higher than that of NLR (AUC = 0.762). Base on N/LPR≥15.48 as a predictor of cut-off value of death in 28 days of sepsis patients, the sensitivity was 75.0% and the specificity was 80.0%, respectively. Base on NLR≥10.65 as a predictor of cut-off value of death in 28 days of sepsis patients, the sensitivity was 75.0% and specificity was 56.7%, respectively. Subgroup analysis showed that the 28-day mortality in the patients with N/LPR≥15.48 ( n = 21) was significantly higher than those with N/LPR < 15.48 ( n = 29; 71.4% vs. 17.2%, χ 2 = 14.901, P < 0.01); and the 28-day mortality in the patients with NLR≥10.65 ( n = 28) was also significantly higher than those with NLR < 10.65 ( n = 22; 53.6% vs. 22.7%, χ 2 = 4.884, P < 0.05). The results were consistent with Kaplan-Meier survival curve analysis. Conclusion:Peripheral blood N/LPR has a good predictive value for 28-day mortality of sepsis patients, and which is better than NLR.
6.Suppressive effect of CORM-2 on platelet α-granule exocytosis in sepsis via SNARE/Munc18b complex formation
Mingfeng ZHUANG ; Bingwei SUN ; Dadong LIU ; Yuan SHI
Chinese Critical Care Medicine 2017;29(2):156-161
Objective To investigate the suppressive effect of carbon monoxide-releasing molecule Ⅱ (CORM-2) on LPS induced platelet α-granule exocytosis in sepsis via soluble N-ethylmaleimide-sensitive factor attached protein receptor/mammalian uncoordinated 18b (SNARE/Munc18b) complex formation.Methods Blood was collected from healthy volunteers' cubital vein, then platelets were isolated by differential centrifugation. Platelets were randomly divided into 5 groups. The control group did not undergo any treatment, the LPS group received 10 mg/L LPS simulation, the CORM-2 group and iCORM-2 group underwent LPS simulation and immediate administration of CORM-2 (10μmol/L and 50μmol/L) or iCORM-2 (50μmol/L), respectively. Samples were incubated in a CO2-incubator at 37 ℃, 95% humidity, and 5% CO2. Platelet α-granule contents were detected by using standard enzyme linked immunosorbent assay (ELISA), including platelet factor 4 (PF4), platelet derived growth factor-BB (PDGF-BB), and matrix metalloproteinase-2 (MMP-2). The expression of P-selectin was detected by flow cytometer. Transmission electron microscope and immunofluorescence microscope was used to assess platelet α-granules distribution. Expressions of Munc18b and SNARE proteins including vesicle-associated membrane protein-8 (VAMP-8), synaptosomal-associated protein-23 (SNAP-23) and syntaxin-11 (STX-11) were detected by Western Bolt. The SNARE/Munc18b complex formation was detected by immunoprecipitation.Results Compared with the control group, levels of PF4, PDGF-BB, MMP-2 and P-selectinin LPS-induced platelets were found to markedly elevated, while CORM-2 (10μmol/L and 50μmol/L) could decrease platelet α-granule contents exocytosis: [PF4 (μg/L): 7.69±0.58, 6.03±0.71 vs. 10.13±0.82; PDGF-BB (μg/L): 112.71±1.79, 102.91±5.86 vs. 128.78±1.39; MMP-2 (ng/L): 32.94±2.73, 27.58±3.36 vs. 53.26±1.21; P-selectin: (17.14±0.57)%, (15.35±0.68)% vs. (23.78±0.62)%; allP < 0.01]. Transmission electron microscope and immunofluorescence microscope showed that the extent of platelet α-granules assembled to platelet plasma membrane was significantly decreased following CORM-2 treatment. Compared with the control group, the expressions of Munc18b and SNARE proteins and SNARE/Munc18b complex formation in LPS-stimulated platelets were significantly increased, while CORM-2 (10μmol/L and 50μmol/L) inhibited these elevations (Munc18b/GAPDH: 0.80±0.08, 0.69±0.01 vs. 0.99±0.09; VAMP-8/GAPDH: 0.72±0.09, 0.50±0.12 vs. 1.18±0.14; SNAP-23/GAPDH: 1.18±0.22, 0.63±0.10 vs. 1.90±0.08; STX-11/GAPDH: 0.76±0.02, 0.57±0.08 vs. 1.16±0.23; VAMP-8/ Munc18b: 0.65±0.09, 0.53±0.07 vs. 1.21±0.20; SNAP-23/Munc18b: 0.85±0.07, 0.55±0.09 vs. 1.26±0.08; STX-11/ Munc18b: 0.78±0.05, 0.61±0.10 vs. 1.39±0.16; allP < 0.01). Above all, the data showed a dose dependent change.Conclusion We could suggest that CORM-2 suppressed α-granule exocytosis in LPS-stimulated platelets and the potential mechanisms might involve SNARE/Munc18b complex formation.
7.The value of combined sampling at multiple sites of gastric mucosa for Helicobacter pylori culture
Jiaochang WANG ; Dadong HUANG ; Xianzuo XU ; Min LIN ; Qiuxiang LIU ; Ningmin YANG ; Fei MENG ; Jiansheng WU
Chinese Journal of Digestive Endoscopy 2017;34(3):200-202
Objective To evaluate combined sampling at multiple sites of gastric mucosa for Helicobacter pylori (HP) culture.Methods A total of 258 patients with upper gastrointestinal symptoms received 13C-urea breath test between August 2014 and May 2015.During endoscopy,gastric mucosa biopsy samples from the lesser curvature of the antrum (A),the greater curvature of the antrum (B),gastric angle (C) and the body of the stomach (D) were collected to isolate HP strains.The positive rates of HP based on combined sampling and single site sampling were compared with a Nemenyi test.Results Consistency between 13C-urea breath test and HP culture was 82.56%.There was significant difference between the single site sampling and two-site sampling in the positive rate of HP,except for the body of the stomach (P<0.05).There was significant difference in the positive rate of HP between the single site sampling and three-site sampling (P < 0.05).There was no significant difference between any two-site sampling in the lesser curvature of the antrum and the body of the stomach,gastric angle and the body of the stomach,the greater curvature of the antrum and the body of the stomach,and any three-site sampling (P>0.05).Conclusion The combined sampling of the lesser curvature of the antrum and the body of the stomach have the highest cost-effectiveness in HP culture compared with the single site sampling and three-site sampling.
8.Regulatory roles of rno-miR-30b-5p in expressions of IL-10 and TLR4 in rats with experimental autoimmune uveitis
Yuanyuan SUN ; Dadong GUO ; Meiqing CHEN ; Shaoyu LI ; Bin LIU ; Kai TANG ; Hongsheng BI
Recent Advances in Ophthalmology 2017;37(4):330-334
Objective To investigate the regulatory role of rno-miR-30b-5p in the expressions of interleukin-10 (IL-10) and toll-like receptor 4 (TLR4) in uveitis.Methods Both IL-10 and TLR4 gene 3'UTR lucfferase vectors and relevant binding site mutant vectors were constructed.Further,both rno-miR-30b-5p mimics and reporter gene vector were co-transferred into 293 T cells to validate the fluorescent alterations of the reporter gene expression to detect the interactions between rno-miR-30b-5p and the related target genes.Moreover,an experimental autoimmune uveitis (EAU) model was induced with IRBP peptide emulsion in rats,and both lymph node and spleen were isolated on day 12 after EAU induction.In order to measure rno-miR-30b-5p levels and IL-1 0,TLR4 expressions in spleen and lymph node,quantitative PCR and ELISA techniques were applied.Results The results of double lucfferase reporter gene expression analysis showed rno-miR-30b-5p mimic apparently down-regulated the fluorescence intensity of both IL-10 and TLR4 in wild type cells.After the mutation of the target site,the fluorescence intensity of the mutant vector was significantly reduced,accompanied by a significantly statistical difference (all P < 0.01).Moreover,animal results revealed the expressions of rno-miR-30b-5p were apparently decreased,whereas IL-10 and TLR4 were markedly increased in both lymph node and spleen (all P < 0.05).Conclusion Target identification shows that rno-miR-30b-5p can obviously regulate the expressions of 3'UTR gene with either IL-10 or TLR4 gene fragment,though its regulation might not be through the predicted site.The down-regulated expression of rno-miR-30b-5p in both spleen and lymph node in EAU rats result in the up-regulated expressions of both IL-10 and TLR4,further influence the development of uveitis.This study paves a way for the modulation of microRNA on the occurrence and development of uveitis,and will provide a new insight on treating uveitis.
9.Recent advances in regulation of autophagy in ocular diseases
Bin LIU ; Dadong GUO ; Yuanyuan SUN ; Hongsheng BI
Recent Advances in Ophthalmology 2017;37(8):797-800
Autophagy is of highly conserved self degradation under physiological and pathological conditions.Recent studies has been shown that autophagy play the important role in the development of ocular diseases.This article reviews the role of autophagy in the development of ocular diseases,and provides new ideals for clinical treatment of ocular diseases.
10.Immunomodulatory effects of Longdan Xiegan Tang on related inflammatory cytokines in rats with experimental autoimmune uveitis
Kai TANG ; Dadong GUO ; Xiuzhen LU ; Yuanyuan SUN ; Bin LIU ; Hongsheng BI
Recent Advances in Ophthalmology 2017;37(7):610-614,618
Objective To study the immunomodulatory effects of Longdan Xiegan Tang on related inflammatory cytokines in rots with experimental autoimmune uveitis (EAU).Methods Lewis rats were randomly divided into control group (6 rats),EAU group (18 rats) and LXT group (18 rats).Rats in EAU and LXT groups were immunized with interphotoreceptor retinoid-binding protein (IRBP) emulsion.The expressions of IFN-γ,IL-17,IL-10 and TNF-α were investigated by quantitative real-time PCR and ELISA,respectively.Results In blood of LXT group,the mRNA level of IFN-γ on day 8 was higher than that in control group (P =0.000),but obviously lower than that in EAU group (P =0.000);The mRNA level of IL-17 was peaked on day 12,but was lower than EAU group (P=0.000);The mRNA level of TNF-α on day 12 was higher than that in control group (P =0.000),but obviously lower than that in EAU group (P =0.000);The mRNA level of IL-10 on day 16 was higher than that in EAU group (P =0.042).In lymph node and spleen of LXT group,the mRNA levels of IFN-γ,IL-17 and TNF-α on day 12 were lower than those in EAU group (all P=0.000);The mRNA level of IL-10 was peaked on day 12,but was lower than EAU group (P =0.000).In serum of LXT group,the mRNA levels of IFN--γ,IL-17 and TNF-α on day 12 and day 16 were lower than those in EAU group;The level of IL-10 in EAU and LXT group on day 12 were higher than that in control group,peaked on day 16,but the LXT group was the highest.Conclusion Longdan Xiegan Tang can reduce the expressions of proinflammatory cytokines including IFN-γ,IL-17 and TNF-α.Meanwhile,it can also promote the production of IL-10 and further accelerate the recovery of EAU,indicating that Longdan Xiegan Tang can play a significant role in treating uveitis.

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