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.

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.

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.

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.

A simple rapid detection of antibody to hepatitis delta virus (anti-HDV) in human serum was developed by using double antigen sandwich ELISA. HDV gene fragment encoding HDAg was isolated from a Chinese patient infected with HDV by RT-PCR, and a high-efficient expression HD-PQE31 strain was constructed with the fragment. We obtained high titer and good quality hepatitis delta virus protein purified by Ni-NTA metal-affinity chromatography, which was identified by Western blot and ELISA, then we set up the double antigen sandwich ELISA for detection of anti-HDV in human serum, and the performance of the sandwich ELISA was evaluated in terms of specificity and sensitivity. Results were: 1) The purified HDAg protein's purity was 90%, and its ELISA titer was 1/100 000. 2) 42 anti-HDV positive sera were detected and showed that the sensitivity of sandwich ELISA was higher than that of competitive ELISA (t=2.44, p<0.01). 3) The inhibitory rates for 2 anti-HDV positive sera by the specific HDAg were 74% and 93% respectively. 4) For the assay of specificity, all 60 samples infected by other hepatitis viruses and 30 normal samples were negative for anti-HDV. These results suggested that the double antigen sandwich ELISA with purified recombinant HDAg showed higher specificity and sensitivity, It can be used in routine laboratories to diagnose the HDV infection.

Objective To compare and analyze the clinical characteristics of acute central cervical spinal cord injury with only upper extremity involvement and with both upper and lower extremity involvement.Methods A retrospective case control study was made on clinical data of 76 patients with acute central cervical spinal cord injury hospitalized from January 2010 to December 2013.Nerve injury involved was only upper extremity in 39 patients (upper extremity group),but both upper and lower extremities in 37 patients (upper-and lower-extremity group).In upper extremity group,there were 35 males and four females,age was 21-80 years [(52.5 ± 13.4) years],injury resulted from traffic accidents in 24 patients,ground-level falls in eight,high-level falls in six and heavy-object hit in one,and level of injury was C3/4 in 16 patients,C4/5 in 14 and C5/6 in nine.In upper-and lower-extremity group,there were 30 males and seven females,age was 36-78 years [(59.6 ± 9.7) years],injury resulted from traffic accidents in 16 patients,ground-level falls in 11,high-level falls in seven and heavyobject hit in three,and level of injury was C3/4in nine patients,C4/sin 18 and C5/6in 10.Sagittal diameter of the cervical spinal canal,maximal canal compromise,maximal spinal cord compression,degenerating factors of the cervical spine and treatment protocols were determined.Upper extremity function was assessed with the American spinal injury association (ASIA) score.Results There were significant differences between upper extremity group and upper-and lower-extremity group in sagittal diameter of the cervical spinal canal [(7.5 ± 1.5)mm ∶ (6.8 ± 1.2)mm],maximal canal compromise [(28.9 ±9.6)％ ∶ (34.9 ± 10.6)％],ASIA score at admission[(31.6 ± 11.8)points ∶ (22.7± 11.3)points)] and ASIA score at last follow-up [(46.2 ± 4.2) points ∶ (40.2 ± 4.0) points] (P ＜ 0.05),while the maximal spinal cord compression in upper extremity group [(15.7 ± 11.9)％] had no significant difference from that in upper-and lowerextremity group [(17.0 ± 10.6) ％] (P ＞ 0.05).Lower prevalence of posterior osteophyte of the vertebral body was noted in upper extremity group than upper-and lower-extremity group (15％ ∶ 51％) (P ＜0.01).Twenty patients (49％) in upper extremity group were surgically treated,while 31 patients (84％) in upperand lower-extremity group (P ＜ 0.05).Conclusions Compared to acute central cervical spinal cord injury with both upper and lower extremity involvement,the injury with only upper extremity involvement is much common in younger patients and is characterized by lowered frequency of osteophyte,large buffer space,mild nerve damage,preferred non-operation treatment and good prognosis.

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.

Objective To characterize the distribution of conjunctival sac flora and antibiotic susceptibility in healthy people of different ages.Methods Totally 1 839 conjunctival sac secretion samples from 1 131 people without infectious eye diseases attending Qingdao Eye Hospital of Shandong First Medical University from 2018 to 2021 were retrospectively analyzed.These people were divided into four groups according to age:children(≤18 years old),young(19-45 years old),middle-aged(46-65 years old),and elderly(＞65 years old).Differences in the distribution of conjunctival sac flo-ra and antibiotic susceptibility among these groups were compared using the chi-square test or Fisher's exact test.The Bonferroni method was used for pairwise comparisons.Results The total bacterial detection rate of the conjunctival sac in the study population aged 1 month to 91 years old(median:11.00 years old;quartiles:5.00 years old,40.00 years old)was 64.93％;the bacterial detection rate in the children group(76.79％)was significantly higher than that in the young,middle-aged and elderly groups(all P＜0.001).Staphylococcus epidermidis(55.97％),Staphylococcus aureus(8.60％)and Streptococcus mitis(7.52％)were the most common bacteria in the conjunctival sacs.Gram-positive bacteria were re-sistant to most penicillins(16.79％-53.47％)and macrolides(17.21％-20.87％)antibiotics,and highly susceptive to van-comycin(99.56％)and gatifloxacin(98.57％).Gram-negative bacteria had low susceptibility rates to cephalosporin(41.38％-80.00％)antibiotics and were susceptive to amikacin(90.48％)and fluoroquinolone(90.91％-97.73％)antibi-otics.Of the common antibiotics used in ophthalmology,the susceptibility rate of fusidic acid was the worst in the children group(54.55％),which was significantly lower than the other three groups(all P＜0.001).Gatifloxacin had a susceptibili-ty rate of more than 90％in different Gram-stained bacteria in all groups,with an overall sensitivity rate of 98.44％.Con-clusion Staphylococcus epidermidis is the most common bacteria in the conjunctival sac in healthy people of all ages.Gatifloxacin is generally susceptive in different Gram-stained bacteria in people at different ages,and may be considered as the first choice for perioperative empiric antibiotics in ophthalmology.

Objective To explore the early clinical efficacy of 3D printing technology-assisted customized prosthesis with preservation of femoral condyles for the reconstruction of bone defects after resection of distal femoral malignancies.Methods From June 2019 to September 2021,our department determined the lesion range of 12 cases of malignant tumors in the distal femur through CT and MRI before surgery.We used computer-aided production of osteotomy guide plates and customized prostheses that preserved the femoral condyle.After the prostheses were customized,the tumor segment was removed and a reconstruction was performed by using the customized prosthesis with preservation of femoral condyles.The knee function was evaluated by the American Musculoskeletal Tumor Society(MSTS)functional evaluation criteria.Results The operative time was 180-270 min,with a mean of 200 min.The intraoperative bleeding was 300-800 ml,with a mean of 450 ml.A total of 12 cases were followed up for 12-50 months(average,21.9 months).Two patients died of lung metastasis at 12 and 16 months after surgery,one patient survived with lung metastasis at 3 months after surgery,and one patient(8.3%)developed periprosthetic infection at 1 year after surgery.In 10 patients,imaging showed that the prosthesis and plate were in a good position without signs of loosening or bone resorption,the proximal femur was fixed in place with the 3D printed collar,and continuous bone scab formation was found in 4 cases.At the final follow-up,the functional status of the affected limb was good,with the knee flexion of 105°-135°(mean,115°)and no limitation of extension.The MSTS score was 22-28 points[mean,(24.3±1.8)points].Conclusion Application of 3D printing-assisted customized prosthetic replacement technology with preservation of the femoral condyles allows for precise resection of malignant distal femoral tumors and effective anatomical reconstruction without increasing surgical trauma and risk,significantly improving postoperative limb function and potentially reducing long-term complications.

Objective:To investigate the effects of ursolic acid (UA) on oxidative stress and inflammatory factors in a rat model of AR after PM2.5 exposure.Methods:Sixty healthy female SD rats were randomly divided into five groups: normal control group (NC group), PM2.5 unexposed AR group (AR group), PM2.5 exposed AR group (ARE group), UA intervention AR group (AR+UA group), and UA intervention PM2.5 exposed AR group (ARE+UA group), with 12 rats in each group. AR model was performed by a basal sensitization with intraperitoneal injection of ovalbumin (OVA) and followed by nasal instillation. PM2.5 exposure was carried out by inhalation exposure system at a concentration of 200 μg/m 3 for 3 h/d for 30 days. UA intervention group was given UA intragastric administration at 20 mg/(kg·d). AR symptoms including sneezing, nasal scratching and nasal secretion of rats in each group were observed. The activities of superoxide dismutase (SOD) and the level of malondialdehyde (MDA) in nasal mucosa were tested. The pathological changes of nasal mucosa were observed by HE staining. The levels of OVA-sIgE, IL-6 and IL-17 in serum were measured by enzyme-linked immunosorbent assay (ELISA). Protein microarray was used to measure the expression of multiple inflammation cell factors in nasal mucosa. Statistical analysis was performed with SPSS 20.0. Results:After UA intervention, the frequency of nasal sneezing, scratching and nasal secretion in ARE+UA group were lower than those of ARE group ( P<0.05). Pathological examination of nasal mucosa showed that ARE+UA group had less inflammatory granulocyte infiltration and less pathological damage to the epithelial layer than ARE group. The activities of SOD in nasal mucosa of ARE+UA group were higher than those of ARE group ((50.10±3.09) U/mg vs (20.13±1.30) U/mg, F value was 597.54, P<0.01). The contents of MDA in nasal mucosa of ARE+UA group were lower than those of ARE group ((57.78±12.36) nmol/g vs (124.12±9.40) nmol/g, F value was 115.51, P<0.01). The expression levels of OVA-sIgE, IL-6 and IL-17 proteins were lower in the ARE+UA group than those in ARE group ((11.61±0.27) ng/ml vs (20.30±0.67) ng/ml, (47.59±15.49) pg/ml vs (98.83±10.98) pg/ml, (623.30±8.75) pg/ml vs (913.32±9.06) pg/ml, F value was 283.42, 80.45, 683.73, respectively, all P<0.01). After UA intervention, protein microarray analysis showed that the expression of IL-4, IL-6, IL-13, chemokine CXCL7, IL-1α, IL-1β, MMP-8 and MCP-1 in ARE+UA group was decreased compared with ARE group while IFN-γ and IL-10 increased (all P<0.01). Conclusion:UA can reduce the aggravated AR symptoms and pathological damage of nasal mucosa, inhibit oxidative stress and release of inflammatory factors after PM2.5 exposure, and thus plays a protective role in the pathological damage of AR induced by PM2.5 exposure.