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Objective:To investigate the effect of licochalcone A on osteoarthritis in rats and its relationship with p38-MAPK inflammatory signaling pathway.Methods:A total of 160 male Wistar rats were randomly divided into blank non-intervention, blank intervention, arthritis non- intervention and arthritis intervention groups with 40 rats in each group. Rats in the arthritis groups were subjected to unilateral anterior cruciate ligament transection, while rats in the blank groups were only subjected to skin incision and suture. Rats in the intervention groups were treated by intra-articular injection of 1 mL 10 μmol/L licochalcone A for 8 successive weeks. Eight weeks later, the cartilage of rats in each group was stained with safranin, and osteoarthritis soft tissue was scored according to Osteoarthritis Research Society International guideline under the optical microscope. The cartilage was cultured in low glucose cell culture medium supplemented with 5% fetal bovine serum for 48 hours. The contents of nitric oxide, prostaglandin E 2, sulfated glycosaminoglycan and collagen II in the medium were determined by the chemiluminescence reaction method. The expression levels of p38, phosphorylated p38 (p-p38) and matrix metalloproteinase in cartilage tissue were detected by western blot assay. Results:The progress of osteoarthritis in rats treated with licochalcone A was slow. The Osteoarthritis Research Society International score in the arthritis intervention group was significantly lower than that in the arthritis non-intervention group [(3.8 ± 1.7) points vs. (9.7 ± 1.2) points, P = 0.0064]. The contents of nitric oxide, prostaglandin E 2, sulfated glycosaminoglycan, and collagen II in the arthritis intervention group were (77.84 ± 17.65) μmol/mg and (6.78 ± 1.76) ng/mg, (89.78 ± 9.76) μg/mg, and (1.78 ± 0.76) μg/mg, respectively, which were significantly lower than those in the arthritis non-intervention group [(107.56 ± 18.74) μmol/mg, (10.756 ± 1.87) ng/mg, (125.75 ± 8.87) μg/mg, (3.76 ± 0.88) μg/mg, (NO: P = 0.002; PGE 2: P < 0.001; sGAG: P < 0.001; Collagen II: P < 0.001). Western blot assay results revealed that the relative expression of p38, p-p38, p-p38 to total p38 ratio, matrix metalloproteinase in the arthritis intervention group were (3 454 ± 421), (2 072 ± 175), (0.65 ± 0.14 )and (1 776 ± 765), respectively, which were significantly lower than those in the arthritis non-intervention group (5 322 ± 323), (4 257 ± 184), (0.89 ± 0.11), (3 865 ± 874)( p38: P < 0.001; p-p38: P < 0.001; p-p38/p38: P = 0.002; MMP: P = 0.001). Conclusion:Licochalcone A can delay the progression of osteoarthritis in rats with osteoarthritis through inhibiting inflammatory reaction and cartilage matrix degradation, and p38-MAPK signaling pathway may be involved in the regulation process.
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Objective@#To investigate the possible pathogenesis of Staphylococcus aureus (S.aureus) corneal ulcer by analyzing the differentially expressed genes (DEGs) of S.aureus isolated from the patients with corneal ulcer and healthy conjunctival sac.@*Methods@#Ten strains of S.aureus isolates were obtained from January to August 2018 in the clinical laboratory of Qingdao Eye Hospital.Five strains of S.aureus isolated from patients with corneal ulcer and five strains of S.aureus isolated from healthy conjunctival sac were included.The gene expression profiles of 10 strains of S.aureus were sequenced by Illumina high-throughput RNA-sequencing (RNA-Seq). P≤0.05 and fold change≥2 were used as the threshold to determine the statistically DEGs.Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were used to determine the biological functions of DEGs.@*Results@#The genome-wide transcriptional analysis demonstrated that 270 genes were differentially expressed with 138 upregulated genes and 132 downregulated genes in the strains from corneal ulcer.Function analysis of DEGs revealed that genes encoding alpha hemolysin, delta hemolysin, virulence factor EsxA and LysR family transcriptional regulators were significantly upregulated in strains isolated from cornea ulcer.GO enrichment analysis showed that most DEGs was involved in the metabolic process with the biosynthesis, the most significantly related process was the metabolism of inosine monophosphate.The KEGG pathways suggested that a number of metabolic pathways had significant changes, such as S.aureus infection, two-component system and pyruvate metabolism, purine metabolism, which were critical to the pathogenesis of S.aureus corneal ulcer.@*Conclusions@#Identification of the DEGs between corneal ulcer isolates and healthy conjunctival isolates of S.aureus is helpful for further investigations on genes or pathways associated with the pathogenesis and therapeutic targets of S.aureus corneal ulcer.
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Objective To investigate the possible pathogenesis of Staphylococcus aureus ( S. aureus) corneal ulcer by analyzing the differentially expressed genes ( DEGs) of S. aureus isolated from the patients with corneal ulcer and healthy conjunctival sac. Methods Ten strains of S. aureus isolates were obtained from January to August 2018 in the clinical laboratory of Qingdao Eye Hospital. Five strains of S. aureus isolated from patients with corneal ulcer and five strains of S. aureus isolated from healthy conjunctival sac were included. The gene expression profiles of 10 strains of S. aureus were sequenced by Illumina high-throughput RNA-sequencing ( RNA-Seq) . P≤0. 05 and fold change≥2 were used as the threshold to determine the statistically DEGs. Gene Ontology ( GO ) and Kyoto Encyclopedia of Genes and Genomes ( KEGG) pathway enrichment analyses were used to determine the biological functions of DEGs. Results The genome-wide transcriptional analysis demonstrated that 270 genes were differentially expressed with 138 upregulated genes and 132 downregulated genes in the strains from corneal ulcer. Function analysis of DEGs revealed that genes encoding alpha hemolysin,delta hemolysin,virulence factor EsxA and LysR family transcriptional regulators were significantly upregulated in strains isolated from cornea ulcer. GO enrichment analysis showed that most DEGs was involved in the metabolic process with the biosynthesis, the most significantly related process was the metabolism of inosine monophosphate. The KEGG pathways suggested that a number of metabolic pathways had significant changes,such as S. aureus infection,two-component system and pyruvate metabolism,purine metabolism, which were critical to the pathogenesis of S. aureus corneal ulcer. Conclusions Identification of the DEGs between corneal ulcer isolates and healthy conjunctival isolates of S. aureus is helpful for further investigations on genes or pathways associated with the pathogenesis and therapeutic targets of S. aureus corneal ulcer.
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Objective To investigate the role of autophagy in the development of diabetic cataract by detecting the expression of autophagy-related factors (BECN1,LC3B,P62) in diabetic mouse lens epithelial cells.Methods Eighty C57BL/6 male mice were selected.Fifty C57 mice were consecutively dosed with streptozotocin (STZ) 50 mg/ (kg · d) by intraperitoneal injection to induce type 1 diabetes mellitus model,and served as the model group;the remaining 30 mice were injected with appropriate dose of citrate buffer,and served as the control group.The fasting plasma glucose was tested by collecting the caudal vein blood in the model group.The morphological changes of autophagy of lens epithelial cells were observed by transmission electron microscopy.The expression and localization of LC3B and P62 protein were detected by immunohistochemistry.PCR was used to detect the expression of BECN1,LC3B and P62 mRNA in the anterior capsule.The relative expression of autophagy-related proteins in the anterior capsule was detected by Western blot.The use of animals complied with Regulations on the Management of Experimental Ainimals from Shandong Eye Institute.Results Compared with the control group,transmission electron microscopy revealed that the autophagosomes of lens epithelial cells in model group was large and contained more mitochondria.Immunohistochemical method showed that the expression of LC3B and P62 proteins in the anterior capsule tissue of experiment group was stronger than that of the control group.The relative expression level of BECN1,LC3B and P62 mRNA in the experiment group was 1.48±0.10,2.62±0.15 and 1.89±0.20,respectively,which was higher than 1.10±0.02,1.10±0.05 and 1.01±0.01 in the control group,with significant differences between the two groups (t =6.64,14.25,6.14;all at P ＜ 0.05).The relative expression of BECN1,LC3B and P62 protein in the experiment group was 1.50±0.10,1.24±0.09 and 3.19± 1.04,respectively,which was higher than 1.00±0.00,1.00±0.00 and 1.00±0.00 in the control group,with significant differences between the two groups (t =8.75,6.10,3.65;all at P＜0.05).Conclusions The phenomenon of autophagy in lens epithelial cells of diabetic mice is abnormal,and autophagy dysfunction may play an important role in the formation of diabetic cataract.
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Background Endophthalmitis is a serious complication of intraocular surgery.Conventional identification methods for bacteria are becterial culture and smear method,but these laboratory tests spend a long time and have low positive rates.16S rDNA is the bacterial chromosome encoding ribosomal RNA sequences,and it is determined that 16S rDNA sequencing has a high specificity for the identification of bacteria.Objective This study was to identify the infectious bacteria from aqueous humor or vitreous body in the eyes with endophthalmitis by 16S rDNA sequencing technique,and to investigate the diagnosis efficency of 16S rDNA sequencing technique on bacterial endophthalmitis.Methods Anterior chamber fluid (0.1-0.2 ml) or vitreous humor (0.5-1.0 ml) specimens were collected from 5 eyes of 5 patients with endophthalmitis in Qingdao Eye Hospital from June to December 2015 and used for high throughput sequencing,bacterial culture and smear,respectively.Bacteria DNA was extracted from the specimen with D3096-01 trace DNA kit for the amplification of V3-V4 region of 16S rRNA gene and sequencing of hypervariable region of all microbes in the samples by MiSeq Illumina Sequencing Platform.Then the bioinformatic analysis including analysis of taxonomy,abundance and alpha diversity were performed.Nucleasefree water of 50 μl in the centrifuge tube was used as control.Results Five aqueous humor or vitreous body samples were collected,and the positive results were exhibited by smear examination,with the Gram positive bacilli in the trumatic endophthalmitis eye and Gram negative bacilli in the filtering bleb infectious endophthalmitis eye,and all culture results were negative.16S rDNA squencing showed the positive outcomes in all the 5 samples.The high abundent nacteral genuses were Staphylococcus (65.28％),Streptococcus (18.90％) and Pseudomonas (12.76％) in the trumatic endophthalmitis eye;the major components of sample were Pseudomonas (53.68％),Acinetobacter (8.62％) and Limnobacter (5.96％) in the eye with acute endophthalmitis occurring at 2 days following cataract surgery;the major components in the filtering bleb infectious endophthalmitis eye were Moraxella (88.89％) and Pseudomonas (9.52％);the Pseudomonas was major components in the later-onset endophthalmitis eye (84.63％) and the eye with acute endophthalmitis occurring at 1 day after cataract surgery (97.89％).Conclusions A distinct advantage is found in 16S rDNA sequencing technique for the indentification of the pathogenic bacteria in endophthalmitis eyes due to its high positive rate in comparison with bacterial culture and smear method.
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Background miRNAs are a group of non-coding small RNA molecules,and they play an important role in regulating the apoptosis of LECs.The biological effects and mechanisams of miRNAs on LECs in agerelated cataract still need to be further elucidated.Objective This study was to investigate the anti-oxidative effects of miR-204 on age-related cataract in vitro.Methods The specimens of anterior lens capsules from agerelated cataract patients and normal donors were collected in Shandong Eye Institution and 20 subjects for each.The expression level of miR-204 was detected and compared between cataractous eyes and normol eyes by real-time fluorescence quantitative PCR (RT-qPCR).HLE-B3 cells,a human LEC line,were cultured,and the oxydative stress models of LECs were established by adding 200 μmol/L H2 O2 in the medium.The models were divided into model control group,miR-204 agomir group,agomir negative control group,miR-204 antgomir group and antagomir negative control group according to the difference of tranfected agents,and normal cells served as the control group.Twenty-four hours after transfection,the expression levels of miR-204 mRNA in the cells of various groups were detected by RT-qPCR to varify the transfection rate.Apoptosis rate of the cells was assayed by flow cytometry.The relative expression levels of TP53INP1 mRNA and p53 mRNA as well as their proteins were detected by RT-qPCR and Western blot,respectively.Results The mean expression level of miR-204 was significantly lower in the anterior lens capsules of cataractous eyes than that in the normal eyes (t=14.21,P＜0.05).The mean apoptosis rate of cells was 1.31±0.12,4.90±0.28,2.60±0.15,4.39±0.20,5.74±0.13 and 4.34±0.63 in the normal control group,model control group,miR-204 agomir group,agomir negative control group,miR-204 antgomir group and antagomir negative control group,respectively.The apoptosis rate was significantly increased in the model control group compared with the normal control group (t =-20.69,P＜0.01) and the apoptosis rate was also increased in the miR-204 antagomir group compared with antagomir negative control group (t =3.79,P＜0.05);while the apoptosis rate in the miR-204 agomir group was significantly declined in comparison with agomir negative control group (t=-12.20,P＜0.01).The relative expression levels of TP53INP1 and p53 mRNA and proteins in the cells were significantly higher in the model control group than those in normal control group (mRNA:t =6.44,11.71,both at P＜0.0I;protein:t =10.72,19.40,both at P＜0.01),and so were between the miR-204 antagomir group and antagomir negative control group (mRNA:t =4.07,3.74,both at P＜ 0.05;protein:t =7.18,10.58,both at P＜0.05).However,the expression levels of TP53INP1 and p53 mRNA and protein were significantly reduced in the miR-204 agomir group in comparison with the agomir negative control group (mRNA:t =-19.28,-10.58,both at P＜0.05;protein:t=-6.50,-6.36,both at P＜0.05).Conclusions miR-204 induces oxidative damage of age-related cataract via targeting TP53INP1,which suggests that the activation of TP53INP1-p53 signaling may be involved in the development of age-related cataract.
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Background Epithelial-mesenchymal transition (EMT) is one of the pathogenesis mechanisms of posterior capule opcification (PCO).Studying EMT is of important significance for the prevention and treatment of PCO.However,EMT model is lack.Objective This study was to establish an in vitro EMT model for the study of human PCO.Methods In vitro mimic cataract enucleation was performed on 40 donor eyes,including anterior capsulorhexis,nucleus hydroexpression,and aspiration of lens fibers.The capsular bag of lens was dissected free during the surgery and pinned flat on a plastic culture dish with DMEM/F12 supplemented containing 10％ fetal bovine serum for 4 weeks.The proliferation of LECs on the capsular bag was observed by phase-contrast and dark-field microscope in 0,3,7,14 and 28 days after culture.The capsular bag tissues were collected in cultured 0,3,7 and 28 days for the preparation of sections and hematoxylin-eosin stain,and the growth and morphology of LECs were examined with optical microscope.The expression and location of α-SMA,E-cadherin and Vimentin were assessed by immunochemistry.The expression levels of α-SMA,E-cadherin and Vimentin mRNA and proteins were detected by using real-time fluorescnce quantitative PCR and Western blot in different time points.Results No LECs were seen on the uncultured capsular bag.LECs appeared in cultured day 3 on the periphery capsular bag and grew toward the center and covered the posterior capsule 7 days after cultured,with a cobblestone-like appearance.Wrinkles occurred and extended gradually along with the enhancement of bag tension.Immunochestry showed that the expression intensity of E-cadherin in the capsular bag gradually weakened,and that of α-SMA or Vimentin was gradually enhanced during the culture duration.The relative expression levels of E-cadherhin mRNA at 0,3,7,14 and 28 days after culture were 3.35±0.13,1.47±0.20,1.13±0.14,1.00±0.85 and 0.23±0.03,and relative expression levels of Vimentin mRNA were 1.00 ± 0.73,1.05 ± 0.14,2.24 ± 0.43,2.84 ± 0.34 and 8.57 ± 0.40,and those of α-SMA mRNA were 1.00 ± 0.06,2.68 ± 0.28,4.24 ± 0.05,2.05 ± 0.90 and 15.30 ± 0.19,showing significant differences among different time points (E-cadherhin mRNA:F =23.430,P =0.000;Vimentin mRNA F =8.915,P =0.002;α-SMA mRNA:F =103.500,P =0.000),with the lowest expression levels in the E-cadherhin mRNA and the highest expression levels in the Vimentin mRNA and α-SMA mRNA at 28 days during the culture period (all at P＜0.01).The gray values of E-cadherin protein expression were 1.443 ± 0.017,1.023 ± 0.003 and 0.568 ± 0.018,and those of Vimentin protein were 0.565±0.012,1.156±0.007 and 1.241±0.009,and those of α-SMA protein were 0.195±0.045,0.693±0.036 and 1.501±0.005 at 0,3 and 28 days,with significnant differences among various time points (E-cadherin:F =2 787.000,P =0.000;Vimentin:F =4 488.000,P =0.000;α-SMA:F =1 173.000,P =0.000).The expression levels were significantly declined in E-cadherhin protein and elevated in Vimentin and α-SMA proteins at 3 and 28 days after culture in comparison with before culture.Conclusions A novel in vitro EMT model of LECs is established in this study.This model can mimic a natural EMT procedure after extracapsular cataract enucleation and therefore is a useful model for the further research of the mechanism and prevention and treatment of PCO.