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Background: To analyze the effects of oral alkali therapy on renal function, nutritional status and bone density in patients of diabetic kidney disease. Material & Methods: A randomized controlled trial was conducted on 60 patients of age>18 years with diabetic kidney disease who were not on dialysis and had plasma bicarbonate levels between 16 and 20 mmol/l. Patients were randomly divided into two groups: Test group (n=30) which received oral alkali therapy as sodium bicarbonate and control group (n=30) who did not receive oral alkali therapy. The patients were followed for 12 months to compare the improvement. Results: In comparison to controls, test group showed a significant improvement in the Hb (0.7 vs. 0.25, P =0.003), significantly less decrease in eGFR (-2.25 vs. -2.9, P=0.049), non-significant less increase in creatinine (-0.26 ± 0.4 vs. -0.43 ± 0.33, P=0.09), significant improvement in bicarbonate levels (7.5 vs. 1, p<0.0001), significant restoration of albumin (0.32 vs. 0.05, P<.0001), significant fall in iPTH levels (50 vs. 25, p=0.007) and ALP levels (32 vs. 12, p=0.015). Bone density (0.28 ± 0.17 vs. 0.01 ± 0.13, P<.0001) and clinical well-being VAS scores improved significantly among the cases (9.83 ± 5.65 vs. -1.67 ± 7.11, P<.0001). Conclusion: In conclusion, oral alkali therapy slows the rate of decline of renal function and the development of end stage renal disease in patients with advanced stages of CKD. This cheap and simple strategy, which is in line with current renal consensus documents, also improves the nutritional status of patients and bone density.
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Objective:To investigate the role of microRNA (miR)-497 in the formation of corneal neovascularization (CNV) induced by alkali burn and its mechanism.Methods:Forty-two wild type (WT) C57BL/6 mice aged 6 to 8 weeks, 42 CRISPR/Cas9 mediated miR-497 knockout (KO) and 42 CRISPR/Cas9 mediated overexpression transgenic (TG) C57BL/6 mice were selected and assigned as WT group, KO group and TG group, respectively.The corneal alkali burn model was established.At 3, 7, 14 and 21 days after modeling, corneal epithelium damage and stromal turbidity were scored according to slit lamp microscopy.The area of neovascularization was measured.Corneal structural changes and expression of inflammatory cells were observed by histopathological staining.The expression of CD31 in corneal tissues was detected by immunohistochemistry staining.The targeted binding relationship between miR-497 and signal transducer and activator of transcription 3 (STAT3) was detected by luciferase reporter assay.The relative expressions of miR-497, vascular endothelial growth factor A (VEGFA), tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-1β and macrophage inflammatory protein (MCP)-1 mRNA were detected by real-time quantitative PCR.At 14 days following modeling, the expression of STAT3 and p-STAT3 proteins in mice corneal tissues was detected by Western blot.The use and care of animals complied with the ARVO statement.The study protocol was approved by the Ethics Committee of Renmin Hospital of Wuhan University (No.2019K-K010).Results:Corneal injury, inflammatory cell infiltration and CNV occurred in mice cornea after alkali burn.Corneal epithelial injury score, corneal stromal turbidity score and CNV area increased first and reached the peak on the 14th day after modeling, and then decreased.There were significant differences in corneal epithelial injury score, corneal stromal turbidity score, CNV area and number of CD31-positive cells among various time points after alkali burn ( Fgroup=49.19, 34.56, 44.56, 77.56; all at P<0.01; Ftime=51.62, 65.62, 71.32, 46.12; all at P<0.01). Corneal epithelial injury score, corneal stromal turbidity score, CNV area and the number of CD31-positive cells were greater in KO group at various time points than in WT and TG groups, and those in WT group were greater than in TG group (all at P<0.05). In WT STAT3 co-transfected cells, the luciferase activity of the miR-497 group was significantly lower than that of the miR-negative control group and normal control group (both at P<0.05). In mutant STAT3-transfected cells, there was no significant difference in luciferase activity among all groups ( F=0.69, P=0.56). On the 14th day after modeling, the relative expression levels of miR-497 in corneal tissue of WT, KO and TG groups were 0.68±0.11, 0.41±0.06 and 1.05±0.14, respectively, which were significantly lower than 1.00±0.04, 0.56±0.07 and 1.34±0.11 before modeling (all at P<0.01). The relative expressions of STAT3 and p-STAT3 were higher in KO group than in WT and TG groups, and were lower in TG group than in WT group, and the differences were statistically significant (all at P<0.05). The expressions of VEGFA, TNF-α, IL-6, IL-1β and MCP-1 mRNA at various time points after modeling in various groups were significantly higher than before modeling, which were higher in KO group than in WT and TG groups and were lower in TG group than in WT group, and the differences were statistically significant (all at P<0.01). Conclusions:MiR-497 inhibits corneal inflammation and CNV formation induced by alkali burn.It might inhibit the activation of the inflammation signal pathway via targeting STAT3.
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Abstract As an important enzyme, xylanase is widely used in the food, pulp, and textile industry. Different applications of xylanase warrant specific conditions including temperature and pH. This study aimed to carry out sodium alginate beads as carrier to immobilize previous reported mutated xylanase from Neocallimastix patriciarum which expressed in E. coli, the activity of immobilization of mutated xylanase was elevated about 4% at pH 6 and 13% at 62 °C. Moreover, the immobilized mutated xylanase retained a greater proportion of its activity than the wide type in thermostability. These properties suggested that the immobilization of mutated xylanase has potential to apply in biobleaching industry.
Resumo Como importante enzima, a xilanase é amplamente utilizada na indústria alimentícia, de celulose e têxtil. Diferentes aplicações de xilanase garantem condições específicas, incluindo temperatura e pH. Este estudo teve como objetivo realizar grânulos de alginato de sódio como carreador para imobilizar xilanase mutada relatada anteriormente de Neocallimastix patriciarum que expressa em E. coli, a atividade de imobilização da xilanase mutada foi elevada em cerca de 4% em pH 6 e 13% a 62 °C. Além disso, a xilanase mutada imobilizada reteve uma proporção maior de sua atividade do que o tipo amplo em termoestabilidade. Essas propriedades sugerem que a imobilização da xilanase mutada tem potencial para aplicação na indústria de biobranqueamento.
Subject(s)
Neocallimastix , Temperature , Escherichia coli/geneticsABSTRACT
【Objective】 To explore the effect of lactate and alkali deficiency on the need for red blood cell transfusion in emergency of patients with traumatic hemorrhagic shock. 【Methods】 A total of 126 patients with traumatic hemorrhagic shock in our hospital from January 2019 to December 2021 were retrospectively analyzed, and the 99 cases with effective treatment were divided into two groups according to the outcome of blood transfusion within 24 hours after admission: non-transfusion group (n=36) and transfusion group (n=63). The changes of lactic acid (Lac), alkali deficiency (BE), hemoglobin (Hb), hematocrit (Hct) at admission, hemoglobin (Hb), hematocrit (Hct) 24 hours after admission and the length of stay in ICU were compared between the two groups. The binary logistic regression was used to analyze the risk factors of whether there was a need for blood transfusion at the time of emergency admission. The correlation between individual and combined indicators of each risk factor and the need for blood transfusion were analyzed by the receiver operating curve (ROC). 【Results】 The mean level of Lac (2.90±1.82) in the non-transfusion group at admission was lower than that in the transfusion group (5.80±2.83) (P0.05)The maximum AUG of Lac and BE(0.875, 0.766) in predicting the need for emergency red blood cell transfusion in patients with traumatic hemorrhagic shock was significantly better than that of Hb and Hct (0.692, 0.682); the optimal threshold for Lac was >3.6 mmol/L, while the optimal threshold for Hb is ≤106 g/L; the maximum AUG obtained by ROC curve analysis combined with Lac, BE, Hb and Hct was 0.910, which was higher than that of the sole virable. Comparative predictive value using the optimal thresholds of Lac and Hb as indications for transfusion showed that Lac had better predictive value than Hb. 【Conclusion】 Lac and be can be instructive for patients with traumatic hemorrhagic shock as to whether they need red blood cell transfusion in an emergency setting, and combination of Lac, BE, Hb and Hct may help to determine the transfusion needs of patients more timely and accurately and optimize the transfusion management of emergency patients.
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AIM: To evaluate the efficacy of transplantation of human umbilical cord mesenchymal stem cells(hUCMSCs)in the treatment of corneal alkali burn in rabbits, and study the infiltration of polymorphonuclear neutrophils(PMNs)and the changes of vascular endothelial growth factor(VEGF)expression.METHODS: Corneal alkali burn models were established in right eyes of 75 healthy Japanese white rabbits, which were divided into three groups(group A, B and C), with 25 rabbits in each group. Group A was treated with amniotic membrane combined with hUCMSCs on the day after corneal alkali burn. Group B was treated with amniotic membrane only. Group C did not give any treatment after corneal alkali burn. At 3, 7, 14, 21 and 28d after corneal alkali burn, the corneal recovery was observed by slit lamp and photographed, the growth of corneal neovascularization(CNV)was scored, and corneal tissue was separated to make pathological sections. PMNs infiltration was observed by hematoxylin-eosin(HE)staining, and the expression of VEGF was determined by immunohistochemical staining.RESULTS: The growth of CNV in group A was much slower than that in group B at 14d after alkali burn. The CNV growth score around lesions of group A was significantly lower than that of group B(P<0.05). The quantity of PMNs increased on the 3d with the stromal layer of cornea infiltrated, relatively decreased on the 7d, shown a peak on the 14d, and then decreased gradually. Early infiltration after alkali burn was in the corneal stroma of the lesion area, and the extent of infiltration was equal to the ulcer area at later stage. The cell densities of corneal PMNs in group A and group B were significantly lower than those in group C at all time points after alkali burns(P<0.05), and those in group A were significantly lower than group B at 14 and 21d(P<0.05). The expression levels of corneal VEGF in all groups after alkali burn reached peak at 7～14d and decreased significantly at 28d, and the expression levels of VEGF in group A and group B at all time points after alkali burn were significantly lower than those in group C(P<0.05), and group A was significantly lower than that in group B at 7, 14 and 21d(P<0.05).CONCLUSION: The transplantation of hUCMSCs after alkali burn cornea can reduce the formation of CNV and inhibit corneal revascularization after alkali burn. The corneal pathological lesions and vascularization are closely related to PMNs and VEGF.
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To improve the quality control methods of Poria and develop and utilize its resources fully, alkaline extraction was used in this study to determine the yield and content of alkali-soluble polysaccharides of Poria. The alkali-soluble extracts of Poria were obtained according to the optimum extraction conditions on the basis of single-factor test, and 30 batches of samples were determined. The structure and chemical composition of the alkali-soluble extracts was characterized by high-performance gel permeation chromatography(HPGPC), Fourier transform infrared spectrometry(FT-IR), nuclear magnetic resonance(NMR) spectroscopy and high-performance liquid chromatography(HPLC) with 1-phenyl-3-methyl-5-pyrazolone(PMP-HPLC). The results showed that the content of the alkali-soluble extracts was in the range of 46.98%-73.86%. The main component was β-(1→3)-glucan, and its molecular mass was about 1.093×10~5. Further, the content of alkali-soluble polysaccharides of Poria was measured by UV-Vis spectrophotometry and HPLC coupled with the evaporative light scattering detector(HPLC-ELSD), and 30 batches of samples were measured. The results indicated that the content of alkali-soluble polysaccharides determined by UV-Vis spectrophotometry was in the range of 73.70%-92.57%, and the content of samples from Hubei province was slightly higher than that from Yunnan province, Anhui province and Hunan province. The content of alkali-soluble polysaccharides determined by HPLC-ELSD was in the range of 51.42%-76.69%, and the samples from Hunan province had slightly higher content than that from the other three provinces. The content determined by UV-Vis spectrophotometry was higher than that by HPLC-ELSD. However, the content determined by HPLC-ELSD was close to that of alkali-soluble extract, which could accurately characterize the content of alkali-soluble polysaccharides in Poria, and the method was simple and repeatable. Therefore, it is recommended that the quantitative analysis method for alkali-soluble extract and alkali-soluble polysaccharides by HPLC-ELSD be used in the quality standards of Poria in Chinese Pharmacopeia.
Subject(s)
Poria/chemistry , Spectroscopy, Fourier Transform Infrared , China , Polysaccharides/chemistry , Reference Standards , Chromatography, High Pressure Liquid/methodsABSTRACT
As an important enzyme, xylanase is widely used in the food, pulp, and textile industry. Different applications of xylanase warrant specific conditions including temperature and pH. This study aimed to carry out sodium alginate beads as carrier to immobilize previous reported mutated xylanase from Neocallimastix patriciarum which expressed in E. coli, the activity of immobilization of mutated xylanase was elevated about 4% at pH 6 and 13% at 62 °C. Moreover, the immobilized mutated xylanase retained a greater proportion of its activity than the wide type in thermostability. These properties suggested that the immobilization of mutated xylanase has potential to apply in biobleaching industry.
Como importante enzima, a xilanase é amplamente utilizada na indústria alimentícia, de celulose e têxtil. Diferentes aplicações de xilanase garantem condições específicas, incluindo temperatura e pH. Este estudo teve como objetivo realizar grânulos de alginato de sódio como carreador para imobilizar xilanase mutada relatada anteriormente de Neocallimastix patriciarum que expressa em E. coli, a atividade de imobilização da xilanase mutada foi elevada em cerca de 4% em pH 6 e 13% a 62 °C. Além disso, a xilanase mutada imobilizada reteve uma proporção maior de sua atividade do que o tipo amplo em termoestabilidade. Essas propriedades sugerem que a imobilização da xilanase mutada tem potencial para aplicação na indústria de biobranqueamento.
Subject(s)
Alginates/pharmacokinetics , Neocallimastix , Xylans/analysisABSTRACT
Abstract As an important enzyme, xylanase is widely used in the food, pulp, and textile industry. Different applications of xylanase warrant specific conditions including temperature and pH. This study aimed to carry out sodium alginate beads as carrier to immobilize previous reported mutated xylanase from Neocallimastix patriciarum which expressed in E. coli, the activity of immobilization of mutated xylanase was elevated about 4% at pH 6 and 13% at 62 °C. Moreover, the immobilized mutated xylanase retained a greater proportion of its activity than the wide type in thermostability. These properties suggested that the immobilization of mutated xylanase has potential to apply in biobleaching industry.
Resumo Como importante enzima, a xilanase é amplamente utilizada na indústria alimentícia, de celulose e têxtil. Diferentes aplicações de xilanase garantem condições específicas, incluindo temperatura e pH. Este estudo teve como objetivo realizar grânulos de alginato de sódio como carreador para imobilizar xilanase mutada relatada anteriormente de Neocallimastix patriciarum que expressa em E. coli, a atividade de imobilização da xilanase mutada foi elevada em cerca de 4% em pH 6 e 13% a 62 °C. Além disso, a xilanase mutada imobilizada reteve uma proporção maior de sua atividade do que o tipo amplo em termoestabilidade. Essas propriedades sugerem que a imobilização da xilanase mutada tem potencial para aplicação na indústria de biobranqueamento.
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Objective:To prepare vorinostat encapsulated hydroxypropyl-β-cyclodextrin (SAHA-CD) eye drops and investigate its inhibitory effect on corneal neovascularization (CNV) induced by alkali burns in mouse.Methods:The SAHA-CD eye drops at concentrations of 0.1%, 0.2%and 0.4%were prepared by inclusion technology with hydroxypropyl-β-cyclodextrin, and the content was assayed by high performance liquid chromatography.Seventy-five SPF mice with alkali burn-induced CNV were randomized into 0.1%SAHA-CD group, 0.2%SAHA-CD group, 0.4%SAHA-CD group, dexamethasone group and normal control group according to a random number table, 15 for each group, among which the SAHA-CD groups and dexamethasone group were treated with corresponding drugs, and model control group was treated with normal saline immediately after modeling, four times a day and five microliters each time, lasting for six days.The healing of corneal epithelium was examined with a slit lamp microscope after fluorescein sodium staining, and the areas of cornea epithelial defects were calculated using Eyestudio software.The corneal flat mount was prepared, and the length and areas of CNV were calculated with ImageJ software.The histology of mouse corneas was observed through hematoxylin and eosin staining.The expression level of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and matrix metalloproteinase-9 (MMP-9) in cornea were measured with enzyme linked immunosorbent assay (ELISA) kits.The use and care of animals complied with the ARVO statement and this study protocol was approved by the Experimental Animal Ethics Committee of Henan Eye Institute (No.HNEECA-2020-01).Results:The actual drug contents of the 0.1%, 0.2% and 0.4%SAHA-CD eye drops were 97.62%, 98.33%and 98.14%of the labeled amount.The cornea showed edema and opacification after modeling.On the sixth day after treatment, significant differences were found in the length and areas of CNV among various groups ( F=7.655, 8.802; both at P<0.01).The areas of CNV in 0.2%SAHA-CD, 0.4%SAHA-CD and dexamethasone groups were significantly smaller than model control group, and the length of CNV in 0.1%SAHA-CD, 0.2%SAHA-CD and dexamethasone groups were significantly smaller than model control group (all at P<0.05).On the third and sixth day following modeling, significant differences in the expression levels of VEGF, bFGF and MMP-9 were found among the five groups (third day: F=6.345, 7.149, 18.650; all at P<0.01; sixth day: F=6.749, 5.105, 5.023; all at P<0.01), and the expression levels of VEGF, bFGF and MMP-9 in 0.2%SAHA-CD group were significantly lower than those in 0.1%SAHA-CD group, 0.4%SAHA-CD group and model control group (all at P<0.05). Conclusions:SAHA-CD eye drops can inhibit alkali burn-induced CNV in mouse.
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Chromium is a harmful contaminant showing mutagenicity and carcinogenicity.Therefore,detection of chromium requires the development of low-cost and high-sensitivity sensors.Herein,blue-fluorescent carbon quantum dots were synthesized by one-step hydrothermal method from alkali-soluble Poria cocos polysaccharide,which is green source,cheap and easy to obtain,and has no pharmacological ac-tivity due to low water solubility.These carbon quantum dots exhibit good fluorescence stability,water solubility,anti-interference and low cytotoxicity,and can be specifically combined with the detection of Cr(Ⅵ)to form a non-fluorescent complex that causes fluorescence quenching,so they can be used as a label-free nanosensor.High-sensitivity detection of Cr(Ⅵ)was achieved through internal filtering and static quenching effects.The fluorescence quenching degree of carbon dots fluorescent probe showed a good linear relationship with Cr(Ⅵ)concentration in the range of 1-100 μM.The linear equation was F0/F=0.9942+0.01472[Cr(Ⅵ)](R2=0.9922),and the detection limit can be as low as 0.25 μM(S/N=3),which has been successfully applied to Cr(Ⅵ)detection in actual water samples herein.
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Objective@#To compare the efficiency and biocompatibility of four different silanes on immobilizing c(RGDfK) peptide on titanium surface.@*Methods @# After alkali-heat treatment (group OH), the titanium surface was treated with 3-aminopropyltriethoxysilane (APTES) (group OHAP), 3-chloropropyltriethoxysilane (CPTES) (group OHCP) (3-mercaptopropyltrimethoxysilane (MPTS) (group OHMPT) and 3-isobutyryloxy propyltrimethoxysilane(γ- MPS) (group OHMPS) to immobilize the c(RGDfK) cyclic peptide and constructa titanium-silane-c(RGDfK) coating. The NT group was the blank control group. The surface morphology and wettability of the coatings were detected using scanning electron microscopy and contact angle measurement. The elemental composition of the titanium surface was analyzed using X-ray photoelectron spectroscopy. After fluorescent staining with 4’,6-diamino-2-phenylindole (DAPI) and phalloidin, the adhesion of mouse preosteoblast MC3T3-E1 cells on the surface of the materials was observed using laser confocal microscopy. Cell counting kit-8 (CCK-8) and alkaline phosphatase (ALP) activity assays were used to evaluate the proliferation and osteogenic differentiation of MC3T3-E1 cells on the surface of the materials, respectively. @*Results @#Scanning electron microscope observation showed a spongy-like 3-dimensional network formed on the titanium surface after alkali-heat treatment with silane-c(RGDfK) coating adhesion. The wettability of each group was greatly improved compared to the untreated titanium surface. The element ratios of Si/Ti and amide-N/Ti in the OHMPS group were the highest. The OHAP group exhibited the best cell adhesion effect. The cell proliferation and ALP activity of the OHAP, OHMPT, and OHMPS groups were significantly higher than the control group (P <0.05); there was no statistical difference between the OHCP group and the control group.@*Conclusion @#MPTS, CPTES and γ-MPS covalently immobilized cyclic peptide c(RGDfK) on the titanium surface, which promoted adhesion, proliferation and osteogenic differentiation of MC3T3-E1 cells. Theγ-MPS conjugated C (RGDfK)cyclic peptide exhibited the best effect. MPTS, CPTES and γ-MPS coupled with c(RGDfK) cyclic peptides had similar biological properties.
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Chemical burns represent blinding ocular injuries and constitute an ocular emergency requiring immediate assessment and initiation of treatment. The majority of patients are of young age groups and exposure will occur anywhere as an accident and in association with criminal assaults too. Alkali injuries occur more frequently. Chemical injuries of the eye produce extensive damage to the conjunctiva, cornea, anterior segment and limbal stem cells resulting in unilateral or bilateral visual impairment. This article reviews the emergency management to improve the prognosis of patients with chemical injuries
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@#AIM: To investigate the effect of L-carnitine(LC)on corneal epithelial repair and its regulatory molecular mechanism in the hypertonic and inflammatory environment caused by alkali burn.<p>METHODS: Ninety-six healthy C57/6J mice were randomly divided into blank control group, phosphate buffered solution(PBS)group and LC group. The blank control group did not receive any treatment, LC group and PBS group were prepared acute alkali burn models. LC group was given 60mmol/L LC eye drops, and PBS group was given PBS eye drops, 6 times/d, for continuous days from one day before alkali burn. The repair of corneal epithelium was observed by fluorescein sodium staining under slit lamp microscope at 0h, 3 and 7d. On the 3d, the expressions of Ki-67 and IL-1β proteins in cornea were detected by immunofluorescence, the total proteins of corneal epithelial were extracted for Western blot to detect the expression of P63, NLRP3, Caspase-1 and phosphorylation level of STAT3.<p>RESULTS: The results of corneal fluorescein sodium staining showed that on the 3 and 7d after alkali burn, the percentage of residual corneal epithelial defect area in PBS group compared with LC group was(29.38±6.83)% <i>vs</i>(17.78±4.11)% and(14.23±4.51)% <i>vs</i>(4.10±2.10)%, respectively(<i>P</i><0.01). The repair of corneal epithelium in LC group was faster than that in PBS group. On the 3d, compared with the blank control group, the expressions of pyroptosis related proteins NLRP3 and Caspase-1 in the corneal epithelium of the alkali burn treated mice were up-regulated, the expression of P63 was decreased, and the p-STAT3/STAT3 level was increased, all the differences were significant except cleaved Caspase-1 of blank control group <i>vs</i> LC group. Compared with PBS group, in LC group, the expression of NLRP3, pro Caspase-1 and cleaved Caspase-1 protein were decreased, P63 was up-regulated, and p-STAT3 /STAT3 was increased, all the differences were significant. Immunofluorescence showed that compared with the blank control group,the expressions of IL-1β and Ki-67 were up-regulated in the alkali burned group. Compared with PBS group, the expression of Ki-67 protein was up-regulated and IL-1β was decreased in LC group.<p>CONCLUSION: LC can promote the proliferation of stem/progenitor cells in the corneal epithelium of mice and further promote the repair of corneal epithelium after alkali burn by inhibiting the pyroptosis signaling pathway and promoting the activation of STAT3 signaling pathway.
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Objective To evaluate the effectiveness of reactive oxygen species (ROS)-responsive nanomedicine in suppressing corneal neovascularization (CNV) in vivo.Methods ROS-responsive nanomedicine (ROS-TK-5/siVEGF),which consists of vascular endothelial growth factor (VEGF) small interfering RNA (siRNA) and thioketal linkage was synthesized by the Michael addition.The cumulative release of siVEGF from nanomedicine under oxidant conditions was assessed by agarose gel electrophoresis.Thirty-nine VEGFR2-1uc-KI transgenic mice were used in this study,of which 30 mice were randomly divided into a normal control group,a PBS control group,an ROS-TK-5/NC group,an ROS-TK-5/siVEGF group,and a ranibizumab group,with 6 mice in each group.The ROS levels in the corneal tissue after alkali burning were tested by dihydroethidium (DHE) staining in the other 9 mice.In each group,alkali-burned mice were subconjunctivally injected with 10 μl of a different formula every two days.The effectiveness of nanomedicine in attenuating CNV was evaluated by slit-lamp microscopy and an in vivo imaging system (IVIS) at 7,14,and 21 days after alkali burning.The use and care of the animals complied with the Statement of the Association for Research in Vision and Ophthalmology (ARVO) and the Guidelines of the Animal Experimental Committee of Liberation Army General Hospital.The study protocol was approved by the Ethics Committee of Liberation Army General Hospital (No.2018-X14-82).Results After treathrent with an aqueous solution without ROS,only 5％-10％ of the siVEGF was released from the nanoparticles within 10 hours.In contrast,about 70％ of the siVEGF was released from the nanoparticles after treatment with 10 mmol/L H2O2 within 10 hours.The relative fluorescent intensities in the corneal stromal layer at 7 days and 14 days after alkali burning were 5.403±0.306 and 2.930±0.255,respectively,which was significantly greater than those in the normal control group (1.003±0.015) (both at P＜0.05).The CNV areas were statistically different among the four groups at various time points (Fgroup =49.855,P＜0.01;Ftime =65.556,P＜0.01).The CNV area was significantly reduced in the ROS-TK-5/siVEGF and ranibizumab groups compared with the PBS control and ROS-TK-5/NC groups at 7 days and 14 days after modeling,and the CNV area was more effectively reduced in the ROS-TK-5/siVEGF group than the ranibizumab group at 7 days and 14 days after modeling (all at P＜0.05).At day 21 after modeling,the CNV area was significantly reduced in the ROS-TK-5/siVEGF and ranibizumab groups compared to the PBS control and ROS-TK-5/NC groups (all at P＜ 0.05).IVIS showed that the corneal fluorescent intensity was statistically different among the four groups at various times (Fgroup =27.193,P =0.003;Ftime =51.062,P ＜ 0.01).The corneal fluorescent intensities were significantly reduced in the ROS-TK-5/siVEGF and ranibizumab groups compared to the PBS control and ROS-TK-5/NC groups at 7 days and 14 days after modeling;in addition,the corneal fluorescent intensity was more effectively reduced in the ROS-TK-5/siVEGF group in comparison with the ranibizumab group at 7 days and 14 days after modeling (all at P＜ 0.05).At 21 days after modeling,the corneal fluorescent intensity was significantly reduced in the ROS-TK-5/ siVEGF and ranibizumab groups compared to the PBS control and ROS-TK-5/NC groups (all at P ＜ 0.05).Conclusions ROS-TK-5/siVEGF nanomedicine effectively attenuates alkali burn-induced CNV formation and appears to have a better effect in comparison with ranibizumab at an early stage.
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@#AIM: To investigate the effect of comprehensive treatment of ocular alkali burn in different periods.<p>METHOD: A retrospective analysis was performed on 124 cases(166 eyes)of ocular alkali burns admitted to our hospital from January 2019 to December 12. According to the severity of the disease, a number of comprehensive measures were taken to treat the ocular alkali burn with drugs and surgery respectively. The patients were followed up for 6-12mo to observe the healing of ocular alkali burn and the final outcome of disease.<p>RESULTS: After treatment, the symptoms of all patients were relieved, the corneal conjunctiva healed, and no infection occurred. The average hospitalization time was 13d, totally 118 eyes were cured(71.1%), 43 eyes were improved(25.9%), 5 eyes were ineffective(3.0%). There was no complication in degree I and degree II of ocular alkali burn patients, degree III was better than degree IV, and the complication rate in degree III was lower than that in degree IV.<p>CONCLUSION: According to the corneal conjunctiva and eyelid injury evaluation of ocular alkali burn degree, choose appropriate time to take corresponding treatment measures, and give systemic and local drug treatment. Combined with ocular surface irrigation, anterior chamber puncture, amniotic membrane transplantation, conjunctival flap covering, corneal transplantation, limbal stem cell transplantation and other comprehensive treatment methods can obtain good clinical effect.
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@#AIM: To evaluate the inhibitory effect of glycyrrhizin(Gly)on acute alkali burn induced corneal neovascularization(CNV)in mice. <p>METHODS: Corneal neovascularization was established in mice by alkali burn. Sixty mice were then randomly distributed into normal group, Gly group and phosphate buffer solution(PBS)group. The mice were treated with subconjunctival injection of 2g/L Gly solution or vehicle alone every other day for 14d. Corneal inflammation and neovascularization were monitored with a slit lamp microscope. At the end of treatment, the corneas were harvested for hematoxylin-eosin(HE)staining as well as immunohistochemical of CD34 and myeloperoxidase(MPO)staining, microvessel density(MVD), neutrophils were then calculated. <p>RESULTS: At the 7 and 14d, the CNV area of Gly group were 4.16±0.00 and 7.33±0.13mm<sup>2</sup> respectively, which were lower than those in PBS group(7.58±0.20 and 9.24±0.08mm<sup>2</sup>; all <i>P</i><0.05). The HE pathological staining showed that there were no changes in morphology as well as no neovascularization or inflammatory cell infiltration in the cornea of control group. In the Gly group, blood vessels and inflammatory cell infiltration nearly diminished with collagen in normal shape. While in the PBS group, extensive infiltration of inflammatory cells and neovascularization was examed in the corneal stroma. The immunohistochemical CD34 staining performed that the MVD in the Gly group was 11.13±1.46 bars per square millimeter, which was lower than that in PBS group(34.08±2.46)bars per square millimeter(<i>P</i><0.001). Additionally, the immunohistochemical MPO staining showed that the number of neutrophils in Gly group was 17.50±1.98 cells per 200-fold field of view, lower than that in PBS group(59.56±4.79, <i>P</i><0.001). <p>CONCLUSION: Gly can eliminate corneal inflammation and inhibit corneal neovascularization in mice with acute corneal alkali burn, which provides a new idea for clinical prevention and treatment of corneal neovascularization.
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Objective: There are some anthraquinones, anthraquinones and flavonones in Sennae Folium which exhibited significant acidity, such as sennoside A/B and sennoside C/D. The current strategies used in separating these components are mainly based on conventional column chromatography which is time consuming, laborious and costly. This study is aimed at exploring a method of precipitation extraction of acid components in Sennae Folium. Using alkaloid as a “hook”, it is reasonable to use the principle of “acid-alkali complexation” to "fish" the acidic components in Sennae Folium. Methods: Isothermal titration calorimeter (ITC) was used to measure the extraction efficiency of different alkaloids. Then, alkaloid determined by ITC was mixed with extracting solution of Sennae Folium to form complex. High performance liquid chromatography coupled with mass spectrometry (HPLC-MS2) was used to investigate the ingredients “fished” by berberine (Ber). The mechanism of “fishing” process was explained by ITC, optical activity, fluorescence spectrometry and scanning electron microscope. Results: The ITC results proved that the choice of “hook” was particularly important in the process of “fishing”. Among the hooks, the fishing efficiency of the isoquinoline alkaloids (Ber) was the highest, reaching 10.3%. Nine ingredients were detected and determined by HPLC-MS2, and the main components were sennoside A/B and sennoside C/D. Based on ITC test of Ber and sennoside A, the combination mechanism of the two ingredients was a chemical reaction with a nearly binding ratio (2:1). Fluorescence and optical properties of the active ingredients were changed after complexation. By scanning electron microscope, we found that two types of components had obviously self-assembled behavior during the formation process. Conclusion: Ber successfully “fished” the main acidic components, sennoside A/B and sennoside C/D, from Sennae Folium. Combined with different characterizations, the “fishing” process was determined as a chemical association reaction induced by electrostatic interaction or π-π stacking. Therefore, with special identification ability, the “fishing” process had the potential of practical application.
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@#AIM: To compare the effect of using autologous serum and deproteinised calf serum eye gel in the treatment of corneal alkali burn through establishing corneal alkali burn models. <p>METHODS: Alkali burn model of cornea was established on the right eyes by putting the filter paper with 1.0mol/L NaOH on the center cornea for 1min in 30 white rabbits. The model rabbits were divided randomly into 3 groups after scoring based on Hughes criteria. Normal saline, calf blood deproteinized eye gel and autologous serum eye drops 4 times/day, atropine eye gel 1 times/night, ofloxacin eye gel 1 times/night for 2wk respectively. The morphology of corneal neovascularization was observed on the 7 and 14d, and the area was calculated. On the 14d, the corneas of each group were removed and routine histopathological examinations were performed according to the groups. The concentration of CD45, IL-10, IFN-γ and VEGF in corneal homogenate were determined. <p>RESULTS:Area of corneal neovascularization: on the 7 and 14d, the area of corneal neovascularization of Group DCS(29.48±2.27, 34.19±2.67mm2), AS(34.19±2.67, 33.89±2.74mm2)(<i>P</i>>0.05). Concentration of CD45, IL-10, IFN-γ, VEGF in cornea homogenate(pg/mL): on the 14th day, the concentration of CD45 Group DCS(0.56±0.04ng/mL), AS(0.54±0.05ng/mL)<Group Ctrl(1.27±0.07ng/mL)(<i>P</i><0.05). The concentration of IL-10 Group AS(452.49±11.40pg/mL)>Group DCS>(332.49±13.67pg/mL)>Group Ctrl(111.05±6.95pg/mL)(<i>P</i><0.05). The Concentration of IFN-γ Group DCS(23.20±2.89pg/mL), AS(22.61±2.72pg/mL)<Group Ctrl(41.77±4.26pg/mL). They have a significant difference(<i>P</i><0.05). The concentration of VEGF Group DCS(151.14±18.21pg/mL), AS(149.11±14.75pg/mL)<Group Ctrl(391.35±28.59pg/mL)(<i>P</i><0.05). <p>CONCLUSION:AS has the same effect as DCS in inhibiting the release of inflammatory factors(CD45, IFN-gamma and VEGF)and the formation of corneal neovascularization after alkali burn in rabbits, and AS has the strongest effect in promoting the release of anti-inflammatory factors(IL-10)and inhibiting the infiltration of inflammatory cells, followed by DCS.
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Objective@#To evaluate the effectiveness of reactive oxygen species (ROS)-responsive nanomedicine in suppressing corneal neovascularization (CNV) in vivo.@*Methods@#ROS-responsive nanomedicine (ROS-TK-5/siVEGF), which consists of vascular endothelial growth factor (VEGF) small interfering RNA (siRNA) and thioketal linkage was synthesized by the Michael addition.The cumulative release of siVEGF from nanomedicine under oxidant conditions was assessed by agarose gel electrophoresis.Thirty-nine VEGFR2-luc-KI transgenic mice were used in this study, of which 30 mice were randomly divided into a normal control group, a PBS control group, an ROS-TK-5/NC group, an ROS-TK-5/siVEGF group, and a ranibizumab group, with 6 mice in each group.The ROS levels in the corneal tissue after alkali burning were tested by dihydroethidium (DHE) staining in the other 9 mice.In each group, alkali-burned mice were subconjunctivally injected with 10 μl of a different formula every two days.The effectiveness of nanomedicine in attenuating CNV was evaluated by slit-lamp microscopy and an in vivo imaging system (IVIS) at 7, 14, and 21 days after alkali burning. The use and care of the animals complied with the Statement of the Association for Research in Vision and Ophthalmology (ARVO) and the Guidelines of the Animal Experimental Committee of Liberation Army General Hospital.The study protocol was approved by the Ethics Committee of Liberation Army General Hospital (No.2018-X14-82).@*Results@#After treathrent with an aqueous solution without ROS, only 5%-10% of the siVEGF was released from the nanoparticles within 10 hours.In contrast, about 70% of the siVEGF was released from the nanoparticles after treatment with 10 mmol/L H2O2 within 10 hours.The relative fluorescent intensities in the corneal stromal layer at 7 days and 14 days after alkali burning were 5.403±0.306 and 2.930±0.255, respectively, which was significantly greater than those in the normal control group (1.003±0.015) (both at P<0.05). The CNV areas were statistically different among the four groups at various time points (Fgroup=49.855, P<0.01; Ftime=65.556, P<0.01). The CNV area was significantly reduced in the ROS-TK-5/siVEGF and ranibizumab groups compared with the PBS control and ROS-TK-5/NC groups at 7 days and 14 days after modeling, and the CNV area was more effectively reduced in the ROS-TK-5/siVEGF group than the ranibizumab group at 7 days and 14 days after modeling (all at P<0.05). At day 21 after modeling, the CNV area was significantly reduced in the ROS-TK-5/siVEGF and ranibizumab groups compared to the PBS control and ROS-TK-5/NC groups (all at P<0.05). IVIS showed that the corneal fluorescent intensity was statistically different among the four groups at various times (Fgroup=27.193, P=0.003; Ftime=51.062, P<0.01). The corneal fluorescent intensities were significantly reduced in the ROS-TK-5/siVEGF and ranibizumab groups compared to the PBS control and ROS-TK-5/NC groups at 7 days and 14 days after modeling; in addition, the corneal fluorescent intensity was more effectively reduced in the ROS-TK-5/siVEGF group in comparison with the ranibizumab group at 7 days and 14 days after modeling (all at P<0.05). At 21 days after modeling, the corneal fluorescent intensity was significantly reduced in the ROS-TK-5/siVEGF and ranibizumab groups compared to the PBS control and ROS-TK-5/NC groups (all at P<0.05).@*Conclusions@#ROS-TK-5/siVEGF nanomedicine effectively attenuates alkali burn-induced CNV formation and appears to have a better effect in comparison with ranibizumab at an early stage.
ABSTRACT
ObjectiveThere are many methods for the detection of hydroxyproline (HYP), but few of them are suitable for the detection of lung tissue in mice. We intend to establish an accurate and reliable method for measuring HYP levels based on mouse lung tissues to assess the degree of fibrosis development more effectively.MethodsBased on the alkali hydrolysis method, the effects of the concentration of alkali hydrolysate and hydrolysis time on the determination results of HYP level in mice lung tissue were compared; the effects of the changes of experimental conditions on the determination results of HYP standard were compared; and the results of the determination of HYP level in mice lung tissue under dry and wet conditions were compared on the basis of the above experimental results.ResultsThe optimum concentration of alkali hydrolysate is 2 mol/L and the optimum hydrolysis time is 20 min. The optimum pH value of citric acid buffer is 6.0-6.5. The optimum solvent for chloramine T is methanol, the optimum reaction time for chloramine T solution is 15 min, the optimum reaction time for perchloric acid solution is 5 min, and the optimum reaction time for 4-(dimethylamino) benzoyl toluene is 5 min. The optimum condition of aldehyde solution color development is that it is bathed in water at 85 for 3 minutes. Some related reagents are stored in suitable environment after preparation, and the experimental data will not be affected within 7 days. Dry lung tissue of mice can improve the detection level of HYP. The improved experimental protocol was applied to the bleomycin-induced mouse pulmonary fibrosis model, and the HYP measurement results were significantly higher than that of the original protocol.ConclusionAn accurate and reliable method for the determination of hydroxyproline in lung tissue of mice was established.