AIM:To observe the toxic effects of zinc oxide nanoparticles(ZnO NPs)on cornea by constructing intoxicated model in vivo and in vitro.METHODS:Human corneal epithelial cells(HCEpiC)were cultured in vitro and exposed to different concentrations(0.5, 5, 12.5, 25, 50, 100, 250 μg/mL)of ZnO NPs for 24h. The cell culture medium without nano-solution was used as the blank control group. The viability of the cells was assessed by MTT assay. Three different concentrations(25, 50 and 100 μg/mL)of ZnONPs dispersions were exposed to the conjunctival sac of anesthetized mice three times a day for 7d consecutively. The phosphate buffered saline(PBS)eye group was the PBS control group. Corneal morphology was observed on 1, 3, 5 and 7d, and the eyes were removed on 8d for various laboratory examinations, including corneal pathological changes and expression levels of inflammatory factors(TNF-α, IL-6).RESULTS:After treatment of HCEpiC cells with different concentrations of ZnO NPs for 24h, the MTT results showed that Zno NPs cause damage to cells at 0.5 μg/mL, and the cell survival rate was about 80%(P<0.05). Half of the cells were killed at a dose of 5 μg/mL, the damaging effect on cells in the concentration range of 5～250 μg/mL was concentration-dependent(P<0.0001). After 7d of conjunctival capsule spotting in mice, dot-like staining of fluorescein was seen in the 25 μg/mL ZnO NPs and 50 μg/mL ZnO NPs groups. Localized circular fluorescein stained areas were seen in the corneas of the 100 μg/mL ZnO NPs group. HE staining showed that the corneal epithelial layer, stromal layer thickness and stromal layer immune cell number did not change significantly in the 25 μg/mL and 50 μg/mL ZnO NPs groups(all P>0.05), while the corneal epithelial layer thinned, the corneal stromal layer thickened and the stromal layer immune cells increased significantly in the 100 μg/mL ZnO NPs group(all P<0.05). Immunohistochemical staining showed that the number of corneal stromal immune cells producing TNF-α and IL-6 and the mean integral optical density(IOD)values of TNF-α and IL-6 were significantly higher in the 100 μg/mL ZnO NPs group than in the PBS control group(P<0.05), and the degree of inflammation response was concentration-dependent. Compared with the PBS control group, no significant increase in immune cell count and IOD values in the 25 μg/mL ZnO NPs and 50 μg/mL ZnO NPs groups(P>0.05).CONCLUSION:The toxic damaging effect of ZnO NPs on the cornea was confirmed from both in vitro and in vivo, which provided a theoretical basis for the ocular safety evaluation of ZnO NPs.

Objective To explore the protective mechanism of RNA binding protein LIN28 on diabetic nephropathy(DN).Methods LIN28 was overexpressed or knocked down by adenovirus in HEK 293T cells.The gene and functional signaling pathway significantly changed after overexpression of LIN28 were obtained through RNA Seq transcriptome sequencing and bioinformatics analysis.HEK 293T cells were divided into the Control group,the Treatment group,the Adv-LIN28-OE+D-ribose group and the Adv-LIN28-NC+D-ribose group in in vitro LIN28 overexpression studies.And HEK 293T cells were divided into the Control group,the Treatment group,the Adv-shRNA LIN28+D-ribose group and the Adv-shRNA NT+D-ribose group in in vitro LIN28 knockdown studies.ELISA was used to detect the levels of human advanced glycation end products(AGEs)of cells supernatants in the above groups.Western blot was used to detect the expression levels of RAGE,NF-κB and MMP-2 of cells in the above groups.Results The results of RNA-Seq transcriptome sequencing,GO analysis and KEGG analysis showed that overexpression of LIN28 resulted in a significant down-regulation of AGE-RAGE signaling pathway in HEK 293T cells(P<0.05).The results of ELISA showed that compared with the Control group,the cell in the Treatment group produced a large amount of AGEs(P<0.05);compared with the Treatment group,the AGEs level of cells in the Adv-LIN28-OE+D-ribose group was significantly decreased(P<0.05),while the AGEs level of cells in the Adv-shRNA LIN28+D-ribose group was increased(P>0.05).The results of Western blot showed that compared with the Control group,the expression levels of RAGE,NF-κB and MMP-2 of cells in the Treatment group were significantly increased(P<0.05);compared with the Treatment group,the expression levels of RAGE,NF-κB and MMP-2 of cells in the Adv-LIN28-OE+D-ribose group were significantly decreased(P<0.05),while the expression levels of RAGE,NF-κB and MMP-2 of cells in the Adv-shRNA LIN28+D-ribose group were significantly increased(P>0.05).Conclusion Overexpression of LIN28 by adenovirus at the cellular level in vitro can lead to significant differential expression of thousands of genes.In particular,it can inhibit the diabetes and complications-related AGE-RAGE signaling pathway,which is critical in the progression of DN disease,and play a role in protecting DN.

AIM:To investigate the correlation between demodex infection with corneal cell density changes and ocular surface function in patients with blepharo kerato conjunctivitis(BKC).

METHODS: Ninety-four patients with BKC(BKC group)at Department of Ophthalmology of our hospital from July 2019 to July 2020 were selected as the research objects, in addition, 80 matched healthy volunteers were selected as control group. The BKC patients were divided into infected group(45 cases)and uninfected group(49 cases)according to whether they were infected with demodex. According to the number of demodex detected in eyelashes, there were 17 cases of suspicious infection, 18 cases of moderate infection and 10 cases of severe infection. All subjects were examined by laser confocal microscopy, and the cell density in the superficial stromal layer of the central cornea and peripheral cornea was calculated. The ocular surface function \〖Schirmer test, Ocular Surface Disease Index(OSDI)\〗, eyelid margin abnormality score, corneal fluorescence stain and tear film break-up time(TF-BUT)of patients with BKC were examined, and the correlation between demodex infection with corneal cell density and ocular surface function in patients with BKC was analyzed.

RESULTS: Compared with those in the control group, the cell density in the superficial stromal layer of the central cornea and peripheral cornea was lower in the BKC group(P<0.05), and the OSDI, eyelid margin abnormality score and corneal fluorescence stain score were higher(P<0.05); the cell density in the superficial stromal layer of the central cornea and peripheral cornea of patients in uninfected group, patients with suspicious demodex infection, moderate demodex infection and severe demodex infection decreased in turn(P<0.05), and the OSDI, eyelid margin abnormality score and corneal fluorescence stain score increased significantly in turn(P<0.05); the degree of demodex infection was negatively correlated with the cell density in the superficial stromal layer of the central cornea and peripheral cornea in patients with BKC(P<0.05), and was positively correlated with OSDI, eyelid margin abnormality score and corneal fluorescence stain score(P<0.05).

CONCLUSION: The severity of demodex infection has a significant negative correlation with the cell density in the superficial stromal layer of the central cornea and peripheral cornea in patients with BKC, has a significant positive correlation in patients with ocular surface dysfunction.

AIM:To investigate the correlation between demodex infection with corneal cell density changes and ocular surface function in patients with blepharo kerato conjunctivitis(BKC).

METHODS: Ninety-four patients with BKC(BKC group)at Department of Ophthalmology of our hospital from July 2019 to July 2020 were selected as the research objects, in addition, 80 matched healthy volunteers were selected as control group. The BKC patients were divided into infected group(45 cases)and uninfected group(49 cases)according to whether they were infected with demodex. According to the number of demodex detected in eyelashes, there were 17 cases of suspicious infection, 18 cases of moderate infection and 10 cases of severe infection. All subjects were examined by laser confocal microscopy, and the cell density in the superficial stromal layer of the central cornea and peripheral cornea was calculated. The ocular surface function \〖Schirmer test, Ocular Surface Disease Index(OSDI)\〗, eyelid margin abnormality score, corneal fluorescence stain and tear film break-up time(TF-BUT)of patients with BKC were examined, and the correlation between demodex infection with corneal cell density and ocular surface function in patients with BKC was analyzed.

RESULTS: Compared with those in the control group, the cell density in the superficial stromal layer of the central cornea and peripheral cornea was lower in the BKC group(P<0.05), and the OSDI, eyelid margin abnormality score and corneal fluorescence stain score were higher(P<0.05); the cell density in the superficial stromal layer of the central cornea and peripheral cornea of patients in uninfected group, patients with suspicious demodex infection, moderate demodex infection and severe demodex infection decreased in turn(P<0.05), and the OSDI, eyelid margin abnormality score and corneal fluorescence stain score increased significantly in turn(P<0.05); the degree of demodex infection was negatively correlated with the cell density in the superficial stromal layer of the central cornea and peripheral cornea in patients with BKC(P<0.05), and was positively correlated with OSDI, eyelid margin abnormality score and corneal fluorescence stain score(P<0.05).

CONCLUSION: The severity of demodex infection has a significant negative correlation with the cell density in the superficial stromal layer of the central cornea and peripheral cornea in patients with BKC, has a significant positive correlation in patients with ocular surface dysfunction.

Berberis amurensis (Berberidaceae) is a traditional Chinese medicine, which is often used to treat hypertension, inflammation, dysentery and enteritis. It contains alkaloids, mainly including berberine, berbamine, magnoflorine, jatrorrhizine and palmatine. Berberis amurensis extracts (BAEs) is often orally taken. Oral herbs might be metabolized by intestinal bacteria in the small intestine. However, the interaction between the herb and the gut microbiota is still unknown. In the current study, UPLC/Q-TOF-MS/MS combined with Metabolitepilot and Peakview software was used to identify the metabolites of BAEs in anti-biotic cocktail induced pseudo germ-free rats and normal rats. As a result, a total of 46 metabolites in normal rats were detected and its main metabolic pathways include demethylation, dehydrogenation, methylation, hydroxylation, sulfation and glucuronidation. Only 29 metabolites existed in pseudo germ-free rats. Dehydrogenated metabolites (M29, M30, M34 and M36), methylated metabolites (M33, M41 and M46) and other metabolites were not detected in pseudo germ-free rats. The result implied that the intestinal bacteria have an influence on the metabolism of BAEs. Furthermore, this investigation might contribute to the understanding of the metabolism of BAEs, and further promote its clinical application.
Alkaloids ; Animals ; Berberis ; Chromatography, High Pressure Liquid ; Drugs, Chinese Herbal ; Rats ; Tandem Mass Spectrometry

Betacoronavirus ; Consensus ; Coronavirus Infections ; complications ; epidemiology ; prevention & control ; Epidemics ; Humans ; Minimally Invasive Surgical Procedures ; Musculoskeletal Diseases ; complications ; therapy ; Pandemics ; prevention & control ; Pneumonia, Viral ; complications ; epidemiology ; prevention & control

OBJECTIVE: To explore the mechanisms of angiogenesis in chronic myeloid leukemia (CML) through detecting the levels of angiogenesis-related factors secreted from K562 cells after overexpression and interference of HIF-1α gene in K562 cells. METHODS: The K562 cells were transfected by lentiviruses carried and interfered HIF-1α gene, then the transtected K562 cells with carried and interfered with HIF-1α gene were enrolled in overexpression and interference groups respectively, at the same time the K562 cells transfected by the empty virus were enrolled in control group. The cells were harvested after culture for 72 hours under normoxid condition. The transfection efficient in 3 groups was detected by fluorescence microscopy; the mRNA expression of HIF-1α gene and angiogenesis-related factors was detected by RT-PCR; the concentration of angiogenesis-related factors in the caltured supernatant was detected by ELISA. RESULTS: The optimal MOI of K562 cells transfected with lentivirus was 10 and the transfection efficiency was about 50%. The positive rate of transfection after screening by puromycin was more than 90%. The mRNA expression of ANG-I, ANG-II, TGF-α and VEGF in the interference group was lower than that in the over-expression group, and the TGF-β1 mRNA expression in the interference group was higher than in the over-expression group. The mRNA expression of ANG-I and VEGF in the interference group was lower than that in the control group. TGF-αdid not could be detected, and the culture supernatant concentration of ANG-I and TNF-α in the interference group was lower than in the over-expression group, while the VEGF concentration in the interference group was higher than that in the over-expression group. All of the above-mentioned differences were statistically significant (P＜0.05). CONCLUSION The positive K562 cells transfected with leutivirus have been harvested by screening with puromycin. The HIF-1α mRNA positively regulates the mRNA expression of ANG-1, ANG-2, TGF-α, VEGF in K562 cells, promotes the antocrine ability of ANG-1 and TNF-α, moreover not stimulates the autocrine of TGF-α, the up-regulation of HIF-1α expression can inhibit the expression TGF-β1 in K562 cells and the autocrine of TGF-β1.
Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; metabolism ; K562 Cells ; RNA, Messenger ; Vascular Endothelial Growth Factor A

Objective To investigate the protective effect of sodium butyrate on the neonatal mouse model of necrotizing enterocolitis and analyze its possible mechanism.Methods Sixty c57BL/6 neonatal mice were randomly divided into two groups (n=30):PBS group and butyric acid group.At the third day after birth,mice in both groups were respectively given PBS and sodium butyrate solution by gavage once a day for 7 days,and neonatal necrotizing enterocolitis (NEC) model was established by hypoxia,cold stimulation and artificial feeding.The newborn mice were sacrificed overnight after modeling.HE staining and double-blind pathological score were performed to observe the pathological changes of ileocecal intestinal tissue.The mRNA expressions of IL-6,IL-10,TGF-β1 and TNF-a were tested by quantitative real-time PCR.The levels of IL-10 and TGF-β1 in intestine tissues were evaluated using ELISA.Flow cytometry was used to analyze the ratio of regulatory T cells (Treg) on CD4+ T cells in both groups.Results When mice were sacrificed overnight after NEC modeling,the body weight was significantly higher in butyric acid group (4.50 ± 0.42g) than in PBS group (4.16 ± 0.60g,P＜0.05);No significant difference (P＞0.05) existed in survival rate of butyric acid group (76.34％) and PBS group (67.95％).The pathological damage score of intestinal tissue showed that the median score of intestinal injury was significantly lower in butyric acid group [1.33(1.33-1.67)] than in PBS group [2.00(1.67-2.25),P＜0.05].qPCR demonstrated that the expressions of IL-6 and TNF-α mRNA were obviously lower in butyric acid group than in PBS group (0.85 ± 0.30 vs.1.77 ± 0.49 and 0.41 ± 0.25 vs.0.96 ± 0.56,respectively,P＜0.05);and the expressions of IL-10 and TGF-β1 mRNA were markedly higher in butyric acid group than in PBS group (1.91 ± 0.82 vs.0.94 ± 0.43 and 1.46 ± 0.57 vs.0.88 ± 0.29,respectively,P＜0.05);Intestinal tissue ELISA results showed that the expressions of IL-10 and TGF-β1 were higher in butyric acid group than in PBS group (68.60 ± 15.06 vs.37.25 ± 5.81 and 424.93 ± 19.34 vs.127.31 ± 60.83,respectively,P＜0.05);Flow cytometry revealed that the proportion of regulatory T cells (Treg) of CD4+ T cells was higher in butyric acid group than in PBS group (12.68％ ± 6.79％ vs.3.57％ ± 0.88％,P＜0.05).Conclusions Butyric acid plays a protective effect in the intestinal injury of neonatal mouse model of necrotizing enterocolitis.The possible mechanism is that butyrate can down-regulate the expressions of cytokines IL-6 and TNF-o,up-regulate the expressions of cytokines IL-10 and TGF-β1,and promote the differentiation of T cells into Treg cells.

· AIM:To investigate the effect of long-term wearing rigid gas permeable (RGP) contact lenses on the stability of tear film,and to observe its safety.· METHODS:Totally 60 myopia patients (120 eyes) wearing RGP in our hospital from October 2014 to January 2016 and 30 low myopia patients (60 eyes) without a history of wearing RGP were selected.There were 26 patients (52 eyes) wearing RGP at daytime were included into daytime wearing group;34 patients (68 eyes) only wearing RGP at night were set as the night wearing group.And patients without wearing RGP were set as control group.Schirmer Ⅰ test (S Ⅰ t),tear film fern experiments,and break-up time (BUT) of tear film were performed before wearing RGP,wearing for 1 mo,wearing for 6mo,wearing for 1a,wearing for 2a,respectively.· RESULTS:After wearing RGP,the BUT and S Ⅰ t of daytime wearing group and night wearing group significantly decreased,and were significantly lower than those of the control group at the time points,the difference was statistically significant (P＜ 0.05).After wearing for 6mo,1a and 2a,the BUT and S Ⅰ t of daytime wearing group was significantly lower than that of the night wearing group,the statistic found significant difference (P＜0.05).The three groups had no statistically significant differences in tear fern classification experiment before wearing RGP and wearing for 1 and 6mo (P＞0.05).The number of the eyes ≥ Ⅲ in daytime wearing group and night wearing group was significantly higher than that of the control group after wearing for 1 and 2a,the difference was statistically significant (P＜ 0.05),but the difference between daytime wearing group and night wearing group found no significant difference (P＞0.05).· CONCLUSION:Wearing RGP can result in reducing tear secretion and decreasing the stability of tear film.

Objective A variety of secondary metabolites can be obtained after the fermentation of medicinal fungi.The con-tent of these metabolites is much higher than that of cultivated medicinal fungi. We aimed to preliminarily study on the apoptosis of fer-mented Inonotus obliquus(FIO) on hepatocellular carcinoma HepG-2 cells. Methods The experiment was divided into 3 groups:the control group(The same volume of drug-free medium),the UIO group(200 mg/L) and the FIO group(200 mg/L). The contents of polysaccharides,terpenoids and polyphenols in unfermented Inonotus Obliquus (UIO) and FIO were determined by sulfuric acid phenol method,Folin-Ciocalteu method and colorimetric method. Effects of Fermented Inonotus obliquus on apoptosis of HepG-2 Cells were measured by MTT Method and Flow Cytometry. Results The contents of polysaccharides,terpenoids and polyphenols were sig-nificantly increased in FIO group [(10.140±0.849)mg/mL,(1.774±0.001)mg/mL, (9.979±0.022)mg/mL] compared with thatin UIO group [(7.161±0.305)mg/mL,(1.358±0.004)mg/mL,(6.314±0.237)mg/mL](P<0.01). Both UIO and FIO group induced apoptosis of HepG-2 cells according to Annexin V/PI double staining. Compared with the control group(4.3%),the apoptosis rate in-creased in UIO and FIO group (23.14%,27.37%) (P<0.05). In addition, the apoptosis rate was higher in FIO group than that in UIO group (P<0.05). Compared with the control group, the ratio of G0/G1phase cells was significantly increased and the ratio of S phase and G2/M phase was significantly decreased in UIO and FIO group (P<0.01). G0/G1phase cell ratio differences were statistically signif-icant compared with the UIO group (P<0.05) Conclusion FIO can better induce the apoptosis of HepG-2 cells than UIO.