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.

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.

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.

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.

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.

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

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.

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.

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.

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.

Reactive oxygen species and senescence modulatory effects of rice bran extract on 4T1 and NIH-3T3 cells co-treatment with doxorubicin

Objective:To determine the effect of rice bran extract (RBE) in combination with doxorubicin on 4T1 triple-negative breast cancer cells and NIH-3T3 cells. Methods:RBE was obtained by maceration with n-hexane. The phytochemical profile of RBE was observed using high-performance liquid chromatography. Cytotoxic effect of RBE was evaluated through MTT assay. In addition, flow cytometry was used for cell cycle and apoptosis analysis. Cellular senescence was observed using SA-β-Gal assay and intracellular reactive oxygen species (ROS) levels were evaluated using DCFDA staining. The pro-oxidant property of RBE was also evaluated through 1-chloro-2,4-dinitrobenzene spectrophotometry and molecular docking. Results:RBE was obtained with a yield of 18.42％ w/w and contained tocotrienols as the major compound. RBE exerted no cytotoxic effect on 4T1 and NIH-3T3 cells. However, RBE in combination with doxorubicin decreased 4T1 cell viability synergistically (combination index<0.9) and induced apoptosis and senescence on 4T1 cells. RBE significantly decreased senescence in doxorubicin-treated NIH-3T3 cells. Additionally, RBE did not increase ROS levels in doxorubicin-treated 4T1 cells. Meanwhile, the combination of RBE and doxorubicin reduced ROS levels in NIH-3T3 cells. RBE significantly reduced glutathione-S-transferase activity and alpha-tocotrienol interacted with glutathione-S-transferase in the glutathione binding site. Conclusions:Rice bran may be used as a co-chemotherapeutic agent to improve the therapeutic effectiveness of doxorubicin while protecting against the cellular senescence effects of doxorubicin on healthy cells.

Osteogenic effects of antihypertensive drug benidipine on mouse MC3T3-E1 cells in vitro / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Hypertension is a prevalent systemic disease in the elderly, who can suffer from several pathological skeletal conditions simultaneously, including osteoporosis. Benidipine (BD), which is widely used to treat hypertension, has been proved to have a beneficial effect on bone metabolism. In order to confirm the osteogenic effects of BD, we investigated its osteogenic function using mouse MC3T3-E1 preosteoblast cells in vitro. The proliferative ability of MC3T3-E1 cells was significantly associated with the concentration of BD, as measured by methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay and cell cycle assay. With BD treatment, the osteogenic differentiation and maturation of MC3T3-E1 cells were increased, as established by the alkaline phosphatase (ALP) activity test, matrix mineralized nodules formation, osteogenic genetic test, and protein expression analyses. Moreover, our data showed that the BMP2/Smad pathway could be the partial mechanism for the promotion of osteogenesis by BD, while BD might suppress the possible function of osteoclasts through the OPG/RANKL/RANK (receptor activator of nuclear factor-κB (NF-κB)) pathway. The hypothesis that BD bears a considerable potential in further research on its dual therapeutic effect on hypertensive patients with poor skeletal conditions was proved within the limitations of the present study.

Effect of Daizongfang on Insulin Resistance of 3T3-L1 Adipocytes Induced by Different Methods / 中国实验方剂学杂志

Objective:To investigate the effects of Daizongfang （DZF） on insulin resistance （IR） of adipocytes induced by different methods. Method:The cocktail induction method was adopted to induce the differentiation and maturity of 3T3-L1 preadipocytes. An IR model in mature adipocytes was established by the induction of palmitic acid （PA）， high-concentration glucose （HG）， and dexamethasone （DEX）. DZF extracts at different concentrations （2.0， 0.5， 0.1 g·L^-1） intervened for 24 hours. A model group， a rosiglitazone （RSG） group， and a blank control group were set up at the same time. The glucose concentration in the culture supernatant was measured by the glucose oxidase-peroxidase （GOD-POD） method. Glucose consumptions under basic conditions （G_Basic） and insulin stimulation （G_Ins） were calculated to evaluate the insulin sensitivity index （ISI）. The mRNA expression of glucose transporter 4 （GLUT4） was detected by the real-time polymerase chain reaction （PCR）. Result:Compared with the model group， the DZF （2.0 g·L^-1） showed increased G_Basic， G_Ins， and ISI in three IR models （<italic>P</italic><0.05， <italic>P</italic><0.01）. In addition， for the PA-induced IR model， G_Basic and G_Ins in the DZF （0.5 g·L^-1） group were elevated （<italic>P</italic><0.01）， and G_Basic， G_Ins， and ISI in the RSG group increased （<italic>P</italic><0.05， <italic>P</italic><0.01）. For the HG-induced IR model， G_Ins and ISI increased in the DZF （0.5 g·L^-1） group （<italic>P</italic><0.05）， and G_Basic， G_Ins， and ISI were elevated in the RSG group （<italic>P</italic><0.01）. For the DEX-induced IR model， G_Ins and ISI increased in the RSG group （<italic>P</italic><0.01）. In the three models， there were differences among groups with different doses. G_Basic， G_Ins， and ISI in the high-dose DZF group increased in varying degrees compared with those in the medium- and low-dose DZF groups （<italic>P</italic><0.05）. In the three models， the DZF （2.0 g·L^-1） group and the RSG group both increased GLUT4 mRNA expression （<italic>P</italic><0.05）. Conclusion:DZF can reduce IR of adipocytes induced by HG， DEX， or PA in a dose-dependent manner and increase glucose uptake in an insulin-independent manner， which may be related to the increase in GLUT4 expression.

Efecto citotóxico y genotóxico in vitro del extracto crudo y etanólico del rizoma de Curcuma longa L / In vitro cytotoxic and genotoxic effect of the crude and ethanolic extract from the rhizome of Curcuma longa L

RESUMEN Objetivos: Determinar el efecto citotóxico y genotóxico in vitro del extracto crudo y etanólico del rizoma de Curcuma longa L. Materiales y métodos: El efecto citotóxico fue evaluado utilizando líneas celulares DU-145, HT-29, 3T3 BALB/c. Se hallaron los porcentajes de crecimiento en 48 horas y se determinó la concentración inhibitoria 50 (CI50). El efecto genotóxico en el ADN genómico humano se determinó mediante el método Tomasevich. Resultados: El extracto crudo produjo una CI50 de 12,98 ± 0,21 μg/mL para la línea celular tumoral HT-29, que es inferior a DU-145 con una CI50 de 36,77 ± 9,12 μg/mL; el extracto etanólico presentó una CI50 de 13,24 ± 0,77 y 20,54 ± 2,58 µg/mL para ambas líneas celulares, respectivamente; el compuesto estándar curcumina presentó una CI50 de 3,96 ± 0,60 y 13,94 ± 2,79 μg/mL, respectivamente. El extracto crudo a concentraciones de 50 y 100 mg/mL fragmentó entre el 40% a 95% de ADN genómico humano; mientras que, a 200 mg/mL, la fragmentación fue mayor al 95%. El extracto etanólico a todas las concentraciones no fragmentó el ADN. La curcumina a 200 mg/mL fragmentó menos del 5% de ADN genómico humano. Conclusiones: Los extractos crudo y etanólico de Curcuma longa L. demuestran efecto citotóxico in vitro diferencial para la línea celular tumoral humana DU-145 y HT29 semejante al compuesto estándar curcumina. El extracto crudo de Curcuma longa L. presenta una potente actividad genotóxica in vitro frente al ADN genómico humano, esta actividad está ausente en el extracto etanólico.

ABSTRACT Objectives: To determine the in vitro cytotoxic and genotoxic effect of the crude and ethanolic extract from the Curcuma longa L. rhizome. Materials and methods: The cytotoxic effect was evaluated using DU-145, HT-29, 3T3 BALB/c cell lines. The growth percentages in 48 hours; and the half maximal inhibitory concentration (IC50) were determined. The genotoxic effect on human genomic DNA was determined using the Tomasevich method. Results: Crude extract produced an IC50 of 12.98 ± 0.21 μg/mL for the HT-29 tumor cell line, which is lower than the value obtained for DU-145, with an IC50 of 36.77 ± 9.12 μg/mL. The ethanolic extract presented an IC50 of 13.24 ± 0.77 and 20.54 ± 2.58 μg/mL for both cell lines, respectively; the curcumin standard compound presented an IC50 of 3.96 ± 0.60 and 13.94 ± 2.79 μg/mL, respectively. Crude extract concentrations of 50 and 100 mg/mL fragmented between 40% to 95% of human genomic DNA; while at 200 mg/mL, fragmentation was greater than 95%. The ethanolic extract at all concentrations did not fragment the DNA. Curcumin at 200 mg/mL fragmented less than 5% of human genomic DNA. Conclusions: The crude and ethanolic extracts of Curcuma longa L. demonstrate different in vitro cytotoxic effects for the human tumor cell lines DU-145 and HT-29; similar to the standard curcumin compound. The crude extract of Curcuma longa L. shows a potent genotoxic in vitro activity against human genomic DNA; this type of effect is not produced by the ethanolic extract.

Caulerpa okamurae extract attenuates inflammatory interaction, regulates glucose metabolism and increases insulin sensitivity in 3T3-L1 adipocytes and RAW 264.7 macrophages / 中西医结合学报

OBJECTIVE@#To examine whether Caulerpa okamurae ethanolic extract (COE) could inhibit obesity-mediated inflammation, improve glucose metabolism and increase insulin sensitivity, using in vitro cell models of RAW 264.7 macrophages and 3T3-L1 adipocytes.@*METHODS@#We cocultured 3T3-L1 adipocytes in direct contact with lipopolysaccharide-stimulated RAW 264.7 macrophages and induced insulin resistance in 3T3-L1 adipocytes with tumor necrosis factor-α (TNF-α) in the presence or absence of 250 µg/mL of COE. We investigated various markers of inflammation, glucose regulation and insulin sensitivity in these models using Griess reagent to measure nitric oxide (NO) production, 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxyglucose to measure glucose uptake, Western blot analysis to quantify protein expression and reverse transcriptase-polymerase chain reaction to evaluate mRNA expression.@*RESULTS@#We found that COE (250 µg/mL) significantly inhibited the lipopolysaccharide-induced inflammatory response in RAW 264.7 macrophages by downregulating NO production, nitric oxide synthase 2 expression and nuclear translocation of nuclear factor-κB. COE also showed similar anti-inflammatory activity in coculture, along with decreased TNF-α, interleukin-6 and monocyte chemoattractant protein mRNA expression. In addition, COE also improved glucose uptake in coculture by upregulating glucose transporter-4 (GLUT-4) and adiponectin and reducing serine phosphorylation of insulin receptor substrate-1 (IRS1). In the TNF-α-induced insulin resistance model of 3T3-L1 adipocytes, COE significantly improved both basal and insulin-stimulated glucose uptake, accompanied by phosphorylation of IRS1 at tyrosine 632, phospho-5' adenosine monophosphate-activated protein kinase α and glycogen synthase kinase-3β (Ser9) as well as upregulation of GLUT-4.@*CONCLUSION@#Together, these findings suggest that COE has potential to treat or prevent obesity-induced metabolic disorders.

Effect of protease inhibitor from Agaricus bisporus on glucose uptake and oxidative stress in 3T3-L1 adipocytes

Objective: To explore the effect of the protease inhibitor from Agaricus bisporus (J.E. Lange) Imbach (AbPI) on glucose uptake and oxidative stress in 3T3-L1 adipocytes. Methods: Adipocytes were differentiated and stained with Oil-Red-O staining to confirm adipogenesis. The toxic/protective effect of AbPI on the adipocytes was determined by MTT assay, intracellular reactive oxygen species generation through flow cytometry, and morphologically through confocal microscopy using propidium iodide, 4,6-diamino-2-phenylindol dihydrochloride, and 2',7'-dichlorofluorescein diacetate dyes. The uptake of fluorescent glucose analog, 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-d-glucose by adipocytes was also studied through confocal microscopy. Results: MTT assay showed that the cell survival rate was (28.00±3.00)％, (92.33±2.60)％, and (71.34±2.10)％ in the presence of 2 mM H2O2, AbPI alone, and AbPI and H2O2 both, respectively, in comparison to the control. Oil-Red-O staining indicated that AbPI enhanced adipogenesis. AbPI stimulated the glucose uptake by adipocytes similar to the drug rosiglitazone, and showed insulin-sensitizing effect in the presence of insulin, but failed to stimulate the uptake in the absence of insulin. Intracellular reactive oxygen species generation was reduced in differentiating adipocytes upon AbPI treatment. Confocal microscopy showed that the damaged cell population rose to 3.50％, 117.84％, and 261.50％ in the presence of AbPI alone, AbPI with H2O2, and H2O2 alone, respectively. Conclusions: The protease inhibitor enhances glucose uptake by adipocytes and exhibits a cytoprotective effect on them.

Correlation between bone protection of 1,2,3,4,6-pentyl-O-galloyl-beta-D-glucose and Nrf2/HO-1 signaling pathway / 药学学报

To study the osteoprotective effect of 1,2,3,4,6-pentyl-O-galloyl-beta-D-glucose (PGG) its anti-osteoblast apoptosis related mechanism was investigated. A model of zebrafish osteoporosis induced by prednisolone (Pred, 25 μmol·L-1) was established in vivo, and calcein staining was used to detect the effect of PGG on the bone area of ​​zebrafish. Bone marrow mesenchymal stem cells were cultured in vitro, and the number of calcified nodules was observed by alizarin red staining, and the relevant indexes of osteoblast differentiation runt-related transcription factor 2 (Runx 2), osteocalcin (OCN) mRNA level were detected by qRT-PCR. The osteoblast cell line MC3T3-E1 cells was cultured in vitro, and 400 μmol·L-1 hydrogen peroxide (H2O2) was used to intervene the injury to detect the effect of PGG on osteoblasts under oxidative stress. The effect of PGG on osteoblast activity was detected by MTT assay. The effect of PGG on apoptosis was observed by Hoechst 33342 staining. Western blot was used to detect the expression of Bcl-2, Bax, nuclear factor erythroid-2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1). DCFH-DA fluorescence staining for detection of reactive oxygen species (ROS) levels. JC-1 staining was used to detect mitochondrial membrane potential levels. The results showed that PGG could significantly increase the vertebral area of ​​the zebrafish model when compared with the model group. On the 14 th day of osteoblast differentiation, the number of calcified nodules in the PGG group was significantly increased when compared with the control group and the mRNA levels of Runx 2 and OCN were also significantly increased. In addition, under oxidative stress, PGG could increase osteoblast viability, significantly reduce the number of apoptotic cells, and increase the ratio of Bcl-2/Bax. Fluorescence staining results show that PGG decreased intracellular ROS fluorescence density and increased mitochondrial membrane potential. Western blot data showed that PGG could promote the expression of Nrf2 in the nuclear and enhance the expression of downstream protein HO-1. In conclusion, PGG could improve osteoporosis in zebrafish, and this effect may be related to the regulation of Nrf2/HO-1 signaling pathway to improve mitochondrial dysfunction, anti-oxidative stress in osteoblast apoptosis and promote bone formation. This study provides new ideas and clues for the discovery of anti-osteoporosis drugs.