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Objective:To investigate the effects of Connexin-43 (Cx43) on osteoblasts proliferation and osteogenic differentiation and its regulatory mechanism.Methods:Osteoblasts were isolated and cultured in vitro. The osteogenic activity of osteoblasts was detected by alizarin red staining and alkaline phosphatase (ALP) staining after dexamethasone treatment. The expression of Cx43, Runt-related transcription factor 2 (Runx2), ALP, collagen I type (COL-I) and proliferation-related proteins PCNA and CDK4 in osteoblasts were detected by Western-blot. The expressions of osteoblast proteins were detected by immunofluorescence staining. The proliferation of osteoblasts was detected by CCK8 assay. The lentivirus-mediated Cx43 gene overexpression plasmid (Lv-Cx43) was constructed and transfected into osteoblasts. The osteogenic activity and proliferation ability of osteoblasts were further detected by the above methods. Cx43 in osteoblasts was overexpressed by pretreating PD98059. The osteogenic activity and proliferation of Cx43 in overexpressed osteoblasts was detected by CCK8 and alizarin red staining.Results:The isolated osteoblasts have osteogenic differentiation ability. Compared with the control group, 1×10 -6 mol/L dexamethasone treatment could reduce the formation of calcium nodules in osteoblasts. With the increase of dexamethasone treatment duration, the protein expression of Cx43, Runx2, ALP and COL-I in osteoblasts decreased gradually, while the expression of PCNA, CDK4 and p-ERK1/2 decreased. The OD values of normal osteoblasts at 0, 1, 2, 3 and 4 d were 0.316±0.043, 0.891±0.623, 1.683±0.154, 2.315±0.721 and 2.891±0.323, respectively. However, The OD values of osteoblasts treated with dexamethasone were 0.376±0.021, 0.657±0.121, 1.124±0.285, 1.521±0.272, 1.987±0.584, respectively. OD values of dexamethasone treated osteoblasts were lower than those of normal group at 2, 3 and 4 days ( P<0.05). The relative expression levels of Cx43 mRNA in control group, Lv-NC group and Lv-Cx43 group were 0.541±0.086, 0.598±0.018 and 1.000±0.082, respectively. The mRNA expression level of Cx43 in Lv-Cx43 group was higher than that in control group and Lv-NC group ( P<0.05). The ratio of Cx43 protein band to the gray value of GAPDH band in control group, Lv-NC group and Lv-Cx43 group were 0.816±0.737, 0.738±0.643 and 1.145±1.101, respectively. The expression level of Lv-Cx43 was higher than that in control group and Lv-NC group ( P<0.05). The expressions of Runx2, ALP, COL-I mRNA and related marker proteins in Lv-Cx43 group were higher than those in control group and Lv-NC group ( P<0.05). The number of calcium nodules in the Lv-Cx43 group was significantly higher than that in the control group and Lv-NC group. The OD value of osteoblasts and the number of calcium nodules in Lv-Cx43+PD98059 group were significantly lower than those in Lv-Cx43 group ( P<0.05). Conclusion:The proliferation and differentiation ability of osteoblasts is significantly decreased after the treatment of dexamethasone with decreased expression of Cx43. Overexpression of Cx43 can promote the proliferation and osteogenic differentiation of osteoblasts, which may be regulated through the ERK1/2 pathway.
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Objective:To investigate the short-term efficacy of sublingual immunotherapy in patients with allergic rhinitis of all ages.Methods:The data of 230 patients with allergic rhinitis of all ages who received sublingual immunotherapy in The Third People's Hospital of Bengbu from November 2020 to September 2021 were included in this study. Patient distribution characteristics were analyzed. Ninety-three patients were randomly selected and divided into child, adolescent, and adult groups according to different ages. Total nasal symptom scores measured before and 4 months after sublingual immunotherapy were compared among patients of different ages to evaluate the short-term efficacy of sublingual immunotherapy. The skin prick test was used to determine the allergic state of patients. Change in total nasal symptom score after treatment relative to that before treatment was used to evaluate the efficacy of sublingual immunotherapy.Results:The age range of patients receiving sublingual immunotherapy was large (3-71 years), but the average age was only 17.70 years. Ninety-three patients were followed up, including 50 children and 43 adolescents or adults. After 4 months of sublingual immunotherapy, total nasal symptom score in children and adolescents or adults were significantly decreased compared with those before treatment [(3.66 ± 1.69) points vs. (6.60 ± 1.96) points, (3.49 ± 1.72) points vs. (6.28 ± 2.28) points, both P < 0.001]. Before and after treatment, there was no significant difference in total nasal symptom score between children and adolescents or adults (both P > 0.05). Conclusion:Patients with allergic rhinitis who receive sublingual immunotherapy tend to be young. Short-term sublingual immunotherapy is effective for allergic rhinitis. There is no remarkable difference in the efficacy of sublingual immunotherapy between patients with allergic rhinitis of all ages.
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Objective:To investigate the expression of connexin-43 (Cx43) in steroid-induced osteonecrosis of femoral head and osteoblasts in rats and its regulation mechanism.Methods:The model of steroid-induced osteonecrosis of femoral head (SIONFH) of rat was established. Micro-CT and HE staining were used to observe the degree of bone trabecular destruction and the incidence of empty lacunae. The expression levels of Cx43 and PI3K/Akt signaling pathway related molecules and osteoblast-related proteins in model group and control group were detected by RT-PCT and Western blot. The osteoblast (OB) of rats was further isolated and cultured in vitro. Under treatment of dexamethasone (Dex), Cx43 expression in OB cells was detected by Western blot and immunofluorescence. Western blot was used to detect the effect of glucocorticoid (GC) on the expression of related molecules of PI3K/Akt/β-catenin signaling pathway. Akt activator (SC79) and PI3K inhibitor (LY294002) were used to study the molecular mechanism of Dex regulation on Cx43 expression in OB cells. The regulatory relationship between β-catenin and Cx43 was investigated by immunoprecipitation and small interfere RNA (siRNA) technology.Results:The model of SIONFH in rats was successfully established, which proved that Cx43 expression level in the SIONFH model group was significantly lower than that in the control group, and the expression level of Cx43 was positively correlated with the expression of PI3K/Akt signaling pathway related molecules and osteoblast-related proteins Runx2, ALP and Collagen I Type (COL). In addition, in vitro culture of isolated rat OB cells, the expression of Cx43, p-PI3K, P-Akt and β-catenin in OB cells decreased gradually as the Dex action time went on. Moreover, SC79 pretreatment could significantly reverse the inhibitory effect of GCs on Cx43 expression, while LY294002 could significantly enhance the inhibitory effect of GCs on Cx43. In addition, the immunoprecipitation results showed that β-catenin expression was closely related to Cx43 expression, and further studies showed that β-catenin-siRNA could significantly down-regulate the expression of Cx43.Conclusion:Under the action of GC, the expression level of Cx43 in bone tissue and OB cells decreased significantly, and the possible mechanism was that GCs inhibited the expression of Cx43 by inhibiting the PI3K/Akt/β-catenin signaling pathway, which laid a new theoretical foundation for the further study of the role of Cx43 in the pathogenesis of steroid-induced femoral head necrosis.
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Periprosthetic femoral fracture (PFF) is one of severe complications after total hip arthroplasty (THA). As the number of patients receiving THA increased recently, the incidence of PFFs also increased dramatically. There are a number of risk factors for PFFs, such as age, sex, falling and prosthesis loosening. The Vancouver classification system is the most commonly used classification method for PFFs. According to the fracture location, PFFs can be divided into type A intertrochanteric fracture, type B fracture around the stem and type C fracture beyond the stem. The Vancouver type B PFF is further subdivided into type B1 with a well-fixed prosthesis, type B2 with a loose prosthesis but with adequate bone stock, and type B3 with a loose prosthesis and poor proximal bone stock simultaneously. Currently, there are some controversies in treating PFFs, mainly including whether the stem is fixed or not, whether the prosthesis needs to be revised, the selection of the stem, the reconstruction of bone defects, and the methods of fracture fixation. We searched literatures related to PFFs after THA. The incidence, risk factors, classification methods, treatment principles and strategies of PFFs were summarized in the present study. Based on our long-term clinical experience, we evaluated the advantages and disadvantages of each treatment method and provided considerations for the clinical research and selection in treating PFFs.
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Periprosthetic femoral fracture (PFF) is one of severe complications after total hip arthroplasty (THA). As the number of patients receiving THA increased recently, the incidence of PFFs also increased dramatically. There are a number of risk factors for PFFs, such as age, sex, falling and prosthesis loosening. The Vancouver classification system is the most commonly used classification method for PFFs. According to the fracture location, PFFs can be divided into type A intertrochanteric fracture, type B fracture around the stem and type C fracture beyond the stem. The Vancouver type B PFF is further subdivided into type B1 with a well?fixed prosthesis, type B2 with a loose prosthesis but with adequate bone stock, and type B3 with a loose prosthesis and poor proximal bone stock simultaneously. Currently, there are some controversies in treating PFFs, mainly including whether the stem is fixed or not, whether the prosthesis needs to be revised, the selection of the stem, the reconstruction of bone defects, and the methods of fracture fixation. We searched literatures related to PFFs after THA. The incidence, risk factors, classification meth?ods, treatment principles and strategies of PFFs were summarized in the present study. Based on our long?term clinical experience, we evaluated the advantages and disadvantages of each treatment method and provided considerations for the clinical research and selection in treating PFFs.
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AIM:To investigate the role of microRNA-132 (miR-132) on alveolar macrophage inflammation. METHODS: Rat alveolar macrophage cell line NR8383 was transfected with miR-132 mimic, mimic negative control ( NC) , miR-132 inhibitor, or inhibitor NC.The cells were divided into transfection group, transfection +lipopolysaccha-ride ( LPS) group, and transfection +LPS +acetylcholine ( ACh) group.The mRNA expression of acetylcholinesterase ( AChE) was detected by real-time PCR.The protein levels of AChE, signal transducer and activator of transcription 3 (STAT3) and phosphorylated STAT3 (p-STAT3) in the cells, and nuclear factor-κB (NF-κB) in the cytoplasm and nu-cleus were analyzed by Western blot.The activity of AChE in the culture supernatant was measured by AChE activity assay kit.The nuclear translocation of NF-κB was detected by immunofluorescence assay.RESULTS: Up-regulation or down-regulation of miR-132 had no effect on the mRNA expression of AChE.However, up-regulation of miR-132 decreased the protein level of AChE compared with mimic NC group (P<0.05).Transfection with miR-132 inhibitor increased the pro-tein expression of AChE compared with inhibitor NC group ( P<0.05 ) .In the alveolar macrophages treated with LPS+ACh, the inhibition of nuclear translocation of NF-κB p65 in miR-132 mimic group was more effective than that in mimic NC group ( P<0.05) .The inhibitory effect in miR-132 inhibitor group was weaker than that in inhibitor NC group ( P<0.05 ) .The inhibitory effect of miR-132 mimic on the protein levels of STAT3 and p-STAT3 was stronger than that of mimic NC (P<0.05).CONCLUSION:miR-132 in LPS-stimulated alveolar macrophages reinforced ACh-mediated anti-inflam-matory reaction by targeting AChE to suppress ACh hydrolyzation, which was related to the suppression of NF-κB and STAT3 activation.
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This study was aimed to establish a HPLC method for the determination of isogarcinol. Daojin Inertsil WP300 C18 (4.6 mm× 150 mm, 5μm) was employed with methanol and water (75?25) as the mobile phase. The flow rate was 1.0 mL·min-1. The column temperature was set at 40°C. The detection wavelengthγ was set at 277 nm. The results showed that the linear range of isogarcinol was 0.005 7-0.039 9μg. The average recovery rate was 99.58%. The relative standard deviation (RSD) was 1.25%. The contents of isogarcinol inGarcinia mangostana,Garcinia oblongifolia,Garcinia oligantha,Cratoxylum cochinchinense andCalophyllum membranaceum were 0.285%, 0.199%, 0.857%, 0.161% and 0.006%, respectively. Isogarcinol was not detected inCratoxylum formosum orCalophyllum inophyllum. It was concluded that the method was convenient, accurate with high sensitivity, good stability and repeatability. It can be used for determination of isogarcinol content in Chinese herbal medicine.
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ObjectiveTo observe the effect of acetylcholine (ACh) on lipopolysaccharide (LPS) induced inflammatory model of rat alveolar macrophages, and to observe the effect of the acetylcholinesterase inhibitor physostigmine (Phy) on the anti-inflammatory effect of ACh.Methods The rat alveolar macrophages NR8383 were cultured in vitro, which were divided into five groups: blank control group, LPS group (stimulated with 1 mg/L LPS for 12 hours), LPS+ ACh group (0.01, 0.1, 1, 10, 100μmol/L of ACh were added for 5 minutes before LPS stimulation), LPS+ Phy group (1 mmol/L Phy was added for 5 minutes before LPS stimulation), and LPS+ ACh+ Phy group (1 mmol/L Phy and 10μmol/L ACh were added for 5 minutes before LPS stimulation). The supernatants were collected in each group, the enzyme-linked immunosorbent assay (ELISA) was used to assay the contents of tumor necrosis factor-α (TNF-α), interleukins (IL-1β, and IL-6). The activity of acetylcholine esterase (AChE ) in the supernatant was also determined.Results① The contents of TNF-α (ng/L: 605.09±57.13 vs. 34.07±8.62), IL-1β (ng/L: 377.09±28.55 vs. 32.33±10.62) and IL-6 (ng/L: 558.04±77.45 vs. 42.62±11.21) in the LPS group were significantly higher than those in the blank control group (allP 0.05). Nevertheless, 10μmol/L and 100μmol/L ACh notably reduced the production of TNF-α (ng/L: 451.19±30.67, 332.19±32.19 vs. 604.96±22.56), IL-1β(ng/L: 261.08±24.78, 143.98±28.39 vs. 367.06±10.44) and IL-6 (ng/L: 342.75±54.60, 235.48±29.75 vs. 562.69±63.34) in the culture supernatants compared with the LPS group (allP< 0.05).③ The activity of AChE in the LPS group was significantly higher than that in the blank control group (kU/L: 5.21±0.63 vs. 3.09±0.10,P< 0.05). The activity of AChE was successfully inhibited by 1 mmol/L acetylcholinesterase inhibitor Phy pretreatment compared with that in the LPS group (1.51±0.12 vs. 5.21±0.63,P< 0.05).④ The level of TNF-α (ng/L: 183.17±35.44 vs. 451.19±30.67), IL-1β (ng/L: 91.49±12.27 vs. 261.08±24.78) and IL-6 (ng/L: 108.17±22.82 vs. 342.75±54.60) in the culture supernatants of LPS+ ACh+ Phy group was significantly decreased as compared with LPS+ ACh group (allP< 0.05).Conclusions ACh with the final concentrations of 10μmol/L and 100μmol/L can inhibit the LPS induced inflammatory reaction in alveolar macrophages. The acetylcholinesterase inhibitor Phy can reinforce the ACh-mediated anti-inflammatory effect on alveolar macrophages inflammatory model.
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[Objective] To observe the therapeutic effect of Qingfei Jianpi Decoction (QJD) for the treatment of acne vulgaris (AV) . [Methods] One hundred and five cases of AV were randomized into two groups: group A (n =64) was treated with QJD, a prescription medicine mainly composed of Herba Houttuyniae, Cortex Lycii, Cortex Moutan, Radix Scutellariae, Radix Fici Simplicissimae, Radix Flemingiae Philippinensis, Rhizoma Atractylodis Macrocephalae, Poria, etc. , and group B (n=41) with tetracycline for 4 weeks. After treatment, the therapeutic effect, changes of facial lesion and signs, and side effects were observed. A 3-month follow-up was made for the cured patients to investigate the recurrence rate. [Results] Facial lesion and seborrheic degree were relieved in group A, the difference being significant as compared with those before treatment (P0.05 ) . The therapeutic effect was better in group A than that in group B (P
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【Objective】To observe the effect of treating psoriasis vulgari(PV)by solving disorders of blood system.【Methods】Two hundred and six patients with PV were randomized into groups A and B.Group B(n=70)was treated with oral use of clarityne,vitamin C,Tripterygium Glycosides Tablets,and roxithromycin,as well as external application of sulphur ointment or clobetasol propionate ointment.Group A(n=136)was additionally treated with basic recipe(composed of Rhizoma Smilacis Glabrae,Radix Isatidis,Folium Isatidis,Radix Rehmanniae,Cortex Moutan,Herba Hedyotis Diffusae,Herba Lobeliae Chinensis,Nidus Vespae,Rhizoma Chuanxiong,Rhizoma Alismatis,Herba Plataginis,Fructus Kochiae,Cortex Dictamni,Radix Glycyrrhizac).Meanwhile,patients in group A were classified into 3 syndrome patterns of blood heat,blood stasis and blood dryness,and treated with corresponding recipes.After three courses of treatment(3 month in total),the therapeutic effect,as well as the side effects and recurrence rate was evaluated in the two groups.【Results】In group A,71 patients were cured,22 markedly effective,26 effective,17 ineffective and the total effective rate was 87.50%,and 23,15,21,11 and 84.28% in group B,respectively.The therapeutic effect in group A was superior to that in group B(P