Alleviation of Diabetic Tendon Injury via Activation of Tendon Fibroblasts Autophagy under Berberine Treatment.

Berberine (BBR) is an isoquinoline alkaloid isolated from Coptis chinensis and possesses valuable pharmacological activities, including anti-inflammatory, anti-tumor, and alleviating several complications of type 2 diabetes mellitus (T2DM). However, the role of BBR in regulating diabetic tendon injury remains poorly understood. In this study, a rat model of T2DM was constructed, and cell apoptosis and autophagy were assessed in tendon tissues after BBR treatment through TdT-Mediated dUTP nick-end labeling (TUNEL) assay and immunohistochemical analysis. Tendon fibroblasts were obtained from the rat Achilles tendon, and the role of BBR in regulating cell apoptosis, the production of inflammatory cytokines, and autophagy activation were assessed using flow cytometry, quantitative real-time PCR (qRT-PCR), and western blot analysis. We demonstrated that BBR treatment significantly increased autophagy activation and decreased cell apoptosis in tendon tissues of T2DM rats. In tendon fibroblasts, BBR repressed High glucose (HG)-induced cell apoptosis and production of proinflammatory cytokines. HG treatment resulted in a decrease of autophagy activation in tendon fibroblasts, whereas BBR restored autophagy activation. More important, pharmacological inhibition of autophagy by 3-MA weakened the protective effects of BBR against HG-induced tendon fibroblasts injury. Taken together, the current results demonstrate that BBR helps relieve diabetic tendon injury by activating autophagy of tendon fibroblasts.

High glucose represses the proliferation of tendon fibroblasts by inhibiting autophagy activation in tendon injury.

Diabetic foot ulcer (DFU) is a kind of common and disabling complication of Diabetes Mellitus (DM). Emerging studies have demonstrated that tendon fibroblasts play a crucial role in remodeling phase of wound healing. However, little is known about the mechanism underlying high glucose (HG)-induced decrease in tendon fibroblasts viability. In the present study, the rat models of DFU were established, and collagen deposition, autophagy activation and cell apoptosis in tendon tissues were assessed using Hematoxylin-Eosin (HE) staining, immunohistochemistry (IHC), and TdT-mediated dUTP Nick-End Labeling (TUNEL) assay, respectively. Tendon fibroblasts were isolated from Achilles tendon of the both limbs, and the effect of HG on autophagy activation in tendon fibroblasts was assessed using Western blot analysis, Cell Counting Kit-8 (CCK-8) assay, and flow cytometry. We found that cell apoptosis was increased significantly and autophagy activation was decreased in foot tendon tissues of DFU rats compared with normal tissues. The role of HG in regulating tendon fibroblasts viability was then investigated in vitro, and data showed that HG repressed cell viability and increased cell apoptosis. Furthermore, HG treatment reduced LC3-II expression and increased p62 expression, indicating that HG repressed autophagy activation of tendon fibroblasts. The autophagy activator rapamycin reversed the effect. More importantly, rapamycin alleviated the suppressive role of HG in tendon fibroblasts viability. Taken together, our data demonstrate that HG represses tendon fibroblasts proliferation by inhibiting autophagy activation in tendon injury.

Ameliorative effects of the traditional Chinese medicine formula Qing-Mai-Yin on arteriosclerosis obliterans in a rabbit model.

CONTEXT: Qing-Mai-Yin (QMY) is a clinically used herbal formula for treating arteriosclerosis obliterans (ASO). OBJECTIVE: To evaluate the chemical constituents and effects of QMY on ASO rabbit model. MATERIALS AND METHODS: Forty-eight New Zealand rabbits were divided into six groups (n = 8): normal (normal rabbits treated with 0.5% CMC-Na), vehicle (ASO rabbits treated with 0.5% CMC-Na), positive (simvastatin, 1.53 mg/kg), and QMY treatment (300, 600, and 1200 mg/kg). ASO rabbit model was prepared by high fatty feeding, roundly shortening artery, and bovine serum albumin immune injury. QMY (300, 600 and 1200 mg/kg) was orally administered for 8 weeks. The effects and possible mechanisms of QMY on ASO rabbits were evaluated by pathological examination, biochemical assays, and immunohistochemical assays. The compositions of QMY were analysed using HPLC-Q-TOF-MS/MS analysis. RESULTS: Compared to the vehicle rabbit, QMY treatment suppressed plaque formation and intima thickness in aorta, and decreased intima thickness, whereas increased lumen area of femoral artery. Additionally, QMY treatment decreased TC, TG and LDL, decreased CRP and ET, and increased NO and 6-K-PGF1α in serum. Furthermore, the potential mechanisms studied revealed that QMY treatment could suppress expression of TNF-α, IL-6, ICAM-1 and NF-κB in endothelial tissues, and increase IκB. In addition, HPLC analysis showed QMY had abundant anthraquinones, stilbenes, and flavonoids. CONCLUSION: QMY has ameliorative effects on ASO rabbit, and the potential mechanisms are correlated to reducing inflammation and down-regulating NF-κB. Our study provides a scientific basis for the future application and investigation of QMY.

[In vivo high-resolution imaging of rat retina with optical coherence tomography and the expression of Bcl-2, Bax, Caspase-3 mRNA during critical period plasticity].

OBJECTIVE: To observe the changes of retinal thickness during critical period plasticity in rat, and to investigate whether apoptosis participates in the structural forming of retina. METHODS: Experimental research. 50 normal newborn pups of SD rat were randomly selected in the experiment. In vivo consecutive scanning of retinal image was taken and the retinal thickness from RPE to ILM was recorded in 10 pups (20 eyes) with spectral domain optical coherence tomography (OCT) at postnatal day 14 (P14), P18, P21, P24 and P42. Bcl-2, Bax, Caspase-3 mRNA was assessed with fluorescent quantitative real-time polymerase chain reaction after total RNA extracted from 4 retinas of 2 pups at each time point from P14 to P42. Histological measurement of retinal thickness of sections with HE staining from 4 pups (8 eyes) at each time point was compared with the results of OCT scanning. TUNEL staining was used to detect the apoptotic cells in retinal cryosections of 2 pups (4 eyes) at the same time point. The data were analyzed by One-way ANOVA and Linear Regression through SPSS 11.5 software. RESULTS: There was significant difference between the retinal thickness measured through OCT in pups from P14 to P42 (F = 15.425, P = 0.001). And the values were (243.42 ± 13.83) µm at P14, (218.78 ± 8.21) µm at P18, (195.42 ± 8.02) µm at P21, (195.74 ± 14.85) µm at P24, (190.79 ± 11.70) um at P42. The retinal thickness measured through OCT decreased significantly during the first 3 weeks after birth. The results of OCT measurement had linear correlation with histology measurement (R = 0.794, P = 0.000). There was significant difference between mRNA expression of Bcl-2, Bax and Caspase-3 in pups from P14 to P42 (F = 18.684, F = 47.307, F = 49.611; P = 0.000). The relative expression of Bax and Caspase-3 peaked at P24 while Bcl-2 was much more stable. There were a lot of apoptotic cells in the ganglion cells layer, the inner nuclear layer and the outer nuclear layer during P18 to P24 by TUNEL staining. And the apoptosis alleviated at P42. CONCLUSIONS: The retinal thickness decreases when the retina continues to develop during critical period plasticity. Cirrus HD-OCT can be used as an effective instrument to show the layers of retina in rat in vivo. Apoptosis participates in the course of retinal development which possibly leads to the thinning of retina.

F-heparin modified intraocular lenses in Rhesus monkeys.

AIM: In order to improve the biocompatibility of intraocular lenses (IOL), the polymethylmethacrylate (PMMA) IOL was modified with F-heparin. METHODS: The PMMA IOL was modified with F ions and heparin by the technique of ion beam combined with low temperature and low pressure plasma. The monkeys (20 eyes) with cataract partly were randomly classified into 2 groups and implanted with PMMA IOL and modified IOL respectively for 180 days. All of the eyes were examined by slit-lamp microscope at postoperative 15, 30, 60, 90, 180 days. The extracted IOL was analyzed with computer image analysis, light microscope (LM) and scanning electron microscope (SEM) at postoperative 180 days. RESULTS: The early inflammatory reactions postoperatively include anterior chamber exudation and aqueous cell count. The modified IOL group showed less than the non-modified IOL group. The late foreign body cell reaction that adhered to the surface of non-modified IOL was more predominant. The morphologic and pathological changes of posterior capsule opacification (PCO) in monkeys' eyes included fibrosis-type, pearl-type and soemmerring's ring. There was a significant difference between the two groups. CONCLUSION: F-heparin modified IOL has good uveal and capsular biocompatibility.

[Combined rapamycin eye drop in nanometer vector and poly (lactic acid) wafers of cyclosporine A effectively prevents high-risk corneal allograft rejection in rabbits].

OBJECTIVE: To evaluate the combined effect of topical rapamycin (RAPA) eye drop in nanometer vector and poly (lactic acid) (PLA) wafers of cyclosporine A (CsA) in the prevention of acute allograft rejection after rabbit corneal transplantation. Methods It was an experimental study. RAPA was incorporated into the nanometer particles and CsA was incorporated into PLA wafers. A was syngeneic control whose both donor and recipient are New Zealand rabbit. Gray donor corneas were implanted into the 102 recipients of New Zealand albino rabbits with corneal neovascularization who were randomly divided into B, C, D, E, F, G 6 groups to receive the different types of therapy: B was no therapy control; C was eye drop of nanometer vector but no RAPA twice a day, 28 days; D was PLA wafers in the anterior chamber of rabbit eyes but no drugs; E was 0.5% RAPA eye drop of nanometer vector twice a day, 28 days; F was PLA wafers of CsA in the anterior chamber of rabbit eyes; G was PLA wafers of CsA in the anterior chamber of rabbit eyes and 0.5% RAPA eye drop of nanometer vector eye drop twice a day for 28 days together. Postoperative evaluation included slit-lamp biomicroscopy, histopathology and immunohistology, Cytokines related with neovascularization and immunosuppression in the corneal tissue by RT-PCR. The graft survival was assessed by One-Way ANOVA and q test. RESULTS: Corneal allograft survival time: A (100.00 +/- 0.00), B (8.44 +/- 1.24), C (8.89 +/- 2.57), D (8.56 +/- 2.30), E (43.11 +/- 5.58), F (43.67 +/- 9.54), G (72.00 +/- 15.34) d. Group G led to a statistically significant prolongation of transplant survival and was superior than group E and F which was a statistical prolongation compared with group B, C and D (qGE = 11.42, qGF = 11.24, qEB = 13.64, qEC = 13.38, qED = 13.46, qFB = 13.82, qFC = 13.56, qFD = 13.64; P < 0.01). Immunohistopathologically, the grafts were subjected to an immune response contained a dense infiltrate of neutrophils, CD4+ and CD8+ T lymphocytes in the group B, C and D. This cellular infiltrate was a significant reduction in group E,F,G. RT-PCR showed that the gene expression of IL-2 was inhibited earlier (3 days) in group F, G and VEGF gene expression being suppressed later (14 days) in group E, G. CONCLUSIONS: Combined therapy with topical application of RAPA eye drop of nanometer vector and CsA PLA wafers can significantly prolong the survival of allograft at high-risk. Moreover, topical combined treatment of them is more effective, lower dosage, less side-effects and cheaper than the treatment with topical individual immunosuppressive drug.

Effects of bone-marrow mesenchymal stem cells transplanted into vitreous cavity of rat injured by ischemia/reperfusion.

OBJECTIVE: To examine the survival, migration, integration, differentiation and the expression of various neurotrophic factors of bone-marrow mesenchymal stem cells (BMSCs) transplanted into the vitreous cavity of rats injured by ischemia/reperfusion(I/R). METHODS: The BMSCs were separated from rat marrow using the wall-sticking method, and cultured in vitro to expand. Flow cytometry detected the surface antigens of BMSCs. Ninety-six rats were randomly divided into four groups: normal control injected PBS(C+P), normal control injected BMSCs (C+B), ischemic/reperfusion injected PBS(I/R+P)and ischemic/reperfusion injected BMSCs(I/R+B). After retinal I/R injury was induced in each group by increasing intraocular pressure, 10 microl PBS and BMSC suspensions labeled by red fluorescence CM-Dil were immediately injected into the vitreous cavity. We observed the survival, migration and integration of BMSCs using confocal microscopy. The differentiation and expression of basic fibroblast growth factor (bFGF), brain-derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF) of CM-Dil-labeled BMSCs were detected by immunofluorescent labeling and reserved by confocal microscopy. The expression of mRNA and proteins of bFGF, BDNF and CNTF were assayed by RT-PCR and Western Blot respectively. RESULTS: After transplantation to normal eyes, BMSCs labeled by CM-Dil were mostly present in the vitreous cavity, and did not migrate. After transplantation to I/R eyes, BMSCs labeled by CM-Dil were mostly present along with the inner limiting membrane. Only a few cells were integrated into the ganglion cell layer. Two or 4 weeks after transplantation, a few BMSCs labeled by CM-Dil were observed to express markers of neuron- neurone specific enolase (NSE), neurofilament (NF) and various neurotrophic factors. The BMSC-injected I/R model eyes showed less reduction in the number of RGCs than that of the I/R eyes with PBS injection. CONCLUSIONS: BMSC transplantation is a valuable neuroprotection tool for the treatment of retina and optic nerve diseases.

[Experimental study of spherical aberrations of intraocular lenses with different materials and designs].

OBJECTIVE: To study the characteristics of spherical aberrations (SA) of the traditional intraocular lenses (IOLs) produced by different materials and designs. METHODS: A special optical metrical system was established in the laboratory to measure the SA of IOLs by using the Hartmann-Shack aberrometer. Eight different types of IOLs were divided into two Groups. Group 1 consisted of 4 types of IOLs made by different materials, Group 2 consisted of 4 types of IOLs made by different designs. SA of these IOLs was measured at 3, 4, 5, 6 and 7 mm apertures separately. All data was analyzed statistically. RESULTS: With the size of aperture increased, SA in these two groups was both increased. There were positive correlations between the size of aperture and the SA (r = 0.6465, 0.7872; P < 0.01). The numbers of IOLs fitted with Rayleigh criterion at apertures of 3, 4, 5, 6, and 7 mm were 8, 5, 3, 2 and 2, respectively. In Group 1, the acrylic IOL (SA60AT) with high refractive index showed the greatest SA, and the acrylic IOL (AR40e) with low refractive index showed the smallest SA. The difference of SA between these two IOLs was statistically significant (P < 0.01). SA of the silicon IOL (SI40NB) and PMMA IOL (PC330UV) lied between these two extremities. In Group 2, the convexo-convex IOL with the posterior surface more curved than the anterior surface had the greatest SA. The convex-plano IOL had the smallest spherical aberration. The difference of SA between these two IOLs was statistically significant (P < 0.01). SA of convexo-convex IOL with the anterior surface more curved and the epuiconvex IOL was lied between these two extremities. CONCLUSIONS: IOLs made by different materials and designs under different apertures show significant difference of SA, which is useful for the improvement of optic quality of IOLs. Further studies on the exact mechanism of these differences are required.

[Early clinical study of bimanual microphacoemulsification with rollable intraocular lens].

OBJECTIVE: To analyze the results of bimanual microphacoemulsification combined with rollable intraocular lens (IOL), to evaluate the feasibility and safety of this procedure and to compare the clinical results of two different types of rollable intraocular lens. METHODS: Thirty-five cases (35 eyes) were divided into 2 groups. Group 1 included 15 eyes used Thinoptx IOL, Group 2 included 20 eyes used Acri. Smart IOL. The data included best corrected visual acuity (BCVA) and intraocular pressure (IOP) (pre-, 1 day, 1 week and 1 month post-operatively); corneal curvature (pre- and 1 month post-operatively); target refraction and post-operation refraction (1 month after the operation). RESULTS: A significant difference was found between pre-operative and 1 month post-operative BCVA (P<0.01). No significant different was found between pre-operative and post-operative IOP, astigmatism, target refraction and refraction. There was no significant difference in pre-operative and post-operative astigmatism, target refraction and post-operation 1-month refraction between these two groups. CONCLUSIONS: Bimanual microphacoemulsification with rollable intraocular lens (IOL) is a feasible and safe procedure for the cataract surgery.

[Cloning, expression and cytotoxin to human lens epithelial cells of targeting toxin DT389-hbFGF].

OBJECTIVE: To construct pGEX-DT(389)-hbFGF plasmid, express and identify the cytotoxicity to human lens epithelial cells (HLECs). METHODS: Extracting DNA of dead diphtheria bacillus and RNA of 12-week fetal brain cortex. The fragments of truncated diphtheria toxin (containing 389 amino acids of N-terminus, DT(389)) and full length human bFGF gene (encoding 18kd protein) were amplified by PCR technique respectively. The two fragments were inserted into prokaryotic expression vector pGEX-4T-1. After testing sequence, the expressing plasmid was transformed into E.Coli BL21 strain and induced expression under IPTG. The expressed fusion protein was purified and identified. MTT experiment tested cytotoxicity of the fusion protein to HLECs in vitro. The way of HLECs death under different dosage was identified by flow cytometry. RESULTS: The gene fragments of DT(389) and human bFGF were accurately amplified. The expression vector including DT(389)-hbFGF fused gene was constructed and expressed successfully. DT(389)-hbFGF fusion protein can induce HLECs apoptosis in a dosage dependence manner during certain range. The LD(50) was about 3.8 x 10(-11) mol/L. CONCLUSION: The successful cloning and expression of DT(389)-hbFGF immunotoxin lay a foundation for accelerating lens epithelial cells apoptosis and the targeting therapy toward posterior capsule opacification.

[Distribution and morphological changes of corneal nerves in type 2 diabetic patients detected by confocal microscopy].

OBJECTIVE: To evaluate corneal nerve distribution and morphological changes in subjects with type 2 diabetes. METHODS: Sixty-five eyes of 59 patients with type 2 diabetes (26 males, 39 females) and 26 eyes (10 males, 16 females) of control subjects were included in the present study. Based on the indirect ophthalmoscopic examination and fluorescein angiography, the diabetic group was divided into three sub-groups according to the stage of retinopathy: without diabetic retinopathy (NDR), non-proliferative diabetic retinopathy (NPDR) and proliferative diabetic retinopathy (PDR). The central cornea was scanned with Confoscan 3.0. The images of subbasal nerve plexus and stromal nerves were analyzed. RESULTS: Corneal branch nerve fiber density of subbasal nerve plexus decreased in diabetic group as compared with control group, the difference was statistically significant. The decrease of nerve fiber density appeared only in the PDR sub-group, while the decrease was not significant in NDR and NPDR sub-groups. No obviously difference was found among control group, NDR and NPDR sub-groups. The proportion of patients with an abnormal morphologic nerve fibers in mid-stroma in diabetes group was greater than that in the control group and the difference was statistically significant (chi(2) = 46.613, P = 0.000). CONCLUSIONS: Corneal confocal microscope allows rapid and noninvasive in vivo evaluation of corneal nerves. The subbasal and stromal nerves of subjects with type 2 diabetes were abnormal in morphology.

[Application of wavefront aberration technique in the study of phakic and pseudophakic eyes].

Modern cataract surgery is routinely combined with the implantation of an intraocular lens (IOL). Although surgical technique as well as IOL materials and designs have been improved dramatically, the problems related postoperative visual acuity still occur, such as glare disability, low visual acuity at night, and so on. Wavefront aberration technique can be used to help us revealing the real reasons of such postoperative visual problems. We reviewed the relative study outcome of the application of wavefront aberration technique in phakic eyes, and summarized the current opinions on the study of pseudophakic eyes.

Sex hormones and their receptors in patients with age-related cataract.

PURPOSE: To determine the sex hormone levels in the serum and aqueous humor in patients with age-related cataract and the corresponding hormone receptors in cataract lens epithelial cells (LECs). SETTING: Research Laboratory, International Intraocular Implant Training Center, Tianjin Medical University, Tianjin, China. METHODS: Serum and aqueous humor were drawn from patients with cataract and a control group. Enzyme-linked immunosorbent assay was used to determine the levels of estradiol, progesterone, and testosterone in the serum and the aqueous. The anterior lens capsules with attached LECs were obtained during cataract surgery, and the expressions of estrogen receptor, progesterone receptor, or androgen receptor were examined by immunohistochemistry. RESULTS: The testosterone level in the serum was significantly higher in the control group than in the cataract group. In the cataract group, there was no difference between sexes in the serum levels of estradiol or progesterone; however, the testosterone level in men was significantly higher than in women. The aqueous level of each hormone was lower than in serum; however, there was no difference between sexes and no association with corresponding serum levels. No estrogen, progesterone, or androgen receptor was detected in the LECs of patients with age-related cataract. CONCLUSIONS: There was no statistical difference between men and women or between the cataract and control groups in the levels of estradiol or progesterone in serum. There was no between-sex difference in the aqueous levels. It appears that sex hormone levels can be regarded only as a risk factor for cataractogenesis, not as a key factor.