The aim of this study is to investigate the apoptotic inhibition and its molecular mechanism of dexamethasone (DEX) acting on cisplatin (CDDP)-induced apoptosis of human lung adenocarcinoma cell SPC-A1. SPC-A1 cells were pre-cultured in vitro for 24 hours with DEX in different concentrations and then CDDP was added in different concentrations for culturing for further 48 hours. The survival rates of the cells were determined by MTT. The expression of serum/glucocorticoid-induced kinase (SGK-1) and mitogen-activated protein kinase phosphatase-1 (MKP-1) in SPC-A1 cells after being cultured by 1 μmol/L DEX at different time was detected by semi-quantitative RT-PCR technology. The expression of glucocorticoid receptor (GR) in SPC-A1 cells was measured by immunohistochemistry (IHC) with biotin-labeled anti-GR. The results of MTT showed that SPC-A1 cells had resistance to CDDP-induced apoptosis with pre-cultured DEX and the resistance intensity presented DEX concentration-dependent. The expressing quantity of SGK-1 in SPC-A1 cells stimulated by DEX could be elevated and increased with intention of time, but the express of MKP-1 was not detected. Up-regulated expression of GR in SPC-A1 cells stimulated by DEX was detected by IHC. The number of cells expressing GR in SPC-A1 cells was significantly higher than that in the control group. The results showed that DEX inhibited apoptosis of SPC-A1 cells induced by CDDP. The possible molecular mechanism is that elevated expression of GR induced by DEX up-regulates the expression of SGK-1 which locates at the downstream of anti-apoptosis pathway. The apoptosis resistance of SPC-A1 cells may account for all above the factors.
In this research a strain of isolated Pseudomonas alcaligenes which causes degradation of dexamethasone was acclimated further and its proteins of every position in the bacterium were separated by the osmotic shock method. The separated intracellular proteins which had the highest enzyme activity were extracted by the salting out with ammonium sulfate and were purified with the cation exchange chromatography and gel chromatography. The purified proteins which was active to cause degradation of dexamethasone had been detected were cut with enzyme and were analyzed with mass spectrometry. The results showed that the degradation rate to dexamethasone by acclimated Pseudomonas alcaligenes were increased from 23.63% to 52.84%. The degrading enzymes were located mainly in the intracellular of the bacteria and its molecular weight was about 41 kD. The specific activity of the purified degrading enzymes were achieved to 1.02 U·mg-1. Its 5-peptide amino acid sequences were consistent with some sequences of the isovaleryl-CoA dehydrogenase. The protein enzyme may be a new kind degrading enzyme of steroidal compounds. Our experimental results provided new strategies for cleanup of dexamethasone in water environment with microbial bioremediation technique.
Objective To investigate the effect of bone morphogenetic protein 2 (BMP-2) and dexamethason (DXM) on proliferation and differentiation of human dental pulp cellsin vitro. Methods Primary human dental pulp cells were cultured in vitro by tissue culture method. The 3rd generation cells were used to identify cell phenotype for vimentin and cytokeratin by immunocytochemistry staining. The 3-5 generations of human dental pulp cells were randomly divided into 4 groups: 100 ng/mL BMP-2 (group A), 1×10–8 mol/L DXM (group B), and both 100 ng/mL BMP-2 and 1×10–8 mol/L DXM (group C) were added; neither BMP-2 nor DXM was added in group D as control group. The cell growth curve was drawn at 1, 3, 5, and 7 days after culture. The expressions of osteo/dentanogenic genes including alkaline phosphatase (ALP), dentin sialophoshoprotein (DSPP), and dentin matrix protein 1 (DMP-1) were detected by RT-PCR analysis at 5 and 7 days after culture, the ratio between the positive staining area and the total area by ALP staining at 14 days, and absorbance (A) value at 562 nm by alizarin red staining at 21 days after culture. Results Human dental pulp cells were successfully isolated and cultured, which were long fusiform and showed a positive reaction for vimentin and a negative reaction for cytokeratin. The growth curve indicated that cells increased with the extending of incubation time, reached a peak at 5 days, then reduced at 7 days to the level at 3 days. At 5 days after culture, the cells were significantly more in groups A, B, and C than group D (P<0.05), in group C than group A (P<0.05), and in group A than group B (P<0.05). RT-PCR analysis showed that the mRNA expressions of ALP, DSPP, and DMP-1 at 5 days were significantly higher in groups A, B, and C than group D (P<0.05), and in group C than groups A and B (P<0.05), but no significant difference was found between groups A and B (P>0.05); the mRNA expression of DSPP in groups A, B, and C was significantly higher than that in group D (P<0.05), but there was no significant difference in mRNA expressions between other groups at 7 days (P>0.05). At 14 days, positive staining in varying degrees was observed in each group, especially in group C; the ratio between the positive staining area and the total area was significantly higher in group C than groups A, B, and D (P<0.05), and in groups A and B than group D (P<0.05), but there was no significant difference between groups A and B (P>0.05). At 21 days, there were a variety of mineralized nodules in groups A, B, and C in nonuniformly scattered or clustered distribution, but no mineralized nodules were observed in group D. TheA values of mineralized nodules showed significant difference between groups (P<0.05). Conclusion BMP-2 may be more effective in promoting proliferation of human dental pulp cells than DXM. Combined application of BMP-2 and DXM can remarkably promote the proliferation and differentiation of human dental pulp cells.
Objective To explore the risk factors associated with interleukin 6 (IL-6) level in serum after total knee arthroplasty (TKA). Methods A retrospective study was made on the clinical data of 273 patients underwent primary unilateral TKA between July 2015 and April 2017. There were 50 males and 223 females with an average age of 66.3 years (range, 36-89 years), and the body mass index (BMI) was (25.5±3.7) kg/m2. Of them, 256 patients suffered with osteoarthritis, and the other 17 patients with rheumatoid arthritis. Univariate analysis was made to find the related factors between IL-6 level in serum at 1 day after operation and preoperative data including gender, age, BMI, diagnosis, comorbidities, preoperative American Society of Anesthesiologists (ASA) grade, preoperative varus or valgus deformity, range of motion of the knee, preoperative level of C-reactive protein (CRP) and IL-6 in serum, operation time, intraoperative blood loss, usage of drainage tube and catheter, and dosage of tranexamic acid and dexamethasone used on day of operation. Furthermore, the multiple linear regression analysis was performed to identify the risk factors. Results The operation time was (79.7±15.6) minutes, and the intraoperative blood loss was (107.8±25.3) mL. Drainage tubes were used in 111 patients and catheters were used in 41 patients after operation. The dosage of tranexamic acid and dexamethasone used on day of operation were (3.2±0.8) g and (15.1±6.6) mg, respectively. The levels of IL-6 in serum were (4.48±3.05), (42.65±37.09), and (28.21±26.44) pg/mL before operation and at 1 and 3 days after operation, respectively. Univariate analysis showed that the level of IL-6 in serum at 1 day after operation was significantly higher in variables as follows: age, diagnosis, history of lung infection, range of motion, preoperative levels of CRP and IL-6 in serum, intravenous dosage of tranexamic acid and dexamethasone on day of operation (P<0.05). Multiple linear regression analysis showed that range of motion less than 90°, intravenous dosage of tranexamic acid on day of operation less than 3 g, and dosage of dexamethasone on day of operation less than 10 mg were significant risk factors (P<0.05). Conclusion Range of motion less than 90°, intravenous dosage of tranexamic acid on day of operation less than 3 g, and dosage of dexamethasone on day of operation less than 10 mg were independent risk factors that resulted in increased level of IL-6 in serum at 1 day after TKA.
ObjectiveTo explore the effect of Kaempferol on bone microvascular endothelial cells (BMECs) in glucocorticoid induced osteonecrosis of the femoral head (GIONFH) in vitro. MethodsBMECs were isolated from cancellous bone of femoral head or femoral neck donated voluntarily by patients with femoral neck fracture. BMECs were identified by von Willebrand factor and CD31 immunofluorescence staining and tube formation assay. The cell counting kit 8 (CCK-8) assay was used to screen the optimal concentration and the time point of dexamethasone (Dex) to inhibit the cell activity and the optimal concentration of Kaempferol to improve the inhibition of Dex. Then the BMECs were divided into 4 groups, namely, the cell group (group A), the cells treated with optimal concentration of Dex group (group B), the cells treated with optimal concentration of Dex+1 μmol/L Kaempferol group (group C), and the cells treated with optimal concentration of Dex+5 μmol/L Kaempferol group (group D). EdU assay, in vitro tube formation assay, TUNEL staining assay, Annexin Ⅴ/propidium iodide (PI) staining assay, Transwell migration assay, scratch healing assay, and Western blot assay were used to detect the effect of Kaempferol on the proliferation, tube formation, apoptosis, migration, and protein expression of BMECs treated with Dex. ResultsThe cultured cells were identified as BMECs. CCK-8 assay showed that the optimal concentration and the time point of Dex to inhibit cell activity was 300 μmol/L for 24 hours, and the optimal concentration of Kaempferol to improve the inhibitory activity of Dex was 1 μmol/L. EdU and tube formation assays showed that the cell proliferation rate, tube length, and number of branch points were significantly lower in groups B-D than in group A, and in groups B and D than in group C (P<0.05). TUNEL and Annexin V/PI staining assays showed that the rates of TUNEL positive cells and apoptotic cells were significantly higher in groups B-D than in group A, and in groups B and D than in group C (P<0.05). Scratch healing assay and Transwell migration assay showed that the scratch healing rate and the number of migration cells were significantly lower in groups B-D than in group A, and in groups B and D than in group C (P<0.05). Western blot assay demonstrated that the relative expressions of Cleaved Caspase-3 and Bax proteins were significantly higher in groups B-D than in group A, and in groups B and D than in group C (P<0.05); the relative expressions of matrix metalloproteinase 2, Cyclin D1, Cyclin E1, VEGFA, and Bcl2 proteins were significantly lower in groups B-D than in group A, and in groups B and D than in group C (P<0.05). Conclusion Kaempferol can alleviate the damage and dysfunction of BMECs in GIONFH.
Objective To explore the effect of corn oligopeptide (COP) on dexamethasone-induced muscle atrophy. Methods Forty-nine male Sprague-Dawley rats aged 8 weeks were divided into blank group (n=10) and model group (n=39). The rats in the model group were intraperitoneally injected with dexamethasone (1.0 mg/kg), and the rats in the blank group were injected with normal saline. After 19 days, one rat in the blank group and three rats in the model group were taken to observe whether the model was successfully constructed. After successful modeling, the rats in the model group were randomly divided into model control group, COP low-dose group (COP-L group, 0.5 g/kg), COP medium-dose group (COP-M group, 1.0 g/kg) and COP high-dose group (COP-H group, 2.0 g/kg), with 9 rats in each group. After 33 days, the grip strength of the rats was measured, and then the gastrocnemius, soleus, tibialis anterior and metatarsal muscles were separated and weighed, and muscle fiber diameter, relative expression of Atrogin-1 and MuRF-1 mRNA were measured. Non-targeted metabolomics of gastrocnemius muscle were measured. Results Compared with that in the blank group, the body weight of rats in the model group reduced (P<0.05), and myofibril rupture was observed, indicating that the model was successful. Compared with those in the model control group, the grip strength increased in the COP-L and COP-M groups (P<0.05); the muscle coefficients of gastrocnemius and soleus in the COP-L and COP-H groups increased (P<0.05), and the muscle coefficients of plantaris in the COP-L and COP-M groups increased (P<0.05); the muscle fiber diameter of the tibial anterior muscle increased in the three doses of COP groups (P<0.05), and the muscle fiber diameter of the plantaris muscle increased in the COP-M and COP-H groups (P<0.05); the relative expression of Atrogin-1 mRNA decreased in the three doses of COP groups (P<0.05), while the relative expression of MurF-1 mRNA in the COP-L and COP-H groups decreased (P<0.05). The amino acid synthesis pathway, glycolysis pathway, and acid metabolism pathway were activated in gastrocnemius muscle. Conclusions COP can significantly improve the muscle atrophy induced by dexamethasone. The mechanism may be related to the decrease of Atrogin-1 and MuRF-1 expression in ubiquitin-proteasome pathway and the increase of amino acid biosynthesis.
Objective To observe the clinical efficacy of pars plana vitrectomy (PPV) combined with dexamethasone intravitreal implant (DEX) in the treatment of proliferative diabetic retinopathy (PDR). MethodsA prospective randomized controlled study. A total of 57 PDR patients with 79 eyes diagnosed by Department of Ophthalmology of The First Affiliated Hospital of Nanjing Medical University from May 2021 to February 2023 were included in the study. Best corrected visual acuity (BCVA) and optical coherence tomography (OCT) were performed in all affected eyes. Central macular thickness (CMT) was measured by OCT. The patients were randomly divided into control group and experimental group, with 27 cases and 35 eyes and 30 cases and 44 eyes, respectively. All eyes were treated with routine 25G PPV and intraoperative whole-retina laser photocoagulation. At the end of the operation, the experimental group was given 0.7 mg DEX intravitreal injection. At 1, 4, 12, and 24 weeks after operation, the same equipment and methods were used for relevant examinations. The improvement after surgery was assessed according to the diabetic retinopathy severity score (DRSS). Mixed analysis of variance was used to compare logarithm of the minimum angle of resolution BCVA and CMT between the two groups and within the two groups before and after operation. ResultsAt 1, 4, 12 and 24 weeks after surgery, BCVA was significantly improved at different time points after surgery, and the differences were statistically significant (P＜0.001). At different time after operation, BCVA and CMT in experimental groups were significantly better than that in control group, with statistical significance (P＜0.05). Compared with the CMT before surgery, the CMT at all time point after surgery in experimental group were significantly decreased, and the difference were statistically significant (P＜0.05). There was no significant difference one week after eye operation in control group (P=0.315). At 4, 12 and 24 weeks after operation, CMT decreased in control group, and the differences were statistically significant (P＜0.05). Compared with before surgery, DRSS increased two steps higher at 1, 4, 12 and 24 weeks after surgery in 20 (45.45%, 20/44), 26 (59.10%, 26/44), 32 (72.73%, 32/44) and 31 (70.45%, 31/44) eyes in the experimental groups, respectively. The control group consisted of 15 (42.86%, 15/35), 15 (42.86%, 15/35), 16 (45.71%, 16/35) and 18 (51.43%, 18/35) eyes, respectively. There was no significant difference in DRSS at 1, 4 and 24 weeks after operation between the control group and the experimental group (P=0.817, 0.178, 0.105). At 12 weeks after surgery, the difference was statistically significant (P=0.020). ConclusionPPV combined with intravitreal injection of DEX in the treatment of PDR can improve postoperative visual acuity, alleviate postoperative macular edema and improve the severity of DR.