Growth differentiation factor-5 modified by bisphosphonate promotes osteogenic differentiation of MC3T3-E1 cells / 中国组织工程研究

BACKGROUND:As a member of bone morphogenetic proteins,growth differentiation factor-5 shows promising potential in the application of cartilage and bone repair.The affinity of growth differentiation factor-5 onto bone tissue determines protein use efficiency,so it is of great significance to prepare growth differentiation factor-5 with bone targeting capability. OBJECTIVE:To modify growth differentiation factor-5 using bisphosphonates and investigate the effects of modified protein on the growth of preosteoblasts in mice. METHODS:Pamidronate disodium/growth differentiation factor-5 complex was prepared using chemical crosslinking to couple growth differentiation factor-5 with pamidronate disodium.The functional groups and structures of the complex were characterized using Fourier transform infrared spectroscopy and circular dichromatography.To determine the bone targeting in vitro,the binding of the modified growth differentiation factor-5 with calcium phosphate and in vitro release amount of growth differentiation factor-5 were measured with an ELISA kit.Growth differentiation factor-5(control group)and the pamidronate disodium/growth differentiation factor-5 complex(experimental group)were co-cultured with preosteoblasts MC3T3-E1.Individually cultured cells were blank controls.The effect of the complex on cell proliferation and differentiation was evaluated. RESULTS AND CONCLUSION:(1)The infrared spectroscopy and circular dichromatography results indicated that the bisphosphonate/growth differentiation factor-5 complex was successfully prepared without significant changes in the protein secondary structure.In vitro protein adsorption results showed that growth differentiation factor-5 adsorption on calcium phosphate was increased by about one time after coupling with a bisphosphonate.In the presence of cysteine,growth differentiation factor-5 could be released from the bisphosphonate/growth differentiation factor-5 complex.(2)CCK-8 assay results showed that the absorbance value of the experimental group cultured for 4 and 7 days was higher than that of the control group and blank control group(P<0.000 1).After 7 days of culture,the expression of alkaline phosphatase in the experimental group was significantly higher than that in the control group and blank control group(P<0.000 1).After 13 days of culture,the content of calcium nodules in the experimental group was significantly higher than that in the control group and the blank control group(P<0.000 1).The results of qRT-PCR showed that the mRNA expression of alkaline phosphatase,osteocalcin and Runx2 in the experimental group was higher than that in the control group and the blank control group after 7 days of culture(P<0.01,P<0.001,P<0.000 1).(3)These findings exhibit that bisphosphonate modification can enhance the binding capacity of growth differentiation factor-5 to calcium phosphate as well as improve its biological activity.

Effects of icariin on proliferation and differentiation of MC3T3-E1 cells in an inflammatory environment / 中国组织工程研究

BACKGROUND:Studies have shown that chronic apical periodontitis is one of the common inflammatory bone destruction diseases.Icariin can promote osteogenic differentiation,inhibit bone resorption,and may play a protective role in bone destruction caused by chronic apical periodontitis. OBJECTIVE:To investigate the effect of icariin on the proliferation and differentiation of MC3T3-E1 cells in the inflammatory environment stimulated by lipopolysaccharides. METHODS:Lipopolysaccharides were used to stimulate MC3T3-E1 cells to establish an inflammatory environment in vitro,and cell counting kit-8 was used to detect the best concentration and optimal action time of lipopolysaccharides.Cell counting kit-8 was used to detect the optimal concentration of icariin under the stimulation of lipopolysaccharides at a concentration of 1 μg/mL.Alkaline phosphatase detection,Real-time PCR and western blot assay were used to detect the effect of icariin on osteogenic differentiation of MC3T3-E1 cells in the inflammatory environment.Real-time PCR and western blot were used to detect the effects of icariin on the expression of interleukin-1β and interleukin-6 in MC3T3-E1 cells in the lipopolysaccharide-stimulated inflammatory environment. RESULTS AND CONCLUSION:Cell counting kit-8 results showed that the optimal concentration of icariin was 0.1 μg/mL.In the inflammatory environment,icariin enhanced the expression of alkaline phosphatase and promoted osteoblast differentiation.Compared with the lipopolysaccharide group,the expression of osteogenesis-related factors alkaline phosphatase and Runx2 was increased in the lipopolysaccharide+icariin group.Compared with the lipopolysaccharide group,the expression levels of inflammation-related factors interleukin-1β and interleukin-6 decreased in the lipopolysaccharide+icariin group.To conclude,lipopolysaccharides weaken the osteogenic ability of MC3T3-E1 cells and aggravate the inflammatory response,but icariin has a protective effect on them.

Eriodictyol accelerates glucocorticoid-induced apoptosis by promoting osteoblast autophagy / 中国组织工程研究

BACKGROUND:Glucocorticoid-induced osteoporosis is a common complication of systemic glucocorticoid therapy,which is mainly characterized by its inhibitory effect on osteoblasts.Eriodictyol inhibits osteoclast differentiation and osteoporosis-induced by ovariectomy.However,it is unclear whether eriodictyol regulates glucocorticoid-induced osteoblasts. OBJECTIVE:To explore whether eriodictyol plays a role in glucocorticoid-induced osteoblast apoptosis and its potential regulatory mechanisms. METHODS:Dexamethasone-pretreated osteoblasts MC3T3-E1 were treated with the different concentrations(0,0.5,1,2.5,5,10 μmol/L)of eriodictyol or 5 μmol/L 3-methyladenine,an autophagy inhibitor,and then transfected with heme oxygenase 1 overexpression vector(pcDNA-HMOX1)and empty vector(pcDNA vector).Cell proliferation and apoptosis were assessed by using cell counting kit-8 assay and flow cytometry,respectively.The activity of caspase-3 was detected with ELISA.Western blot assay was used to detect the protein expression of autophagy-related proteins LC3-Ⅱ/LC3-Ⅰ,p62,Atg5 and Atg12,the expression of apoptotic related proteins Bax and Bcl-2,as well as the protein expression of AMPK and p-AMPK. RESULTS AND CONCLUSION:Low concentrations of eriodictyol were non-toxic to MC3T3-E1 cells and promoted cell proliferation,as well as increased the expression of autophagy related proteins LC3-Ⅱ/LC3-Ⅰ,p62,Atg5 and Atg12,decreased caspase-3 enzyme activity,inhibited Bax protein expression,promoted Bcl-2 protein expression and reduced dexamethasone-induced apoptosis in MC3T3-E1 cells in a dose-dependent manner.Moreover,eriodictyol significantly promoted heme oxygenase 1 expression in osteoblasts,whereas overexpression of heme oxygenase 1 promoted AMPK phosphorylation,activated autophagy,and inhibited dexamethasone-induced osteoblast apoptosis.While 3-methyladenine treatment counteracted the effects of heme oxygenase 1 overexpression on MC3T3-E1 cells.To conclude,low concentration of Eriodictyol is non-toxic to osteoblasts and activates AMPK signaling pathway by upregulating the expression of heme oxygenase 1,thereby promoting autophagy and inhibiting dexamethasone-induced osteoblast apoptosis.Eriodictyol has great potential for the treatment of glucocorticoid-induced osteoporosis.

Atf7ip is a negative regulator of bone morphogenetic protein 2 promoting osteogenic differentiation in mouse embryonic adult cells / 中国组织工程研究

BACKGROUND:Whether activating transcription factor 7 interacting protein(Atf7ip)is involved in the regulation in osteogenic differentiation is still controversial,and studying its impact on osteogenic differentiation and its specific mechanisms is of great significance. OBJECTIVE:To investigate the effect of Atf7ip on bone morphogenetic protein 2 promoting osteogenic differentiation of mouse embryonic osteoblast precursor cells(MC3T3-E1). METHODS:MC3T3-E1 cells cultured in vitro were divided into three groups:normal group,interference group(NC-siRNA group,Atf7ip-siRNA group),and high expression group(CMV-VC group and CMV-Atf7ip group),and were transfected for 24 hours,and then treated with 200 ng/mL bone morphogenetic protein 2 for 0,12,24,and 48 hours,respectively.qRT-PCR was used to detect the mRNA expression levels of Atf7ip,alkaline phosphatase,osteocalcin,type I collagen α1 in the cells of each group.Western blot assay was used to detect the protein expression of osteogenic differentiation markers Sp7 and Runx2,and the expression of Atf7ip binding molecule SETDB1,histone H3 and H3K9me3.Alkaline phosphatase activity was detected by alkaline phosphatase staining. RESULTS AND CONCLUSION:(1)With the increase of bone morphogenetic protein 2 treatment time,the protein and mRNA expression of Atf7ip decreased,while the protein expression of Sp7,Runx2 and the mRNA expression of osteocalcin and alkaline phosphatase increased significantly(P<0.05).There was no significant change in the protein expression of Atf7ip binding molecule SETDB1.(2)Compared with the NC-siRNA group,the protein expression of Sp7,Runx2 and the mRNA expression of osteocalcin and type I collagen α1 were significantly up-regulated(P<0.05),and alkaline phosphatase activity was significantly enhanced;and H3K9 methylation significantly decreased in the Atf7ip-siRNA group(P<0.05).(3)Compared with the CMV-VC group,the protein expression of Sp7 and Runx,as well as mRNA expression of osteocalcin,alkaline phosphatase,and type I collagen α1 was significantly downregulated(P<0.05),and the alkaline phosphatase activity was significantly reduced in the CMV-Atf7ip group,while the H3K9 methylation protein in the CMV-Atf7ip group was significantly upregulated compared to the control group(P<0.05).(4)In conclusion,Atf7ip expression was decreased during bone morphogenetic protein 2-induced osteogenic differentiation of MC3T3-E1,and osteogenic differentiation was significantly increased after knockdown of Atf7ip.Overexpression of Atf7ip significantly weakened osteogenic differentiation,indicating that Atf7ip is a negative regulatory factor of bone morphogenetic protein 2 promoting osteogenic differentiation of MC3T3-E1 cells.

Effects of filament B knockdown on proliferation,migration and apoptosis of mouse MC3T3-E1 cells / 中国组织工程研究

BACKGROUND:Filamin B(FLNB)can crosslink the actin cytoskeleton into a dynamic structure that is essential for the directional movement of cells.It can regulate the proliferation,differentiation and apoptosis of chondrocytes.However,the effect of FLNB on osteoblast proliferation,migration and apoptosis has not been reported. OBJECTIVE:To investigate the effect of FLNB on the proliferation,migration and apoptosis of MC3T3-E1 cells. METHODS:The adenoviral vectors for knockdown of FLNB expression(sh-FLNB1,sh-FLNB2,sh-FLNB3)were constructed and infected with MC3T3-E1 cells.After screened by puromycin drug,the efficiency of FLNB knockdown was detected by western blot and RT-PCR.The MC3T3-E1 cell line with the best efficiency of FLNB knockdown was selected as the stable transient cell line of MC3T3-E1 for subsequent experiments.The cells were divided into blank group,mc3t3 group,sh-NC group(empty vector),and sh-FLNB group(sh-FLNB lentivirus).The blank group was cultured in cell-free α-MEM complete medium;the mc3t3 group was cultured in α-MEM complete medium alone;and the sh-NC and sh-FLNB groups were cultured with α-MEM medium containing 2.5 μg/mL puromycin.After 3 days of culture,cell counting kit-8 assay and cell scratch assay were used to detect the proliferation and migration ability of MC3T3-E1;flow cytometry was used to detect cell apoptosis;and RT-PCR was used to detect the expression of apoptosis-related genes. RESULTS AND CONCLUSION:Western blot and RT-PCR results showed that the efficiency of FLNB knockdown was the best in the sh-FLNB3(P<0.000 1),which was used as a stable cell line for subsequent experiments.Cell counting kit-8 data showed that the proliferative ability of MC3T3 cells was significantly weakened after knockdown of FLNB(P<0.05).Cell scratch assay results showed that the migration ability of MC3T3 cells was significantly decreased after knockdown of FLNB.Flow cytometry and RT-PCR results showed that the apoptotic rate of MC3T3-E1 cells increased after knockdown of FLNB,the expression of pro-apoptotic factor Bax increased significantly,and the expression of anti-apoptotic factor Bcl-2 decreased significantly(P<0.05).To conclude,knockdown of FLNB can reduce the proliferation ability of MC3T3-E1 cells,decrease the migration ability of the cells,and increase cell apoptosis.

Effect of temperature on the inhibitory effect induced by hydrogen peroxide on cell proliferation and osteogenic differentiation in preosteoblast MC3T3-E1 cells / 新乡医学院学报

Objective To investigate the effect of temperature on cell proliferation and osteogenic differentiation inhibition of preosteoblast induced by hydrogen peroxide(H2 O2).Methods The MC3T3-E1 cells in the logarithmic phase were randomly divided into 0,450,500,550,600,650 μmol·L-1 H2O2 intervention groups and incubated with 0,450,500,550,600,650 μmol·L-1 H2O2 for 2 h,respectively.Other MC3T3-E1 cells in the logarithmic phase were selected and randomly divided into the control group,model group,low-temperature group,and high-temperature group.Cells in the control group were cul-tured in an incubator with 5％CO2 for 24 h at 37 ℃;cells in the model group were incubated with H2O2 for 2 h and cultured in an incubator with 5％CO2 for 24 h at 37 ℃;cells in the low-temperature group were incubated with H2O2 for 2 h and cultured in an incubator with 5％CO2 for 24 h at 32 ℃;cells in the high-temperature group were incubated with H2O2 for 2 h and cultured in an incubator with 5％CO2 for 24 h at 40 ℃.The cell proliferation in all groups was detected by cell counting kit-8.The expression levels of Runt-related transcription factor 2(RUNX2),osteopontin(OPN)and osteocalcin(OC)mRNA were detected by real-time fluorescence quantitave polymerase chain reaction;and the expression levels of RUNX2,OPN and OC protein were detected by Western blot.Results There was no statistically significant difference in cell proliferation among the 0,450 and 500 μmol·L-1 H2O2 intervention groups(P＞0.05);the cell proliferation rate in the 550,600 and 650 μmol·L-1 H2O2 intervention groups was significantly lower than that in the 0,450 and 500 μmol·L-1 H2O2 intervention groups,showing a significant decrease in cell proliferation with the increase of H2O2 concentrations(P＜0.05).In order to ensure that there were enough cells to perform the following experiments,550 μmol·L-1 H2 O2 was chosen.The cell proliferation rate in the model group and the low-temperature group was significantly lower than that in the control group and high-temperature group(P＜0.05);there was no significant difference in the cell proliferation rate between the control group and high-temperature group(P＞0.05).The relative expression of RUNX2 mRNA in the model group and high-temperature group were significantly higher than that in the control group and low-temperature group(P＜0.05);the relative expression of RUNX2 mRNA in the low-temperature group was significantly lower than that in the control group(P＜0.05);there was no significant difference in the relative expression of RUNX2 mRNA between the model group and high-temperature group(P＞0.05).The relative expression of OPN mRNA in the model group,low-temperature group and high-temperature group was significantly higher than that in the control group(P＜0.05);the relative expression of OPN mRNA in the low-temperature group and high-temperature group was significantly higher than that in the model group(P＜0.05);the relative expression of OPN mRNA in the low-tem-perature group was significantly higher than that in the high-temperature group(P＜0.05).The relative expression of OC mRNA in the model group,low-temperature group and high-temperature group was significantly than that in the control group(P＜0.05);the relative expression of OC mRNA in the low-temperature group and high-temperature group was significantly higher than that in the model group(P＜0.05);there was no significant difference in the relative expression of OC mRNA between the low-temperature group and high-temperature group(P＞0.05).The relative expressions of RUNX2,OPN and OC protein the model group,low-temperature group and high-temperature group were significantly lower than those in the control group(P＜0.05);the relative expressions of RUNX2 and OPN protein in the low-temperature group were significantly lower than those in the model group and high-temperature group(P＜0.05);the relative expression of OC protein was significantly lower than that in the high-temperature group(P＜0.05);and there was no siqnificantly difference in the relatiwe experesson of OC protein between the low-temperature group and model group(P＞0.05);the relative expressions of RUNX2,OPN and OC protein in the high-temperature group were significantly higher than those in the model group(P＜0.05).Conclusion The inhibitory effects of H2O2 on cell proliferation and osteogenic differentiation are observed in MC3T3-E1 cells;low-tempera-ture incubation can enhance the inhibition of H2O2 on cell proliferation and osteogenic differentiation in MC3T3-E1 cells,while high-temperature incubation can relieve its inhibitory effect on cell proliferation and osteogenic differentiation.RUNX2,OPN and OC protein might play an important role in cell proliferation and osteogenic differentiation mediated by temperature.

Inverse correlation of CD3 & vascular endothelial growth factor in incisional oral squamous cell carcinoma biopsies predicts nodal metastasis & poor survival of patients

Background & objectives: Oral squamous cell carcinoma (OSCC) is widely prevalent in the Indian subcontinent mainly due to habit-associated aetiologies. Immune regulation and angiogenesis are the part of tumourigenesis that play a crucial role in metastasis and survival. However, the concurrent expression of vascular endothelial growth factor (VEGF) and CD3 (immune regulator receptor on T-lymphocyte) in the same OSCC tissue samples has not been reported in the Indian population. The present study evaluated the expression of CD3+ T-cells and VEGF in OSCC tissue samples and studied the clinicopathological correlation and survival analysis in an Indian population. Methods: This was a retrospective study conducted on 30 formalin-fixed and paraffin embedded sections which were histologically diagnosed as OSCC cases comprising of 15 metastatic OSCC and 15 non- metastatic OSCC with available clinical data and survival status. Results: Reduced expression of CD3+ T-cells and increased VEGF expression were observed in metastatic OSCC samples. The correlation of expression of CD3+ T-cells and VEGF with clinicopathological parameters showed a significant association between these markers with age, nodal status, site of the lesion and survival. Interpretation & conclusions: Reduced expression of CD3+ T-cells in OSCC was found to be associated with a significantly poor survival. VEGF was found to be over expressed in metastatic OSCC as compared to that in non-metastatic OSCC. The study findings suggest that the evaluation of CD3 and VEGF in incisional OSCC biopsies can be considered for predicting the survival outcome and metastasis

Allosteric modulation on specific gene mutated Vitamin D receptor by essential PUFAs and its distinct molecular effects on type 2 diabetes mellitus

A serious health threat affecting the T2DM group is evident more cases T2DM are diagnosed. In this research, we choose to research into all of this possible mechanism of 3T3-L1 Cell lines and Molecular Docking studies Schrodinger software identified Vitamin D, Omega-3, and 6 PUFAs (EPA DHA & AA) Compounds of hydrophilic and hydrophobic pocket throughout molecular modeling besides T2DM. A group of three analog VDRs is being developed for discovery treatment with T2DM. Its use as it was agreed to run a molecular cell culture and docking study. Recognize the binding method involving the compound in T2DM through ADME prediction. The molecular dynamics simulation was enhanced by confirmation of the strength of the possible composite binding. Based on the computational results, the Omega-3 and 6 PUFAs compound encourages energy interaction. The composite contains an in vitro anti-diabetic activity; the compounds have clearly shown that they are active on T2DM. Our studies provide vital information on the findings of the bimolecular T2DM inhibitors.

Application of the 3t sensorial saturation in femoral vein blood collection of premature infants / 中国实用护理杂志

Objective:To investigate the effect of the 3t sensorial saturation in the application of relieving pain and comfort due to femoral vein blood sampling in preterm infants, so as to provide the reference for the selection of clinical nursing plans.Methods:This was a quasi experimental study. A total of 110 preterm infants admitted to the neonatal unit of Shanxi Children′s Hospital from August 2021 to March 2022 were selected and divided into the control group and intervention group with 52 cases respectively by the random number table method. The control group implemented conventional care, and the intervention group implemented the 3t sensorial saturation method including taste, touch and talk on the basis of the control group. The pain and comfort of two groups were evaluated by the Premature Infant Pain Profile (PIPP) and COMFORTneo Scale at 3 min before, during, and 3 min after blood, and the heart rate and SpO 2 of the two groups were compared. Results:Finally, 52 premature infants were included in both groups. The PIPP score, the total score of the COMFORTneo Scale, the heart rate and SpO 2 were 2.00 (1.00, 3.00), 6.50 (6.00, 7.75), 4.00 (3.00, 5.00), 7.00 (6.00, 8.00), 17.00 (15.00, 19.00), 9.50 (9.00, 10.00) points, (137.29 ± 8.58), (148.31 ± 8.89), (143.06 ± 7.61) times/min, 0.980 (0.970, 0.990), 0.960 (0.950, 0.970), 0.980 (0.970, 0.990) in the intervention group, 2.00 (1.25, 3.00), 12.00 (11.00, 13.00), 7.00 (6.00, 8.00), 7.00 (6.00, 9.00), 25.00 (23.00, 27.00), 20.00 (19.00, 22.00) points, (141.54 ± 10.57), (179.71 ± 14.62), (162.00 ± 14.32) times/min, 0.980 (0.960, 0.990), 0.940 (0.920, 0.958), 0.960 (0.940, 0.978). The results of generalized estimating equation analysis showed that the PIPP score, total COMFORTneo Scale score and SpO 2 via different time points, subgroups, and subgroups with time points were statistically significant (Wald χ2 values were 16.72-2 489.71, all P<0.05). The results of two-factor repeated measures ANOVA showed that the interaction effects of heart rate via different time points, subgroups, and subgroups with time points were statistically significant ( F=253.08, 105.02, 77.17, all P<0.05). Conclusions:The 3t sensorial saturation method can effectively reduce pain during femoral vein blood sampling in preterm infants, can improve the comfort level of preterm infants, is conducive to the stabilization of vital signs in preterm infants, and is suitable for promotion and application in clinical care.

Long-term prognostic analysis of different treatment strategies for T 3-T 4 nasal sinus adenocarcinoma / 中华放射肿瘤学杂志

Objective:To investigate the prognosis and influencing factors of different treatment strategies in T 3-T 4 nasal sinus adenocarcinoma. Methods:The data of 93 cases of T 3-T 4 stage nasal sinus adenocarcinoma diagnosed from 2006 to 2018 were retrospectively analyzed. All patients were divided into combined operation group and non-operation group. The survival status and failure mode after corresponding treatment were analyzed. The enumeration data were analyzed by Chi-square test or Fisher's exact test. Survival analysis was performed by Kaplan-Meier method. Univariate analysis was conducted by log-rank test. Multivariate prognostic analysis was performed by Cox model. Results:The average follow-up time in the whole cohort was 81.3 months (18-156 months). By the end of follow-up, a total of 38.7% (36/93) of patients had local recurrence, 14.0% (13/93) had distant metastasis, 17.2% (16/93) had local recurrence complicated with distant metastasis, and 28.0% (26/93) were stable. The overall 2-, 5-, and 10-year overall survival (OS) and progression free survival (PFS) rates were 83.5%, 59.3%, 31.8% and 73.6%, 40.7% and 25.3%, respectively. In univariate analysis, the PFS and OS of patients aged 46-64 years old (all P<0.001), male ( P=0.022, P=0.001), patients with lesions located in the maxillary sinus ( P=0.001, P<0.001), adenoid cystic carcinoma ( P=0.001, P<0.001), non-invasion of orbital / clivus ( P=0.041, P<0.001), GTV P dose>64 Gy ( P=0.003, P=0.006) and N 1 stage ( P=0.014, P=0.014) were statistically different among different treatment modes. Multivariate analysis showed that age ≥65 years old ( P=0.012, P=0.005), orbital / clival invasion ( P<0.001, P=0.005), and GTV p dose ≤64 Gy ( P<0.001, P=0.011) were the independent adverse prognostic factors affecting PFS and OS in T 3-T 4 stage nasal sinus adenocarcinoma. Conclusions:The local failure rate of T 3-T 4 stage nasal sinus adenocarcinoma is high after treatment. Age, orbital / clival invasion, and GTV p dosage are the independent adverse prognostic factors. Surgery based intervention is superior to other treatment strategies.

Effect of Rubus irritans Focke extract on differentiation of 3T3-L1 preadipocytes / 中国药理学通报

Aim To study the effect of Rubus irritans Focke extract on adipogenic differentiation of 3T3-L1 preadipocytes, and to further explore the potential mechanism of Rubus irritans Focke on adipocyte metabolism, so as to provide a new basis for the prevention and treatment of obesity. Methods Rubus irritans Focke extract was separated and prepared by MCI medium pressure chromatographic column. MTT method was used to detect cell proliferation, and oil red 0 staining and kit test was used to to detect the level of lipid accumulation. Western blot was employed to detect the expressions of peroxisome proliferator-activated receptor ¦y (PPAR7), CCAAT enhancer binding protein a (C/ EBPa), adenosine 5'-monophosphate activated protein kinase (AMPK) and phosphorylated AMPK (p-AMPK) protein. Results When the concentration of Rubus irritans Focke extract was less than 100 mg •L

Rutin Promotes Browning of 3T3-L1 Preadipocytes and Its Mechanism / 中国实验方剂学杂志

ObjectiveTo investigate the effect of rutin on the browning of 3T3-L1 preadipocytes and the mechanism. MethodCell counting kit-8 （CCK-8） assay was used to detect the effect of different concentration of rutin （3.125， 6.25， 12.5， 25， 50， 100， 200 μmol·L-1） on 3T3-L1 cell activity， and Western blot to examine the effect of rutin （12.5， 25， 50 μmol·L-1） on the expression of thermogenesis-associated proteins uncoupling protein 1 （UCP1）， PR domain containing 16 （PRDM16） and peroxisome proliferator-activated receptor γ coactivator-1α （PGC-1α） in adipocytes. After the optimal concentration of rutin was determined， the effect of rutin on lipid droplet formation in adipocytes was observed based on oil red O staining， and the expression of nuclear respiratory factor 1 （NRF1）， nuclear respiratory factor 2 （NRF2） and mitochondrial transcription factor A （TFAM）， which were the landmark proteins of mitochondrial biosynthesis， was detected by Western blot. ResultCompared with the blank group， 200 μmol·L-1 rutin inhibited 3T3-L1 cell activity （P<0.01）. Compared with the blank group， at the concentration of 12.5， 25， 50 μmol·L-1 rutin significantly promoted the expression of thermogenesis-associated proteins （UCP1， PRDM16， and PGC-1α） （P<0.01）， which was determined as the optimal concentration. Compared with the blank group， 50 μmol·L-1 rutin significantly increased the immunofluorescence intensity of mitochondrial UCP1 protein in 3T3-L1 cells （P<0.01） and the expression of the markers of mitochondrial biosynthesis （NRF1， NRF2， and TFAM） （P<0.01）. In addition， 50 μmol·L-1 rutin significantly inhibited lipid droplet formation of 3T3-L1 adipocytes （P<0.01）. ConclusionRutin inhibited lipid droplet deposition in 3T3-L1 adipocytes and increased the expression of thermogenesis-related proteins （UCP1， PRDM16， and PGC-1α） and markers of mitochondrial biosynthesis （NRF1， NRF2， and TFAM）， thereby inducing the browning of 3T3-L1 adipocytes. This lays a basis for the development of drugs that safely regulate the browning of white cells.

Effect and mechanism of Zexie Decoction in promoting white adipose tissue browning/brown adipose tissue activation based on GLP-1R/cAMP/PKA/CREB pathway / 中国中药杂志

This study investigated the mechanism of Zexie Decoction(ZXD) in promoting white adipose tissue browning/brown adipose tissue activation based on the GLP-1R/cAMP/PKA/CREB pathway. A hyperlipidemia model was induced by a western diet(WD) in mice, and the mice were divided into a control group, a model group(WD), and low-, medium-, and high-dose ZXD groups. An adipogenesis model was induced in 3T3-L1 cells in vitro, and with forskolin(FSK) used as a positive control, low-, medium-, and high-dose ZXD groups were set up. Immunohistochemistry and immunofluorescence results showed that compared with the WD group, ZXD promoted the expression of UCP1 in white and brown adipose tissues, and also upregulated UCP1, CPT1β, PPARα, and other genes in the cells. Western blot analysis showed a dose-dependent increase in the protein expression of PGC-1α, UCP1, and PPARα with ZXD treatment, indicating that ZXD could promote the white adipose tissue browning/brown adipose tissue activation. Hematoxylin-eosin(HE) staining results showed that after ZXD treatment, white and brown adipocytes were significantly reduced in size, and the mRNA expression of ATGL, HSL, MGL, and PLIN1 was significantly upregulated as compared with the results in the WD group. Oil red O staining and biochemical assays indicated that ZXD improved lipid accumulation and promoted lipolysis. Immunohistochemistry and immunofluorescence staining for p-CREB revealed that ZXD reversed the decreased expression of p-CREB caused by WD. In vitro intervention with ZXD increased the protein expression of CREB, p-CREB, and p-PKA substrate, and increased the mRNA level of CREB. ELISA detected an increase in intracellular cAMP concentration with ZXD treatment. Molecular docking analysis showed that multiple active components in Alismatis Rhizoma and Atractylodis Macrocephalae Rhizoma could form stable hydrogen bond interactions with GLP-1R. In conclusion, ZXD promotes white adipose tissue browning/brown adipose tissue activation both in vivo and in vitro, and its mechanism of action may be related to the GLP-1R/cAMP/PKA/CREB pathway.

The commencement and practice of parenteral nutrition globally and in China / 中华临床营养杂志

The aim of this work is to delineate the odyssey of parenteral nutrition (noun number: 01.080), which is also called intravenous nutrition. Robert Elman (a surgeon) administered fibrinogen hydrolysate intravenously to dogs and observed the positive nitrogen balance in 1937. Corresponding report about this clinical research was published later on. Oscar Schuberth (a surgeon) and Arvid Wretlind (an internist) developed the soybean oil emulsion with good tolerability and infused a mixture of glucose, amino acids and fat emulsion into patients for the first time. Stanley Dudrick (a surgeon) carried out laboratory studies, such as infusion of 30% glucose through the central vein, selection of materials for silicone rubber catheter, monitoring of nitrogen balance, and so on. He validated that the growth and development of beagle could be supported with parenteral nutrition. Douglas Wilmore (a surgeon) applied parenteral nutrition to a baby girl with congenital stenosis of alimentary tract and thus initially validated the effectiveness of parenteral nutrition in human after 18 months' observation. All mentioned above are landmarks in the history of parenteral nutrition.The development of it is a rugged but interesting journey parenteral nutrition, exemplifying the general principles and methodology of scientific researches. Fifty-six years passing by since the commencement, now, it is time to march into Phase 3 translational (T3) research for parenteral nutrition. T3 clinical researches in parenteral and enteral nutrition is now in the ascendant in China. By looking back at the odyssey, we would like to highlight the necessity of incessant optimization and standardization of parenteral and enteral nutrition practice, to eventually achieve the rational use of nutrition support therapy and bring about patient benefits.

Cytotoxicity evaluation of haloperidol, clozapine and a new molecule with antipsychotic potential, PT-31, in NIH-3T3 cells

Abstract Schizophrenia is an illness that affects 26 million people worldwide. However, conventional antipsychotics present side effects and toxicity, highlighting the need for new antipsychotics. We aimed to evaluate the cytotoxicity of haloperidol (HAL), clozapine (CLO), and a new molecule with antipsychotic potential, PT-31, in NIH-3T3 cells. The neutral red uptake assay and the MTT assay were performed to evaluate cell viability and mitochondrial activity, morphological changes were assessed, and intracellular reactive oxygen species (ROS) detection was performed. HAL and CLO (0.1 µM) showed a decrease in cell viability in the neutral red uptake assay and in the MTT assay. In addition, cell detachment, content decrease, rounding and cell death were also observed at 0.1 µM for both antipsychotics. An increase in ROS was observed for HAL (0.001, 0.01 and 1 µM) and CLO (0.01 and 1 µM). PT-31 did not alter cell viability in any of the assays, although it increased ROS at 0.01 and 1 µM. HAL and CLO present cytotoxicity at 0.1 µM, possibly through apoptosis and necrosis. In contrast, PT-31 does not present cytotoxicity to NIH-3T3 cells. Further studies must be performed for a better understanding of these mechanisms and the potential risk of conventional antipsychotics

Ginseng used for bone tissue scaffold

The scaffold based tissue engineering materialized for bone tissue therapy. Gelatin-glutaraldehyde cross linked scaffold was prepared by solvent casting -porogen leaching method. It was characterized by FTIR and SEM microphotograph analysis. Absence of peak at waves no. 1625 cm?1 in ATR-FTIR indicated formation of cross-linking. FE-SEM micrograph showed honeycomb pad like structure with high porosity. Methanolic extract of Withania somnifera (Ashwagandha) root extract induced MC3T3 E1 osteoblast cell adhesion and proliferation on porous gelatin scaffold. GC-MS analysis pointed out presence of 4-amino- 2-ethyl-3-methylquinoline, an active phyto-chemicals having tissue regeneration potential. High anti-oxidant capacity down regulates cell death mechanism by scavenging free radical. The biocompatible gelatin scaffold has RGD moiety that attune the MC3T3 E1 osteoblast cell adhesion. Withania somnifera root extract may boost up cell proliferation on scaffold. Therefore treatment with Withania somnifera root extract may be the new approaches for designing bone tissue scaffold for bone tissue therapy.

Characterization and biocompatibility analysis of different silanes coupling c(RGDfK) cyclic peptide on titanium surfaces / 口腔疾病防治

Objective@#To compare the efficiency and biocompatibility of four different silanes on immobilizing c(RGDfK) peptide on titanium surface.@*Methods @# After alkali-heat treatment (group OH), the titanium surface was treated with 3-aminopropyltriethoxysilane (APTES) (group OHAP), 3-chloropropyltriethoxysilane (CPTES) (group OHCP) (3-mercaptopropyltrimethoxysilane (MPTS) (group OHMPT) and 3-isobutyryloxy propyltrimethoxysilane(γ- MPS) (group OHMPS) to immobilize the c(RGDfK) cyclic peptide and constructa titanium-silane-c(RGDfK) coating. The NT group was the blank control group. The surface morphology and wettability of the coatings were detected using scanning electron microscopy and contact angle measurement. The elemental composition of the titanium surface was analyzed using X-ray photoelectron spectroscopy. After fluorescent staining with 4’,6-diamino-2-phenylindole (DAPI) and phalloidin, the adhesion of mouse preosteoblast MC3T3-E1 cells on the surface of the materials was observed using laser confocal microscopy. Cell counting kit-8 (CCK-8) and alkaline phosphatase (ALP) activity assays were used to evaluate the proliferation and osteogenic differentiation of MC3T3-E1 cells on the surface of the materials, respectively. @*Results @#Scanning electron microscope observation showed a spongy-like 3-dimensional network formed on the titanium surface after alkali-heat treatment with silane-c(RGDfK) coating adhesion. The wettability of each group was greatly improved compared to the untreated titanium surface. The element ratios of Si/Ti and amide-N/Ti in the OHMPS group were the highest. The OHAP group exhibited the best cell adhesion effect. The cell proliferation and ALP activity of the OHAP, OHMPT, and OHMPS groups were significantly higher than the control group (P <0.05); there was no statistical difference between the OHCP group and the control group.@*Conclusion @#MPTS, CPTES and γ-MPS covalently immobilized cyclic peptide c(RGDfK) on the titanium surface, which promoted adhesion, proliferation and osteogenic differentiation of MC3T3-E1 cells. Theγ-MPS conjugated C (RGDfK)cyclic peptide exhibited the best effect. MPTS, CPTES and γ-MPS coupled with c(RGDfK) cyclic peptides had similar biological properties.

Aqueous extract of freeze-dried Protaetia brevitarsis larvae promotes osteogenesis by activating β-catenin signaling

@# Objective: To investigate the effect of an aqueous extract of Protaetia brevitarsis (AEPB) on osteogenesis using preosteoblast MC3T3-E1 cells and zebrafish larvae. Methods: Flow cytometric analysis was used to measure the cytotoxicy. Alkaline phosphatase activity was detetmined using p-nitrophenyl phosphate as a substrate. Calcium deposition was detected using alizarin red staining along with osteogenic marker expression in preosteoblast MC3T3E1 cells. In addition, vertebral formation in zebrafish larvae was detected using calcein staining and osteogenic gene expression. Results: AEPB highly promoted the expression of osteogenic markers including runt-related transcription factor 2, osterix, and alkaline phosphatase, along with elevated levels of mineralization in MC3T3-E1 cells. Moreover, AEPB accelerated vertebral formation in zebrafish larvae accompanied by upregulated expression of osteogenic genes. FH535, an inhibitor of Wnt/β-catenin, suppressed AEPB-induced osteogenic gene expression and vertebral formation, indicating that AEPB stimulates osteogenesis by activating the Wnt/β-catenin signaling pathway. Conclusions: AEPB stimulates osteoblast differentiation and bone formation by activating β-catenin. Therefore, AEPB is a promising material that induces osteogenesis, and is useful for the treatment of bone resorption diseases.

Functions of key genes involved in TGF-β/Smad signaling pathway in progression of pulmonary fibrosis / 环境与职业医学

Background Although transforming growth factor-β (TGF-β)/Smad signaling pathway is important in regulating the occurrence and development of pulmonary fibrosis, the pathogenesis of pulmonary fibrosis remains elusive. Objective To explore the functions of genes associated with TGF-β/Smad signaling pathway in the progression of pulmonary fibrosis. Methods A NIH-3T3 fibroblast model induced by TGF-β1 was established. The experiment samples were divided into a control group and a TGF-β1 treatment group. The control group was exposed to normal saline, while the TGF-β1 treatment group was exposed to 10 ng·mL−1 TGF-β1 for 12 h. The RNAs of the two groups were extracted, sequenced, and analyzed by bioinformatics methods to identify seven key genes in TGF-β pathway, including Dcn, Smad3, Smad7, Fbn1, Thbs1, TGF-β1, and TGF-β3. The gene expression levels of five markers [Collagen1α1, Collagen1α2, α-smooth muscle actin (α-SMA), TGF-β1, and TGF-β3] and the seven key genes were detected by quantitative real-time PCR (qRT-PCR). The proteins of the two groups were extracted. The important marker protein expression levels of Smad3, the phosphorylation of Smad3 (P-Smad3), and α-SMA were detected by Western blotting. At the same time, 30 healthy SPF-grade C57BL/6 mice were randomly divided into three groups, with 10 mice in each group: a control group, a SiO2 inhalation exposure group for 28 d (10 mice), and a SiO2 inhalation exposure group for 56 d (10 mice). The mice in the two treatment groups were exposed to a natural SiO2 environment for 4 h per day with a 10-min pause for breathing fresh air at 2 h intervals. The lung tissues of the mice were taken after execution. The changes of pulmonary fibrosis were detected by Masson staining, and mRNAs and proteins were extracted to detect the expression of the above key genes and proteins. Results The expression levels of the five marker genes Collagen1α1, Collagen1α2, α-SMA, TGF-β1, and TGF-β3 were significantly increased in the TGF-β1-induced NIH-3T3 fibroblasts than those in the control group (P < 0.01); the expression levels of P-Smad3 and α-SMA proteins increased significantly (P < 0.01); the expression results of the seven key genes screened in the TGF pathway were that Dcn and Smad3 were obviously down-regulated (P < 0.01), and Smad7, Fbn1, Thbs1, TGF-β1, and TGF-β3 were obviously up-regulated (P < 0.01). The changes in gene expression levels of the transcriptome sequencing showed the same trend. The results of Masson staining showed that the content of collagen fibers in the lung tissues also increased in the SiO2 inhalation exposure groups over time. In the mouse experiment, five marker genes were obviously up-regulated compared with the control group (P < 0.01); no obvious change was found in the expression of Smad3 protein, and the expression levels of P-Smad3 and α-SMA were obviously higher in the SiO2 exposure groups than those in the control group (P < 0.01); the expression levels of Dcn and Smad3 showed a down-regulated trend, while the expression levels of Smad7, Fbn1, Thbs1, TGF-β1, and TGF-β3 showed an up-regulated trend with the increase of SiO2 inhalation exposure days (P < 0.01). The expression levels of the above five marker genes, three important marker proteins, and seven key genes were consistent with the expression trends of TGF-β1-induced NIH-3T3 fibroblasts. Conclusion The expression levels of pulmonary fibrosis-related marker genes and proteins change significantly in TGF-β1-induced fibroblast cells, and the lung tissues of mice under natural SiO2 inhalation exposure has obvious fibrosis characteristics. Seven genes (Dcn, Smad3, Smad7, Fbn1, Thbs1, TGF-β1, and TGF-β3) may be involved in the regulation of pulmonary fibrosis by the TGF-β/Smad signaling pathway.

The role of CSE1L expression in cervical lymph node metastasis of larynx tumors / O papel da expressão de CSE1L na metástase em linfonodos cervicais de tumores laríngeos

Abstract Introduction: According to international reports, 30-40% of all head and neck cancers are larynx cancers, comprising 1-2.5% of all cancer types. Cervical nodal involvement has been reported to be 40% and 65% in T3 and T4 cases, respectively. Five-year survival in patients with cervical lymph node metastasis has been demonstrated to be 50% lower compared to patients with no metastasis. Chromosome segregation like 1 protein; is a DNA fragment isolated by Brinkmann et al. in 1995 that corresponds to yeast chromosome segregation protein. Studies on the effect of chromosome segregation like 1 protein expression in head and neck tumors are rare and it has been shown that nuclear chromosome segregation like 1 protein is over-expressed in these studies where gastrointestinal and breast tumors over-expressed cytoplasmic chromosome segregation like 1 protein. Objective: Chromosome segregation like 1 protein may regulate the proliferation and metastasis of T3-T4 glottic larynx cancer. The aim of this study is to show the relationship between chromosome segregation like 1 protein expression and cervical lymph node metastasis of T3-T4 glottic larynx cancer. Methods: A total of 57 male patients who were operated for T3-T4 glottic cancer in a tertiary referral hospital was included in this study. There were 28 patients with cervical lymph node metastasis and 29 patients without lymph node metastasis. Immunohistochemistry was carried out on formalin-fixed, paraffin-embedded archival glottic larynx tumour tissue. According to the percentage of immunoreactive cells, chromosome segregation like 1 protein status was analyzed. Results: Among the patients, who had no cervical lymph node metastasis, 15 patients showed weak nuclear staining, 12 patients showed moderate nuclear staining and only 2 patients showed high nuclear staining for chromosome segregation like 1 protein. Among the patients who had cervical lymph node metastasis, 18 patients showed high nuclear staining, 9 patients showed moderate staining and only one patient showed weak staining for chromosome segregation like 1 protein. None of the metastatic patients showed cytoplasmic staining and only one patient in the non-metastatic group showed cytoplasmic staining for chromosome segregation like 1 protein. There was a positive correlation between nuclear chromosome segregation like 1 protein expression and cervical lymph node metastasis (r = 0,668) and it was statistically significant (p < 0,001). Conclusion: Chromosome segregation like 1 protein expression is correlated with lymph node metastasis in T3-T4 glottic cancers. This may change the approach to cervical node treatment in patients with glottic cancers in future.

Resumo Introdução: De acordo com relatos internacionais, 30% a 40% de todos os casos de câncer de cabeça e pescoço são na laringe, compreendem 1% a 2,5% de todos os tipos de câncer. O envolvimento linfonodal cervical foi relatado em 40% e 65% nos casos T3 e T4, respectivamente. A sobrevida em cinco anos em pacientes com metástase linfonodal cervical demonstrou ser 50% menor em comparação com os pacientes sem metástase. A proteína chromosome seg-regation like 1 é um fragmento de DNA isolado por Brinkmann et al. em 1995 que corresponde à proteína de segregação cromossômica de levedura. Estudos sobre o efeito da expressão da proteína chromosome segregation like 1 em tumores de cabeça e pescoço são raros e os poucos estudos demonstram que a proteína chromosome segregation like 1 nuclear é superexpressa no núcleo, enquanto tumores gastrointestinais e de mama superexpressam a proteína chromosome segregation like 1 citoplasmática. Objetivo: A proteína chromosome segregation like 1 pode regular a proliferação e metástase do câncer glótico de laringe T3-T4. O objetivo deste estudo é mostrar a relação entre a expressão da proteína chromosome segregation like 1 em metástase de linfonodo cervical no câncer glótico de laringe T3-T4. Método: Foram incluídos neste estudo 57 pacientes do sexo masculino submetidos a cirurgias por câncer glótico T3-T4 em um hospital terciário. Havia 28 pacientes com metástase de linfonodos cervicais e 29 pacientes sem metástase linfonodal. A análise imunohistoquímica foi realizada em tecido de tumor glótico de laringe embebido em parafina e fixado em formol. De acordo com a porcentagem de células imunorreativas, analisou-se a expressão da proteína chromosome segregation like 1. Resultados: Entre os pacientes, que não tinham metástase linfonodal cervical, 15 apresentaram coloração nuclear fraca, 12 apresentaram coloração nuclear moderada e apenas 2 apresentaram coloração nuclear elevada para proteína chromosome segregation like 1. Entre os pacientes que apresentavam metástase linfonodal cervical, 18 pacientes apresentaram coloração nuclear elevada, 9 apresentaram coloração moderada e apenas um paciente apresentou coloração fraca. Nenhum dos pacientes com metástase apresentou coloração citoplasmática e apenas um paciente no grupo não-metastático mostrou coloração citoplasmática para a proteína chromosome segregation like 1. Houve uma correlação positiva entre a expressão nuclear da proteína chromosome segregation like 1 e a metástase de linfonodo cervical (r = 0,668), que foi estatisticamente significante (p < 0,001). Conclusão: A expressão da proteína chromosome segregation like 1 está correlacionada com metástases linfonodais em casos de câncer glótico T3-T4 e isso pode mudar a abordagem do tratamento cervical de câncer glótico no futuro.