BACKGROUND:Studies have found that erythropoietin has a protective effect on embryonic development of the retina, but there are rare studies concerning the embryonic development of the lens. OBJECTIVE: To observe the difference in erythropoietin expression in the lens from Wistar rats at different embryonic periods and to investigate the effect and significance of erythropoietin in the embryonic development of the lens. METHODS:Clean Wistar rats with pregnancy for 10 days (n=5), 12 days (n=5), 14 days (n=5), 16 days (n=5), 18 days (n=5), 20 days (n=5) were randomly colected and divided into six groups. Every two embryonic ratsof the different 30 pregnant rats were obtained randomly by the caesarean operation under ketamine-induced anesthesia. The eye tissues of al the embryonic rats were isolated and cut into sections. The expression of erythropoietin protein and mRNA in rat lens was detected by immunohistochemistry and reverse t ranscription-PCR, respectively. RESULTS AND CONCLUSION:Erythropoietin was distinctly expressed at the six different embryo stages, and the expression of erythropoietin protein and mRNA gradualy increased from embryonic day 10 to embryonic day 16, and decreased from embryonic day 10 to embryonic day 20. There were significant differences between the six groups. These findings indicate that the expression of embryonic appears in a low to high to low fashion during the embryonic development of Wistar rats, which may be closely associated with the developing procedure of lens.

Objective To observe the effect of different concentration netrin-1 on retinal vascular permeability in diabetes mellitus (DM) rats.Methods Eighty adult Sprague-Dawley rats were randomly divided into 8 groups,10 rats in each group,including normal control group (group A),normal+balanced salt solution (BSS) group (group B),normal+netrin-1 (500 μg/ml) group (group C) and DM group (50 rats in 5 subgroups).DM rats were induced by intraperitoneal injection of streptozocin.Three months after intraperitoneal injection,10 DM rats in the control group were injected with BSS (group D).Forty DM rats were injected with 5 μl of different concentrate netrin-1,and were divided into DM+netrin-1 10 μg/ml group (group E),DM+netrin-1 50 μg/ml group (group F),DM+netrin-1 100 μg/ml group (group G),DM+netrin-1 500 μg/ml group (group H)according to the different concentration.Non-DM rats in group C were injected with netrin-1 500 μg/ml.The expression of occludin was determined by immunohistochemistry for protein,and by real-time fluorescence quantitative reverse transcription polymerase chain reaction for mRNA level.Retinal vascular permeability was measured by Evans blue infusion.Results The expression of occludin protein and mRNA in group D were less than group A (t=27.71,8.59;P=0.00,0.00).However,the retinal vascular permeability increased in group D (t=-42.72,P=0.00).The expression of occluding protein,occludin mRNA and retinal vascular permeability showed significant differences between group D,E,F,G and H (F=146.31,16.54,67.77;P=0.00,0.00,0.00).Compared the group B with group C,there was no significant differences between the expression of occludin protein,occludin mRNA and the retinal vascular permeability (t=-1.13,0.93,1.04;P=0.27,0.36,0.31).The concentrate of netrin-1 showed a significant positive correlation to the expression level of occludin and occludin mRNA (r=0.73,0.81;P=0.00,0.00),but negative correlation to the vascular permeability (r=-0.61,P=0.00).Conclusion Netrin-1 can reduce the DM rats' retinal vascular permeability,which depended on the concentration of netrin-1.

Objective To observe the effect of netrin-1 on retinal Müller cells in diabetes mellitus (DM) rats. Methods Fifty Sprague-Dawley rats were randomly divided into the normal control group (group A), normal + balanced salt solution (BSS) group (group B), normal+netrin-1 group (group C), DM+BSS group (group D) and DM+netrin-1 group (group E), with 10 rats in each group. DM rats were induced by intraperitoneal injection of Streptozotocin (60 mg/kg). The expression level of glial fibrillary acidic protein (GFAP) on retinal Müller cells was determined by immunohistochemistry, the level of GFAP mRNA was analyzed by real-time fluorescence quantitative reverse transcription polymerase chain reaction. Results Immunohistochemistry showed that GFAP was distributed in retinal ganglion cells and retinal nerve fiber layer in group A, B and C. Compared to group B, GFAP staining was brighter in the group D. There were significant differences in the expression of GFAP protein and mRNA among groups A-E (F=203.43, 72.91; P=0.00, 0.00), they were higher in group D than group A (t=-26.01, 22.26; P=0.00, 0.00), and group E (t=-10.78, 3.93; P=0.00, 0.00). They were higher in group E than group A (t=7.00, -9.82; P=0.00,0.00). There were no significant differences in between group A and group C (t=-0.29, 0.50; P=0.77, 0.62). Conclusion The expression of GFAP in Müller cells of DM rats could be decreased by injecting netrin-1 into vitreous.

OBJECTIVETo develop zoledronic acid (ZA)-loaded collagen membranes, and to study its effect on osteoclast and osteoblast so as to investigate whether ZA-loaded membranes can inhibit local bone resorption and promote bone formation.

METHODSZA-loaded double-layer (Bio-Gide(®)) and single-layer (BME-10X(®)) collagen membranes were prepared and divided into eight groups according to the concentrations of ZA in the membrane, namely Group BG0, BG1, BG2, BG3 and BM0, BM1, BM2, BM3 (BG refers to Bio-Gide(®), BM refers to BME-10X(®), 0, 1, 2, 3 refer to the concentrations of ZA, 0, 1 × 10(-4), 1 × 10(-3), 1 × 10(-2) mol/L respectively). Blank control group was set without using collagen membrane. The effects of ZA-loaded membranes on osteoclast and osteoblast were assessed using in vitro cell culture models.

RESULTSIn vitro coculture of ZA-loaded membrane with osteoclast for seven days showed that the percentage of bone resorption area in BG1, BG2, BG3, BM1, BM2, BM3 were 18.80%, 14.75%, 14.28%, 20.51%, 15.77%, 15.12% respectively, which were lower than that in BG0 (31.53%) and BM0 (32.22%, P < 0.05), and the higher ZA loading was, the stronger its inhibition to osteoclast was. In vitro coculture of ZA-loaded membrane with osteoblast for four days indicated that alkaline phosphatase (ALP) activities in BG2 (154.67 U/g), BM2 (154.33 U/g), BG3 (155.33 U/g), BM3 (152.00 U/g) were higher than that in BG0 (129.33 U/g) and BM0 (127.67 U/g, P < 0.05). What's more, results from seven-day coculture showed that proliferation index in BG2 (7.00) was higher than that in BG0 (6.90).

CONCLUSIONSZA-loaded collagen membrane can not only inhibit osteoclastic bone resorption but also improve proliferation of osteoblast.

Alkaline Phosphatase ; metabolism ; Animals ; Biocompatible Materials ; pharmacology ; Bone Density Conservation Agents ; administration & dosage ; pharmacology ; Bone Resorption ; pathology ; Cell Proliferation ; drug effects ; Cells, Cultured ; Collagen ; pharmacology ; Diphosphonates ; administration & dosage ; pharmacology ; Dose-Response Relationship, Drug ; Imidazoles ; administration & dosage ; pharmacology ; Membranes, Artificial ; Osteoblasts ; cytology ; drug effects ; enzymology ; Osteoclasts ; cytology ; drug effects ; Osteogenesis ; Rabbits

Objective To observe the influence of human umbilical cord mesenchymal stem cells (hUCMSC) transplanted into the tail vein of diabetic rats on apoptosis of retinal neurons and the retinal expression level of glial fibrillary acidic protein (GFAP).Methods Seventy clean male Sprague-Dawley rats were randomly divided into the normal control group (group A),diabetes mellitus (DM) only group (group B),DM + balanced salt solution (BSS) group (group C),DM + hUCMSC group (group D),with 10 rats in each group.DM rats were induced by intraperitoneal injection of streptozotocin.Apoptosis of retinal cells was assayed by dUTP nick end labeling.Immunohistochemistry and Western blot was performed to detect the retinal expressions of GFAP in rats.Results Compared with group A,large numbers of apoptotic cells could be found in the retinal ganglion cell layer (GCL) and inner nuclear layer (INL) of group B and group C,however the apoptotic cells in group D were significantly reduced than group B and C.The expression of GFAP was mainly located in the retinal GCL and retinal nerve fibre layer (RNFL) in group A,throughout the inner plexiform layer (IPL) in group B and C,only distributed in RNFL and GCL in group D.It was obvious that the expression of GFAP in group B and C was higher than group A.Compared with group B and C,the expression of GFAP in group D was significantly reduced.The difference of GFAP expression among the 4 groups was significant (F=79.635,P＜0.05).Conclusion hUCMSC could inhibit the apoptosis of retinal cells and activation of glial cells in early DM rats.

Objective To observe the effect of Delta-like ligand 4 (Dll-4) on the pathological structure of retina in early diabetic rats (DM) and its relationship with vascular endothelial growth receptor-2 (VEGFR-2).Methods A total of 70 male Sprague-Dawley rats were randomly divided into normal group and DM group,with 10 and 60 rats in each group,respectively.The rats of DM group was induced by intraperitoneal injection of streptozotocin to established DM model.The rats with blood glucose recovery and death were excluded,and the final 60 rats were included in the statistics.Rats in the normal group were injected with an equal volume of citric acid-sodium citrate buffer.Rats in the DM group were divided into DM 1 month (DM lm) group,DM 2 months (DM 2m) group,DM 3 months (DM 3m) group and DM 3m + Anti group,DM 3m + phosphate buffer solution (PBS) group by random number table method,and 10 rats in each group.In the DM 3m+Anti group,4 μl ofantiDll-4 polyclonal antibody was injected into the vitreous cavity,and the antibody concentration was 0.25 mg/ml.The DM 3m+PBS group was intravitreally injected with an equal volume of PBS.Five days after the injection,the rats were sacrificed.Rats in the DM 3m group and the normal group were not treated,and were sacrificed 3 months after the model was established.The structure and microvascular changes of the retina were observed by hematoxylin-eosin staining,and the total thickness of the retina was measured.The expression of Dll-4 and VEGFR-2 in the retina was detected by immunohistochemistry and fluorescence quantitative polymerase chain reaction (PCR).One-way analysis of variance was used to compare the expression of Dll-4 and VEGFR-2 in the retina of each group.The least significant difference t test was used to compare the two groups.Results Light microscopy showed that the retinal ganglion cells layer in the DM 3m group were obviously edematous,the inner and outer nuclear layers were thinner,the number of cells was reduced,the arrangement was disordered,the edema of outer plexiform layer was obvious,and the microvessels were abnormally dilated.In the DM 3m+Anti group,the edema of outer plexiform layer was lessened than that of the DM 3m group,and the other layers were not significantly different from the DM 3m group.Compared with the normal group,the total retinal thickness of the DM 3m group,the DM 3m+Anti group and the DM 3m+PBS group increased (t=5.596,3.290,4.286;P=0.000,0.008,0.002).Immunohistochemical staining showed that a small amount of Dl14 was positively expressed in the retinal ganglion cell layer of the normal group;a small amount of VEGFR-2 was positively expressed in the ganglion cell layer and the inner and outer nuclear layers.The positive expression of Dll-4 and VEGFR-2 in retinal vascular endothelial cells of DM 3m group increased significantly.The expression of Dll-4 was significantly decreased in the retinal layers and vascular endothelial cells ofDM 3m+Anti group,while the expression of VEGFR-2 was significantly increased.There was no significant difference between the positive expression of Dll4 and VEGFR-2 in the DM 3m+PBS group and the DM 3m group.The results of real-time PCR showed that the relative expression of Dll-4 and VEGFR-2 mRNA in the DM 3m group was significantly higher than that in the normal group (t=6.705,20.871;P＜0.05).Compared with DM 3m group,the relative expression of Dll-4 mRNA in DM 3m+Anti group decreased,and the relative expression of VEGFR-2 mRNA increased (t=2.681,3.639;P＜0.05).The relative expressions of Dll-4 and VEGFR-2 mRNA in the DM 3m+PBS group and DM 3m group were not statistically significant (t=0.513,0.657;P＜0.05).Conclusions The expression of Dll-4 in retinal vascular endothelial cells is gradually increased during the early retinopathy of DM rats.The expression of Dll-4 is inhibited,the expression of VEGFR-2 is up-regulated,and the plexus edema is alleviated.

AIM To investigate the effects of asiatic acid (AA) on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinson's disease (PD)-like motor symptoms in mice and its neuroprotective mechanism.METHODS Forty-five male C57BL/6 mice,except the nine mice in control group,were induced to be the PD models by peritoneal injection of 25 mg/kg MPTP for seven days and then were randomly assigned to model group,low-dose,high-dose AA groups and positive control group.Both the control group and the model group were administered with 0.5％ sodium carboxyl methyl cellulose (CMC-Na) solution,the AA groups were dosed with 12.5 mg/kg and 25 mg/kg AA,respectively,and the positive control group was given 75 mg/kg daily intragastric gavage of levodopa for eleven days.On the twelfth day,behavioral tests were performed.Tyrosine hydroxylase (TH) positive cells in substantia nigra were detected by immunohistochemistry.The mRNA expressions of iNOS,COX-2,TNF-α,IL-1β,and malonaldehyde (MDA) content in midbrain were measured.The levels of IL-1 β and TNF-α in the serum were detected using ELISA kits.RESULTS The mice treated with asiatic acid performed better in behavior tests than those in the model group (P ＜0.05,P ＜0.01).In addition,asiatic acid effectively protected the dopaminergic neurons in the substantia nigra due to upregulated TH expression and increased number of TH positive cells (P ＜ 0.05).The asiatic acid-treated mice had their mRNA expressions of IL-1β,TNF-α,iNOS and COX-2 in midbrain markedly suppressed (P ＜0.05,P ＜0.01),and a significant MDA level decrease in the midbrain tissue as well (P ＜ 0.01).Furthermore,reductions of IL-1 β and TNF-α contents in the serum were observed (P ＜ 0.05,P ＜ 0.01).CONCLUSION Asiatic acid attenuates motor dysfunction and dopaminergic neuronal deficits in PD mice,and the neuroprotective mechanisms may attribute to its anti-oxidative and anti-inflammatory activities.

Yinqiaojiedu honeyed pills were equally divided into 1/4, 1/8, 1/12, and 1/16 parts. The materiomics release rates within 12 h of the intact Yinqiaojiedu honeyed pills and the divided granules were determined by the paddle method with a rotate speed at 100 r x min(-1), and the materiome was quantified by UV-scan and Kalman filter methods. The intact Yinqiaojiedu honeyed pills behaved typical sustained release profiles, while the well-divided portions also maintained a sustained release profile over 2-4 h. The release rates were well correlated with the extents for the divisions of the pills. The Weibull distribution parameters, Td and T50, were reduced in line with the particle size, indicating that the ways of administration of the pills may play a role in the in vivo pharmacokinetics of the pills. The visualization results showed obvious difference of materiomic release synchronicities between the intact pills and the equally divided particles, and the divisions enhanced the asynchronization. Therefore the novel theory of materiomic release/dissolution kinetics of traditional Chinese medicines (TCMs) quantitatively proved the traditional dosage form, namely, honeyed pills, as a prototype of the sustained-release dosage form with a visualization of the scientific connotation to the old saying in the classics of TCM, Pills, the moderate ones in action. In terms of materiome increase for each period of the release profiles, the materiomic release synchronicity was visually demonstrated. The novel theories provided methodological basis for the evaluation of traditional dosage forms and the design of the modern drug delivery systems for TCMs.
Delayed-Action Preparations ; Drug Combinations ; Drug Delivery Systems ; Drugs, Chinese Herbal ; administration & dosage ; chemistry ; isolation & purification ; pharmacokinetics ; Kinetics ; Medicine, Chinese Traditional ; Particle Size ; Plants, Medicinal ; chemistry ; Solubility ; Technology, Pharmaceutical ; methods

BACKGROUND: Increasing evidence has shown that visit-to-visit variability (VVV) of blood pressure (BP) is associated with an increased risk of cardiovascular disease (CVD). The objective of this study was to evaluate the impact of VVV of systolic blood pressure (SBP) and diastolic blood pressure (DBP) on the risk of CVD among patients with type 2 diabetes mellitus (T2DM) in China. METHODS: We conducted a retrospective cohort study of 10,163 T2DM patients who were not previously diagnosed with CVD from January 2008 to December 2012 in Ningbo, China. The VVV of BP was calculated using five metrics, including standard deviation (SD), coefficient of variation (CV), variation independent of mean, average real variability, and successive variability (SV) of measurements, obtained over a 24-month measurement period. Hazard ratios and 95% confidence intervals (CIs) were estimated by Cox proportional hazards regression models for the associations of variability in BP with risk of CVD. RESULTS: A total of 894 CVD events were observed during a median follow-up of 49.5 months. The hazard ratio in the highest quintile of SD of SBP was 1.24 (95% CI, 1.01 to 1.52) compared with patients in the lowest quintile. The association between higher VVV of DBP and risk of CVD was not consistent across different metrics and sensitivity analyses. CONCLUSION: Higher VVV of SBP was associated with an increased risk of CVD, irrespective of the mean SBP level. Future studies are needed to confirm these findings.
Blood Pressure ; Cardiovascular Diseases ; China ; Cohort Studies ; Diabetes Mellitus, Type 2 ; Follow-Up Studies ; Humans ; Retrospective Studies

Objective:To observe and investigate the effect of HIF-2α in the process of neovascularization of proliferative diabetic retinopathy (PDR).Methods:Retrospective clinical study. From July 2014 to July 2015, 60 eyes of 57 PDR patients diagnosed in Ophthalmology Department of Affiliated Hospital of Qingdao University were included in the study. Twenty-eight eyes of 27 patients received intravitreal injections of 0.5 mg ranibizumab (0.05 ml) at 2-7 days before surgery (ranibizumab group) and other 32 eyes of 30 patients did not (group without ranibizumab). Eighteen eyes of 18 patients with epiretinal membranes were included as controls. Pathological specimens of PDR fibrovascular membrane and premacular membrane were obtained during vitrectomy. The immunohistochemical staining and real-time PCR (RT-PCR) were used to detecting the expression of HIF-2α, Dll4 and VEGF. Kruskal-wallis test was used to compare the expression differences of correlation factors between groups. Spearman correlation analysis was used to analyze the relationship between the two variables.Results:The immunohistochemical staining revealed that there were positive expression of HIF-2α, Dll4 and VEGF in all PDR membranes, regardless of the injection of the ranibizumab. The levels of HIF-2α, Dll4 and VEGF protein in the group without ranibizumab were higher than those of the ranibizumab group ( t=4.36, 6.01, 4.82; P=0.000, 0.008, 0.016). RT-PCR showed that the differences of the mRNA expression of HIF-2α, Dll4 and VEGF were all statistically significant among the PDR patients and controls ( H=18.81,19.60, 20.50; P=0.000, 0.000, 0.000). The expression of HIF-2α, Dll4 and VEGF in the PDR membranes was higher than that of epiretinal membranes from non-diabetic patients. In the PDR patients,the expression of HIF-2α, Dll4 and VEGF of the group without ranibizumab was higher than that of the ranibizumab group. The spearman correlation analysis showed that the expression of mRNA between HIF-2α and Dll4, HIF-2α and VEGF were both significantly correlated ( r=0.95, 0.87; P<0.05). Conclusions:The expression of HIF-2α in the PDR membranes was higher than that of the controls. It is positively correlated with the expression of the DLL4 and VEGF.