Differentially expressed genes and signalling pathways are involved in mouse osteoblast-like MC3T3-E1 cells exposed to 17-β estradiol

Oestrogen is essential for maintaining bone mass, and it has been demonstrated to induce osteoblast proliferation and bone formation.In this study, complementary DNA（cDNA） microarrays were used to identify and study the expression of novel genes that may be involved in MC3T3-E1 cells’ response to 17-b estradiol. MC3T3-E1 cells were inoculated in minimum essential media alpha（a-MEM）cell culture supplemented with 17-b estradiol at different concentrations and for different time periods. MC3T3-E1 cells treated with1028mol？L2117-b estradiol for 5 days exhibited the highest proliferation and alkaline phosphatase（ALP） activity; thus, this group was chosen for microarray analysis. The harvested RNA was used for microarray hybridisation and subsequent real-time reverse transcription polymerase chain reaction（RT-PCR） to validate the expression levels for selected genes. The microarray results were analysed using both functional and pathway analysis. In this study, microarray analysis detected 5 403 differentially expressed genes,of which 1 996 genes were upregulated and 3 407 genes were downregulated, 1 553 different functional classifications were identified by gene ontology（GO） analysis and 53 different pathways were involved based on pathway analysis. Among the differentially expressed genes, a portion not previously reported to be associated with the osteoblast response to oestrogen was identified. These findings clearly demonstrate that the expression of genes related to osteoblast proliferation, cell differentiation, collagens and transforming growth factor beta（TGF-b）-related cytokines increases, while the expression of genes related to apoptosis and osteoclast differentiation decreases, following the exposure of MC3T3-E1 cells to a-MEM supplemented with 17-b estradiol. Microarray analysis with functional gene classification is critical for a complete understanding of complementary intracellular processes. This microarray analysis provides large-scale gene expression data that require further confirmatory studies.

Breathing Enable the Magnetic Properties of Erythrocytes （Hem Fe） Oxygen, Cells and Carbondioxide

To prove that magnetic characteristics （MC） of participants in breathing are crucial in respiration; to explain the impact of Earth＇s magnetic field （EMF）, cosmic radiation （CR） and MC of cells on evolution and respiration; to show MC of cells, i.e. development of cytoplasm and membrane of nucleus and cell; to explain crossing over; to show in detail the impact of EMF on cell division; to explain the exchange of oxygen and carbon dioxide in the lungs and cells MC; to explain how the nervous system works as a regulator of respiration. Studying the literature about ceil, respiration and the role of nervous system in breathing; linking natural EMF, CR and MC of cells with evolution and respiration. Every cell has MC. They are very important in every cell metabolism. Crossing over provides polymorphism, enabled by EMF. Crucial factor in cell division is EMF. Electrical pulses （action potentials） are generated by CR. Breathing is enabled by MC of oxygen, carbon dioxide, hemoglobin and cells. MC enables respiration. EMF, CR and MC of cells are crucial factors in evolution processes generally. EMF is the initiator of cell division. Micro electrical pulses from central nervous system are regulators of respiration. Conductivity of micro electrical pulses in the body is fast-electronic and slow-ion （chemical）.

雌二醇通过雌激素受体α/糖原合酶激酶-3β/β联蛋白信号通路调节成骨细胞增殖

目的研究观察雌二醇对小鼠成骨细胞前体细胞增殖影响并研究经典wnt通路在其增殖过程中的作用。方法本研究利用小鼠胚胎成骨细胞前体细胞Mc3T3-E1为细胞模型,不同浓度雌二醇处理Mc3T3-E1细胞,四甲基偶氮唑蓝(MTT)检测细胞增殖率,蛋白印迹法和免疫荧光技术检测经典Wnt信号通路信号分子表达。结果研究表明不同浓度雌二醇处理Mc3T3-E1细胞,MTT显示随着雌二醇浓度增加,Mc3T3-E1细胞增殖率增加,其中以10-7 mol/L浓度处理后细胞获得最佳增殖率。蛋白印迹法结果显示随着雌激素浓度增加,小鼠Mc3T3-E1细胞中p-GSK-3β,β联蛋白(catenin),细胞周期蛋白(Cyclin)D1的表达增加。经雌激素受体(ER)α沉默后,相对于空白对照组小鼠Mc3T3-E1细胞p-GSK-3β,β-catenin及Cyclin D1表达下降,而ERβ沉默后,同对照组相比成骨细胞中各分子表达则无明显变化。结论雌二醇可通过ERα/GSK-3β/β-catenin信号通路调节小鼠成骨细胞增殖。

黄连素通过PI3K/Akt/mTOR信号通路抑制高糖条件下的肾脏系膜细胞增殖

目的：探讨黄连素（berberine,BBR）对高糖条件下的大鼠肾脏系膜细胞 HBZY-1 cells （MCS）增殖的影响及其作用机制。方法将对数生长期细胞分成正常糖处理组（NG 组）,高糖组（HG组）和黄连素组（BBR组）。NG组细胞体系常规培养培养,HG 组细胞体系加入40 mmol/L 葡萄糖,BBR组细胞培养体系中加入40 mmol/L葡萄糖＋黄连素30μmol/L。各组细胞培养48 h 后,利用MTT检测各组HBZY-1细胞增殖；RT-PCR检测各组细胞p85,Akt和mTOR基因表达；West-ern blotting检测细胞中 p85、p-p85、Akt、p-Akt,mTOR 和 Collagen-Ⅳ蛋白的变化。结果 NG 组与HG组增值率分别为（25%±3%）和（75%±5%）,与 NG组增值率比较,HG组肾脏系膜细胞增殖率明显增高（P〈0.05）,p85与 Akt mRNA表达水平和蛋白水平均无明显变化（P＆gt;0.05）,但 p-p85、p-Akt,Collagen-Ⅳ蛋白表达水平和 mTOR mRNA 表达与蛋白水平均明显增加（P〈0.05）；BBR 组增值率为（42%±5%）,与 HG 组比较,BBR 组肾脏系膜细胞增值率明显降低（P〈0.05）,但 p-p85、p-Akt、Collagen-Ⅳ蛋白表达水平和 mTOR mRNA 表达与蛋白水平均明显下降（P〈0.05）,而 p85和Akt mRNA和蛋白表达水平均无统计学差异（P〈0.05）。结论黄连素能抑制高糖导致的系膜细胞增殖,其作用可能是通过抑制PI3K/Akt/mTOR信号通路激活。

Sustained release of Semaphorin 3A from a-tricalcium phosphate based cement composite contributes to osteoblastic differentiation of MC3T3-E1 cells

The reinforcement of calcium phosphate materials with silk fibroin （SF） has been one of the strategies to overcome the brittleness. However, the lack of osteoinductivity may still restrict their further use. This study aimed to investigate the biocompatibility and osteogenesis capacity of a novel Semaphorin 3A-loaded chitosan microspheres/SF/a-tricalcium phosphate composite （Sema3A CMs/SF/a-TCP） in vitro. Sema3A was first incorporated into CMs, and the Sema3A CMs/SF/a-TCP composite was then prepared. The morphology of the CMs was observed using SEM. The in vitro release kinetics, cytotoxicity, and cell compatibility were evaluated, and the real-time quantitative polymerase chain reaction （RT-qPCR） and activity of alkaline phosphatase （ALP） were used to evaluate the osteogenesis capacity of the composite. The in vitro release of Sema3A from the Sema3A CMs/SF/a-TCP composite showed a temporally controlled manner. The extract of the Sema3A CMs/SF/a-TCP composite presented no obvious side effect on the MC3T3-E1 cell proliferation, nor promote cell proliferation. The MC3T3-E1 cells were well-spread and presented an elongated shape on the Sema3A CMs/SF/a-TCP composite surface; the ALP activity and the osteogenic-related gene expression were higher than those seeded on the surface of the CMs/SF/a-TCP and SF/a-TCP composites. In conclusion, Sema3A CMslSF/a-TCP has excellent biocompatibility and contributes to the osteoblastic differentiation of MC3T3-E1 cells.

Influence of Moxibustion of Complementary Acupoints on Analgesic Effect and Mast Cell Function in CIA Rats

[Objectives]To explore the moxibustion effect and mechanism of"Complementary Acupoints"on rheumatoid arthritis(RA).[Methods]A total of 32 Wistar rats were randomly divided into 4 group:normal control(K),model group(M group),"the Complementary Acupoints"Yanglingquan and Yinlingquan group(AB group),Shenshu and Zusanli group(CD group),with 8 rats in each group.Moxibustion was performed using refined moxa floss(2 mg/pc)on acupoints in both sides every other day,5 pieces for each acupoint,and implemented for 10 times.Before and after the modeling and after the intervention,the left foot sole thermal pain threshold of each group was measured.After the intervention,the rats'left paws were examined by X-ray to detect the number of mast cells(MCs)and degranulation in the affected area of the foot.[Results]The thermal pain threshold of the model group was lower than that of the K group(P<0.05).The number of MC in the affected area and the degranulation were increased.After the moxibustion intervention,the thermal pain threshold of the AB and CD groups was higher than that of the M group(P<0.05);the number of MCs and degranulation were decreased compared with M group,and the degree of reduction was better than that of CD group.The data of foot and claw showed that the foot and paw soft tissue swelling,bone and joint deformity and bone destruction were better in AB and CD groups were more relieved than that in M group.[Conclusions]The moxibustion of Complementary Acupoints can effectively intervene with RA of rats,and its mechanism may be closely related to the MC degranulation.

In Vitro Biocompatibility of MC3T3-E1 Osteoblast-like Cells on Arg-Gly-Asp Acid Peptides Immobilized Graphite-like Carbon Coating on Carbon/Carbon Composites

Carbon/carbon （C/C） composites were deposited with graphite-like carbon （GLC） coating, and then, Arg-Gly- Asp acid （RGD） peptides were successfully immobilized onto the functionalized GLC coating. GLC coating was utilized to prevent carbon particles releasing and create a uniform surface condition for C/C composites. RGD peptides were utilized to improve biocompatibility of GLC coating. Surface chemical characterizations of functionalized GLC coating were detected by contact angle measurement, X-ray photoelectron spectroscopy and Raman spectra. Optical morphology of GLC coatings was observed by confocal laser scanning microscopy. In vitro biological performance was determined using samples seeded with MC3T3-E1 osteoblast-like cells and cultured for 1 week. Surface characterizations and morphological analysis indicated that C/C composites were covered by a dense and uniform GLC coating. Contact angle of GLC coating was reduced to 27.2° when it was functionalized by H202 oxidation at 40 ℃ for 1 h. In vitro cytological test showed that the RGD peptides immobilized GLC coating had a significant improvement in biocompatibility. It was suggested that RGD peptides provided GLC coating with a bioactive surface to improve cell adhesion and proliferation on C/C composites.

Mast cell degranulation-triggered by SARS-CoV-2 induces tracheal-bronchial epithelial inflammation and injury

SARS-CoV-2 infection-induced hyper-inflammation is a key pathogenic factor of COVID-19.Our research,along with others',has demonstrated that mast cells(MCs)play a vital role in the initiation of hyper-inflammation caused by SARS-CoV-2.In previous study,we observed that SARS-CoV-2 infection induced the accumulation of MCs in the peri-bronchus and bronchioalveolar-duct junction in humanized mice.Additionally,we found that MC degranulation triggered by the spike protein resulted in inflammation in alveolar epithelial cells and capillary endothelial cells,leading to subsequent lung injury.The trachea and bronchus are the routes for SARS-CoV-2 transmission after virus inhalation,and inflammation in these regions could promote viral spread.MCs are widely distributed throughout the respiratory tract.Thus,in this study,we investigated the role of MCs and their degranulation in the development of inflammation in tracheal-bronchial epithelium.Histological analyses showed the accumulation and degranulation of MCs in the peri-trachea of humanized mice infected with SARS-CoV-2.MC degranulation caused lesions in trachea,and the formation of papillary hyperplasia was observed.Through transcriptome analysis in bronchial epithelial cells,we found that MC degranulation significantly altered multiple cellular signaling,particularly,leading to upregulated immune responses and inflammation.The administration of ebastine or loratadine effectively suppressed the induction of inflammatory factors in bronchial epithelial cells and alleviated tracheal injury in mice.Taken together,our findings confirm the essential role of MC degranulation in SARS-CoV-2-induced hyper-inflammation and the subsequent tissue lesions.Furthermore,our results support the use of ebastine or loratadine to inhibit SARS-CoV-2-triggered degranulation,thereby preventing tissue damage caused by hyper-inflammation.

A study on the differences of effect on mast cells and serum 5-HT in the acupoint area of "CV 4" of rats by acupuncture stimulus and thermal stimulus

Objective: To observe the influence of acupuncture stimulus and thermal stimulus on the expression of mast cells(MCs) and 5-hydroxytryptamine(5-HT) in local acupoint area of Guanyuan(关元 CV 4) and serum 5-HT in rats.Methods: Thirty-nine male C57 BL/6 rats were randomly divided into blank control group, acupuncture stimulus group and thermal stimulus group, with 13 rats in each group. The rats were stimulated by manual acupuncture at CV 4 for 5 min in acupuncture stimulus group, while those in thermal stimulus group were stimulated by adopting a thermal moxibustion apparatus on CV 4 for 30 min. The expression of MC and 5-HT in the skin in the acupoint area of CV 4 before and after acupuncture stimulus and thermal stimulus was observed and analyzed via adopting toluidine blue staining method and immunofluorescence histochemical method(5 rats were selected from each group), and the 5-HT content in serum before and after stimulus was determined through enzyme-linked immunosorbent assay(ELISA)(8 rats were selected from each group). The influence of acupuncture stimulus and thermal stimulus on the contents of MC and 5-HT in the skin and serum 5-HT in rats was analyzed and compared.Results: ① After acupuncture stimulus or thermal stimulus on CV 4 of C57 BL/6 rats, the number of MC in the acupoint area significantly increased when compared with that in blank group(the acupuncture stimulus group 12.40 ± 2.07 vs. the blank group 3.00 ±5.96;thermal stimulus group 26.20 ± 10.85 vs. the blank group 12.40 ± 2.07, both P< 0.05), and MC aggregation and degranulation were observed(the acupuncture stimulus group 17.80 ±4.55 vs. the blank group 8.00 ±3.16;the thermal stimulus group24.00±9.05 vs. the blank group 8.00±3.16. P<0.05, P< 0.01).② After acupuncture stimulus or thermal stimulus, 5-HT was released by MCs in the acupoint area, which aggregated around the blood vessels,and the number of 5-HT in the acupoint area significantly increased when compared with that in blank group(the blank group 3.00 ±1.28 vs. the acupuncture stimulus group 10.02 ±3.21;the blank group3.00 ±1.28 vs. the thermal stimulus group 14.00 ±3.94, both P< 0.01).③ Compared with blank group,both acupuncture stimulus and thermal stimulus could reduce the 5-HT content in serum(the blank group 0.72 ±0.2372 vs. acupuncture stimulus group 0.43 ±0.21: the blank group 0.72 ±0.24 vs. thermal stimulus group 0.32 ±0.18, both P<0.01), and the effect in thermal stimulus group was slightly superior to that in acupuncture stimulus group(P<0.05).Conclusion: Both acupuncture stimulus and thermal stimulus can cause the aggregation and degranulation of MCs and high expression of 5-HT in the acupoint area. The effect of thermal stimulus was superior to that of acupuncture stimulus in degranulation.

Eucommia ulmoides Oliv.antagonizes H_2O_2-induced rat osteoblastic MC3T3-E1 apoptosis by inhibiting expressions of caspases 3,6,7,and 9

Eucommia ulmoides Oliv.(EuO),also known as Duzhong,native to China,has been reported to have antioxidative function,but its cellular mechanism is not fully examined yet.We investigated inhibitory effects of EuO leaf ethanol extracts on H2O2-induced apoptosis in rat osteoblastic MC3T3-E1 cells and underlying mechanisms.Locally-grown Duzhong leaves were extracted with ethanol.MC3T3-E1 cells were treated with EuO (6.25,12.5,25,50,and 100 μg/ml) for 24 h,and then H2O2 (800 μmol/L) for an additional 24 h.Cell survival rate,percentage of apoptosis,and expressions of caspases 3,6,7,and 9 were examined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay,microscopic analysis,Western blotting,and reverse transcription polymerase chain reaction (RT-PCR).The final EuO leaf ethanol extract powder was detected to contain caffeotannic acid at 58 mg/g and geniposide at 3.45 mg/g by high performance liquid chromatography (HPLC).EuO remarkably restrained cell oxidative damage and increased cell survival rate in a dose-dependent manner: 0 μg/ml,0.21;6.25 μg/ml,0.28;12.5 μg/ml,0.31;25 μg/ml,0.48;50 μg/ml,0.54;and 100 μg/ml,0.66 (P<0.05),with the half-effective concentration being around 25 μg/ml.MTT results were confirmed by microscopic analysis.Western blotting and RT-PCR analyses showed that the expressions of caspases 3,6,7,and 9 were significantly decreased in the EuO-treated cells compared with the control (EuOand H2O2-free) (P<0.05),with the half-effective concentration of EuO ranging from 12.5 to 25 μg/ml.We conclude that the ethanol-extracted EuO leaf extracts promoted the growth of MC3T3-E1 cells,and suppressed the H2O2-induced apoptosis in a rat MC3T3-E1 osteogenic cell model,likely due to the inhibition of caspases’ activities.The results indicate that EuO is a potent antioxidant,which may contribute to its many cellular protective functions,including the promotion of bone growth.

Alcoholic liver disease and mast cells:What's your gut got to do with it?

Alcoholic liver disease(ALD)remains one of the leading causes of liver injury and death when left un-treated.The gut microbiota has been recognized as a key regulator of a number of pathologies,including ALD.The role of mast cells(MCs)during liver disease progression has been demonstrated in a number of animal models and in human liver diseases.The interaction between the gut microbiota and MCs has been investigated,and links between the gut and these immune cells are being uncovered.The interplay between the gut microbiota and MCs during ALD has been evaluated and studies suggest that there could be an important link between MCs,their mediators and gut inflammation during the progression of ALD.

白藜芦醇抑制高糖诱导的肾脏系膜细胞增殖的机制研究

目的探讨白藜芦醇(resveratrol,RSV)对高糖诱导的大鼠肾脏系膜细胞HBZY-1 cells(MCS)增殖的影响,并探讨其潜在作用机制。方法将对数生长期细胞随机分为对照组、高糖组、RSV组。各组细胞培养48 h后,利用MTT检测各组HBZY-1细胞增殖;RT-PCR检测Wnt、?-catenin、Collagen-Ⅳ、TNF-?、IL-6和MCP-1基因表达;ELISA检测TNF-?、IL-6和MCP-1表达;Western blotting检测Wnt、?-catenin和Collagen-Ⅳ蛋白的变化。结果与对照组相比,高糖组细胞增殖率、Wnt、?-catenin、Collagen-Ⅳ、TNF-?、IL-6和MCP-1表达水平均明显增高(P<0.05)。与高糖组相比,RSV组细胞增殖率、Wnt、?-catenin、p-p65、Collagen-Ⅳ、TNF-?、IL-6和MCP-1表达水平均明显降低(P<0.05)。结论 RSV可通过抑制炎症而减轻高糖诱导的肾脏系膜细胞增殖,其机制与抑制Wnt/?-catenin/p65信号通路激活有关。

In vitro Biological Effects of Ti2448 Alloy Modified by Micro-arc Oxidation and Alkali Heatment

The purpose of this study was to test the hypothesis that the combination of micro-arc oxidation and alkali heatment （MAH） would improve the cytocompatibility of a newly designed Ti-24Nb-4Zr-8Sn alloy. In this study, commercially pure titanium （cp Ti） and Ti-24Nb-4Zr-8Sn were used. Surface modification of Ti-24Nb- 4Zr-8Sn by a two-step treatment of micro-arc oxidation （MAO） and alkali heatment was reported. Surface characterizations were performed by scanning electron microscopy （SEM）, thin film X-ray diffraction （TF-XRD） and X-ray photoelectron spectroscopy （XPS）. The MAH layer consisted of finer crystals and possessed a higher degree of crystallity and stability than the MAO layer. A biocompatibility study on treated and untreated Ti- 24Nb-4Zr-8Sn in comparison with cp Ti was carried out to investigate the effect of the different surfaces on the bone integration property in vitro. The cellular assays revealed that the MAO and MAH layer favored the initial adhesion of MC3T3-E1 cells and that the growth rate of MC3T3-E1 cells on MAH layer was significantly higher than that on the conventional MAO-treated layer after 3-day and 5-day incubation, demonstrating the greater potential of the hybrid treatment of micro-arc oxidation followed with alkali heatment as a novel surface modification method for implanting materials.