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Objective:To investigate the role of integrin-linked kinase (ILK)-small interfering RNA (siRNA)-adeno-associated virus (AAV) in scar formation after glaucoma filtering surgery in rat eyes.Methods:Forty-eight Sprague Dawley rats of SPF grade, aged 8 to 9 weeks old, were selected and divided into blank control group, ILK-siRNA-AAV group, NC-siRNA-AAV group and mitomycin C (MMC) group by random number table method, with 12 rats in each group.Left eyes of the rats were taken as experimental eyes, and no intervention was administered to fellow eyes.The bulbar conjunctival filtering bleb after glaucoma filtration surgery in rats was established by anterior chamber drainage tube implantation.One day after operation, phosphate buffer solution, ILK-siRNA-AAV, and NC-siRNA-AAV were injected into the filtering bleb of blank control group, ILK-siRNA-AAV group and NC-siRNA-AAV group, 5 μl each group, respectively.Cotton tablets containing 0.4 mg/ml MMC were placed under conjunctival flap for 5 minutes during operation in MMC group.Intraocular pressure (IOP) was measured with a handheld tonometer before surgery and at 1, 2, 3, 7, 14, 21, 28 days after surgery.Formation of filtering blebs in rats was observed with a surgical microscope at 1, 2, 3, 7, 14, 21, 28 days after operation, and the bleb survival time was calculated by Kaplan-Meier survival analysis.The mRNA and protein expression levels of ILK in conjunctival and subconjunctival tissues at the surgical sites were detected by reverse transcription PCR and Western blot, respectively, on the 28th day after operation.Silencing of ILK gene was identified.Effect of ILK gene silencing on the morphology of drainage pathway was observed by hematoxylin-eosin staining.Effect of ILK gene silencing on collagen fiber deposition in the bulbar conjunctiva at filtration area was examined by Masson staining, and the percentage of positive area of collagen fiber staining in the total tissue visual field was calculated.The use and care of the animals complied with the ARVO Statement.This research protocol was approved by an Ethics Committee of Xi'an Jiaotong University (No.2013-772). Results:There were statistically significant differences in IOP at different time points between before and after surgery among four groups ( Fgroup=76.84, P<0.001; Ftime=114.49, P<0.001). The IOP of ILK-siRNA-AAV group on the 1st, 7th, 14th and 28th day after operation and the IOP of MMC group on the 2nd, 3rd, 7th, 14th and 28th day after operation were lower than those of blank control group, and the differences were statistically significant (all at P<0.05). The IOP of ILK-siRNA-AAV group was lower on 7th, 14th, 21st and 28th day after operation than those of NC-siRNA-AAV group, with statistically significant differences (all at P<0.05). The bleb survival time of blank control group, NC-siRNA-AAV group, ILK-siRNA-AAV group and MMC group was (3.50±1.51), (5.00±3.41), (31.50±3.15) and (31.33±2.46) days, respectively, with a significant difference among them ( F=395.83, P<0.05). The bleb survival time of ILK-siRNA-AAV group and MMC group was higher than that of blank control group and NC-siRNA-AAV group, and the differences were statistically significant (all at P<0.05). There were statistically significant differences in the relative expression levels of ILK mRNA and protein among four groups ( F=222.32, 752.69; both at P<0.05), and the relative expression levels of ILK mRNA and protein were significantly lower in ILK-siRNA-AAV group than blank control group and NC-siRNA-AAV group, and the differences were statistically significant (all at P<0.05). Proliferative fibrous connective tissue and a large number of cells at surgical sites were found in blank control group and NC-siRNA-AAV group, and the fibroblasts were of a high density and grew in clumps.In ILK-siRNA-AAV group, the bulbar conjunctiva was thin, and the arrangement of fibrous connective tissue was loose, and a few proliferative fibroblasts were found.In MMC group, the conjunctival fibrous layer was loose and thin, forming cavities, and scarce cells were found.There was statistically significant difference in the percentage of collagen fiber positive staining area among four groups ( F=741.66, P<0.05). The positive staining percentage of ILK-siRNA-AAV group and MMC group was significantly lower than that of blank control group, among which there was lower positive staining percentage in ILK-siRNA-AAV group than NC-siRNA-AAV group, and the differences were statistically significant (all at P<0.05). Conclusions:Silencing of ILK can inhibit the scar formation after glaucoma filtering surgery and maintain low IOP in rats.
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OBJECTIVE@#To investigate the the effects of leptin on the proliferation, differentiation and PTEN expression of rat retinal progenitor cells (RPCs) cultured under hypoxic condition.@*METHODS@#SD rat RPCs were cultured in normoxic conditions or exposed to hypoxia in the presence of 0, 0.3, 1.0, 3.0, 10, and 30 nmol/L leptin for 12, 48 and 72 h, and the cell viability was assessed using cell counting kit 8 (CCK 8) assay. The RPCs in primary culture were divided into control group, hypoxia group, and hypoxia+leptin group, and after 48 h of culture, the cell medium was replaced with differentiation medium and the cells were further cultured for 6 days. Immunofluorescence staining was employed to detect the cells positive for β-tubulin III and GFAP, and Western blotting was used to examine the expression of PTEN at 48 h of cell culture.@*RESULTS@#The first generation of RPCs showed suspended growth in the medium with abundant and bright cellular plasma and formed mulberry like cell spheres after 2 days of culture. Treatment with low-dose leptin (below 3.0 nmol/L) for 48 h obviously improved the viability of RPCs cultured in hypoxia, while at high concentrations (above 10 nmol/L), leptin significantly suppressed the cell viability (P < 0.05). The cells treated with 3.0 nmol/L leptin for 48 h showed the highest viability (P < 0.05). After treatment with 3.0 nmol/L leptin for 48 h, the cells with hypoxic exposure showed similar GFAP and β-tubulin Ⅲ positivity with the control cells (P>0.05), but exhibited an obvious down-regulation of PTEN protein expression compared with the control cells (P < 0.05).@*CONCLUSION@#In rat RPCs with hypoxic exposure, treatment with low dose leptin can promote the cell proliferation and suppress cellular PTEN protein expression without causing significant effects on cell differentiation.
Subject(s)
Animals , Rats , Cell Differentiation/drug effects , Cell Hypoxia/drug effects , Cell Proliferation/drug effects , Cells, Cultured , Leptin/pharmacology , PTEN Phosphohydrolase/metabolism , Rats, Sprague-Dawley , Retina/metabolism , Stem Cells/metabolism , TubulinABSTRACT
Aberrant activation of NLRP3 inflammasome in colonic macrophages strongly associates with the occurrence and progression of ulcerative colitis. Although targeting NLRP3 inflammasome has been considered to be a potential therapy, the underlying mechanism through which pathway the intestinal inflammation is modulated remains controversial. By focusing on the flavonoid lonicerin, one of the most abundant constituents existed in a long historical anti-inflammatory and anti-infectious herb
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p21-activated kinase 5 (PAK5), a kind of the PAK family of serine / threonine kinases, modulates various cellular processes, including cytoskeletal remodeling, cell proliferation, migration and metastasis. Although studies have suggested that PAK5 is a key regulator of breast cancer progression, the link of PAK5 to senescence has not been reported yet. In this study, the CRISPR/ Cas9 lentivirus infection method was used to construct two PAK5 stably knockdown cell lines of human breast cancer (MDA-MB-231 and BT474). The effect of PAK5 knockdown on the proliferation of breast cancer cells was detected by CCK-8 and clone formation assays. The effect of PAK5 knockdown on cell cycle was detected by flow cytometry. The effect of PAK5 knockdown on cell senescence was observed by β-galactosidase staining. Western blotting was performed to detect the expression of senescence-related proteins including p53, p21 and p16 in breast cancer cells. Cells were treated by CHX and MG132 to explore the possible mechanism of PAK5 regulating p53 protein expression. The results showed that knockdown of PAK5 inhibited cell proliferation and blocked cells in the G
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Glioma is the most common primary intracranial tumor, among which glioblastoma (GBM) is the most malignant subtype. Because of its high heterogeneity and invasiveness, GBM can't be completely removed by surgical resection and is also resistance to chemotherapy and radiotherapy. Even after a standard therapy, the median survival time is only 14.6 months, the five-year survival rate is less than 10%, and the relapse of GBM is common. Immunotherapy, a new treatment paradigm, treats cancer through regulating the autologous immune system and the tumor microenvironment. As a promising method to improve the prognosis of GBM, immunotherapy has attracted more and more attention. This paper gives a review to the difficulty, the mainly existing strategies and the bottlenecks in GBM immunotherapy, aiming at providing new direction to improve the prognosis of GBM patients.
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Objective@#To understand the emotional and behavioral problems among preschool children in Ezhou city, and to analyze its association with screen time,to provide a reference for making preventive measures.@*Methods@#1 720 children were selected from 9 kindergartens in Ezhou by cluster sampling method. Questionnaires were completed by the children’s parents or primary caregivers.@*Results@#High total difficulty score accounted for 9.8%(169/1 720) of all participants,the abnormal proportion of peer communication problems is the highest. The average screen time was (1.95±1.88) hours, among them 2.6% spent more than 2 hours. The average screen time on weekends was (2.84±2.58) hours, among them 19.1% spent more than 2 hours. The results of multiple linear regression analysis showed that weekday, weekend and total screen times was associated with children’s total difficulty score, emotional problems, conduct problems, peer interaction and hyperactivity(P<0.05). Furthermore, with the duration of screen time increased, the susceptibility of emotional and behavioral problems in these four dimensions increased.@*Conclusion@#Screen time is associated with emotional and behavioral problems among preschool children. Schools and parents should pay attention to interventions that target family TV viewing practices, and parent-child interaction and communication.
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Objective To investigate the bystander effect injury to lung-heart-liver-spleen caused by 12C6+ beam radiation,and explore the prevention and treatment effect of Guiqiyiyuan ointment on the injury and its mechanism.Methods Ninety healthy male Wistar rats were randomly divided into 3 groups:NC group (normal control,normal saline 2ml/kg,n=30),SR group (simple radiation,8Gy,2ml/kg,n=30),GO group (Guiqiyiyuan ointment 11.83g/kg,radiation,8Gy,n=30).All the rats received intragastric administration for 7 days.The right side of the lung was modeled by 12C6+ beam radiation.After modeling,the rats were killed at 48h.The heart,liver and spleen were taken.The malonaldehyde (MDA),glutathione (GSH),glutathione peroxidase (GSH-Px),superoxide dismutase (SOD) contents were measured by colorimetry,DNA methylation rate was assayed by ELISA,and the expressions of Dnmt1,Dnmt3a and Dnmt3b were detected by immunohistochemistry.Results Compared with NC group,the contents of SOD,GSH and GSH-Px decreased (P＜0.01),MDA increased (P＜0.01),the level of DNA methylation decreased (P＜0.01),and the expressions of Dnmt1,Dnmt3a and Dnmt3b increased in SR group (P＜0.01).Compared with SR group,the contents of SOD,GSH and GSH-Px increased (P＜0.01),MDA decreased (P＜0.01),the level of DNA methylation increased (P＜0.01),and the expressions of Dnmt1,Dnmt3a and Dnmt3b decreased in GO group (P＜0.01).Dnmtl,Dnmt3a and Dnmt3b proteins were expressed in the cytoplasm of myocardial cells,hepatocytes and peripheral B cells of the white pulp in spleen,in all the groups.The color of NC group was light brown-brown,showing a weak positive expression.The color of SR group was brown-brown,showing a strong positive expression.The color of GO group was light brown-tan,showing a moderate positive expression.Conclusion The Guiqiyiyuan ointment can reduce the bystander effect caused by the 12C6+ beam radiation,and its mechanism is related to improving the oxidative stress reaction and the level of DNA methylation.
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<p><b>BACKGROUND</b>Retinal degenerative diseases are the leading causes of blindness in developed world. Retinal progenitor cells (RPCs) play a key role in retina restoration. Triamcinolone acetonide (TA) is widely used for the treatment of retinal degenerative diseases. In this study, we investigated the role of TA on RPCs in hypoxia condition.</p><p><b>METHODS</b>RPCs were primary cultured and identified by immunofluorescence staining. Cells were cultured under normoxia, hypoxia 6 h, and hypoxia 6 h with TA treatment conditions. For the TA treatment groups, after being cultured under hypoxia condition for 6 h, RPCs were treated with different concentrations of TA for 48-72 h. Cell viability was measured by cell counting kit-8 (CCK-8) assay. Cell cycle was detected by flow cytometry. Western blotting was employed to examine the expression of cyclin D1, Akt, p-Akt, nuclear factor (NF)-κB p65, and caspase-3.</p><p><b>RESULTS</b>CCK-8 assays indicated that the viability of RPCs treated with 0.01 mg/ml TA in hypoxia group was improved after 48 h, comparing with control group (P < 0.05). After 72 h, the cell viability was enhanced in both 0.01 mg/ml and 0.02 mg/ml TA groups compared with control group (all P < 0.05). Flow cytometry revealed that there were more cells in S-phase in hypoxia 6 h group than in normoxia control group (P < 0.05). RPCs in S and G2/M phases decreased in groups given TA, comparing with other groups (all P < 0.05). There was no significant difference in the total Akt protein expression among different groups, whereas upregulation of p-Akt and NF-κB p65 protein expression and downregulation of caspase-3 and cyclin D1 protein expression were observed in 0.01 mg/ml TA group, comparing with hypoxia 6 h group and control group (all P < 0.05).</p><p><b>CONCLUSION</b>Low-dose TA has anti-apoptosis effect on RPCs while it has no stimulatory effect on cell proliferation.</p>
Subject(s)
Animals , Rats , Apoptosis , Physiology , Caspase 3 , Metabolism , Cell Cycle , Physiology , Cell Hypoxia , Physiology , Cell Proliferation , Physiology , Cell Survival , Physiology , Cells, Cultured , Cyclin D1 , Metabolism , NF-kappa B , Metabolism , Proto-Oncogene Proteins c-akt , Metabolism , Rats, Sprague-Dawley , Retina , Cell Biology , Stem Cells , Cell Biology , Triamcinolone Acetonide , PharmacologyABSTRACT
<p><b>OBJECTIVE</b>To compare the curative effects of primary osteoporosis treated with heat-sensitive point moxibustion and Gaitianli (Oyster Shell and Calcium Carbonate Chewable) tablets for oral administration and explore the treatment mechanism.</p><p><b>METHODS</b>Sixty cases of primary osteoporosis were randomly divided into a heat-sensitive point moxibustion group (moxibustion group) and a Gaitianli tablets group (medication group), 30 cases in each group. In the moxibustion group, the heat sensitized points were searched around Zusanli (ST 36), Pishu (BL 20), Shenshu (BL 23) and Mingmen (GV 4) and treated by heat-sensitive point moxibustion; in medication group, Gaitianli tablets were taken by oral administration, 3 pills for once and 3 times a day. The curative effects, bone mineral density (BMD), alkaline phosphatase (S-AKP) and urinary calcium to creatinine ratio (U-Ca/Cr) in both groups were observed before and after treatment.</p><p><b>RESULTS</b>The total effective rate was 86.7% (26/30) in moxibustion group, superior to that of 76.7% (23/30) in medication group (P < 0.05). After treatment, the BMD of lumbar vertebrae (L2-L4) mean was improved (P < 0.05), and the S-AKP and U-Ca/Cr were reduced (all P < 0.05); in medi cation group, the indexes above were no obvious changes (all P > 0.05).</p><p><b>CONCLUSION</b>The therapeutic effect of primary osteoporosis treated with heat-sensitive point moxibustion is superior to that with Gaitianli tablets for oral administration. The mechanism is restraining bone resorption, increasing bone strength, keeping balance of bone metabolism, in order to increase bone mineral density and improve the clinical symptoms.</p>
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Aged , Female , Humans , Male , Middle Aged , Acupuncture Points , Alkaline Phosphatase , Blood , Bone Density , Calcium , Urine , Creatinine , Urine , Moxibustion , Methods , Osteoporosis , Metabolism , TherapeuticsABSTRACT
<p><b>OBJECTIVE</b>To investigate the effects of tamoxifen (TMX) combined with coenzyme Q10 (CoQ10) on idiopathic oligoasthenospermia.</p><p><b>METHODS</b>A total of 183 patients with idiopathic oligoasthenospermia were randomly divided into a TMX + CoQ10 group (n = 63), a TMX group (n = 61) and a CoQ10 group (n = 59). At the end of 3 and 6 months of treatment, semen analyses and hormone tests were performed, and the results were compared with those obtained before the treatment.</p><p><b>RESULTS</b>Compared with the pre-treatment results, the levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH) and testosterone (T) and sperm concentration were significantly elevated in the TMX + CoQ10 and TMX groups (P < 0.05), but showed no significant difference in the CoQ10 group (P > 0.05); sperm motility and morphologically normal sperm were increased significantly in the TMX + CoQ10 and CoQ10 groups (P < 0.05), and slightly in the TMX group but with no statistically significant difference (P > 0.05).</p><p><b>CONCLUSION</b>Tamoxifen combined with CoQ10 can significantly improve sperm concentration, motility and morphology in patients with idiopathic oligoasthenospermia.</p>
Subject(s)
Adult , Humans , Male , Young Adult , Oligospermia , Drug Therapy , Tamoxifen , Therapeutic Uses , Treatment Outcome , Ubiquinone , Therapeutic UsesABSTRACT
<p><b>OBJECTIVE</b>To compare clinical therapeutic effect of surrounded needling therapy and medication on acute gouty arthritis.</p><p><b>METHODS</b>Sixty cases of acute gouty arthritis were randomly divided into a surrounded needling therapy group and a western medicine group, 30 cases in each group. The surrounded needling therapy group was treated with shallow needling on local affected area as main and 4-5 distant acupoints as adjuvant, once each day; the western medicine group was treated with oral administration of Indomethacin and Allopurinol, thrice each day. They were treated for 15 days. The clinical therapeutic effects, the changes of serum uric acid content and the adverse reaction were observed in the two groups.</p><p><b>RESULTS</b>The total effective rate was 93.3% in the surrounded needling therapy group and 80.0% in the western medicine group, with a significant difference between the two groups (P < 0.01); the serum uric acid contents before and after treatment were (516.85 +/- 48.63) micromol/L and (293.77 +/- 68.45) micromol/L in the surrounded needling therapy group, and (509.66 +/- 51.11) micromol/L and (333.66 +/- 89.22) mciromol/L in the western medicine group, respectively, with significant differences before and after treatment in the two groups (both P < 0.05), and with a significant difference in the serum uric acid content after treatment between the two groups (P < 0.01). The surrounded needling therapy group had no adverse reaction, and the adverse reaction rate of the western medicine group was 46.7%, with a significant difference between the two groups (P < 0.01).</p><p><b>CONCLUSION</b>Surrounded needling therapy is superior to the western medicine in the therapeutic effect on acute gouty arthritis, and it is a safe and effective method for acute gouty arthritis.</p>
Subject(s)
Adult , Aged , Humans , Male , Middle Aged , Acupuncture Points , Acupuncture Therapy , Acute Disease , Therapeutics , Allopurinol , Therapeutic Uses , Arthritis, Gouty , Blood , Drug Therapy , Therapeutics , Indomethacin , Therapeutic Uses , Treatment Outcome , Uric Acid , BloodABSTRACT
<p><b>OBJECTIVE</b>To study the optimal medium composition for xylanase production by Aspergillus niger XY-1 in solid-state fermentation (SSF).</p><p><b>METHODS</b>Statistical methodology including the Plackett-Burman design (PBD) and the central composite design (CCD) was employed to investigate the individual crucial component of the medium that significantly affected the enzyme yield.</p><p><b>RESULTS</b>Firstly, NaNO(3), yeast extract, urea, Na(2)CO(3), MgSO(4), peptone and (NH(4))(2)SO(4) were screened as the significant factors positively affecting the xylanase production by PBD. Secondly, by valuating the nitrogen sources effect, urea was proved to be the most effective and economic nitrogen source for xylanase production and used for further optimization. Finally, the CCD and response surface methodology (RSM) were applied to determine the optimal concentration of each significant variable, which included urea, Na(2)CO(3) and MgSO(4). Subsequently a second-order polynomial was determined by multiple regression analysis. The optimum values of the critical components for maximum xylanase production were obtained as follows: x(1) (urea)=0.163 (41.63 g/L), x(2) (Na(2)CO(3))=-1.68 (2.64 g/L), x(3) (MgSO(4))=1.338 (10.68 g/L) and the predicted xylanase value was 14374.6 U/g dry substrate. Using the optimized condition, xylanase production by Aspergillus niger XY-1 after 48 h fermentation reached 14637 U/g dry substrate with wheat bran in the shake flask.</p><p><b>CONCLUSION</b>By using PBD and CCD, we obtained the optimal composition for xylanase production by Aspergillus niger XY-1 in SSF, and the results of no additional expensive medium and shortened fermentation time for higher xylanase production show the potential for industrial utilization.</p>
Subject(s)
Aspergillus niger , Culture Media , Endo-1,4-beta Xylanases , Fermentation , Nitrogen , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To establish a new method on stratification analysis when the stratification limits of confounding factors was not clear or contradictory.</p><p><b>METHOD</b>Data on a study of diabetes mellitus in Guangdong province collected in the year of 1997 and 1998 was analyzed using cluster-stratification analysis.</p><p><b>RESULTS</b>The efficiency of stratification analysis was improved and the confounding bias was effectively controlled with information bias avoided when the clusters-stratification analysis was applied.</p><p><b>CONCLUSION</b>The problem was logically solved using cluster analysis as an assistant stratification means.</p>