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italic>γ-Aminobutyric acid (GABA) is a crucial inhibitory neurotransmitter found in various cells in the human body. While the GABAergic system is typically associated with the nervous system, recent research has revealed that immune cells and tumor cells also express components of this system. In the tumor microenvironment (TME), GABA is secreted to act extracellularly on other cells. GABA is metabolized via the GABA shunt and is involved in the tricarboxylic acid (TCA) cycle by generating succinate, which can provide energy for tumor cells. Activation of GABA receptors (GABARs) is a major pathway through which GABA participates in the regulation of antitumor immune responses. The activation of GABA type A receptors (GABAARs) can inhibit the activation and proliferation of T cells, elicit anti-inflammatory macrophages, and promote tumor cell growth and migration, while activation of GABA type B receptors (GABABRs) is generally considered to inhibit cancer cell migration and induce cancer cell apoptosis. In general, receptor activation inhibits immune cells, but the effect on tumor cells varies. Additionally, the downregulation of the expression levels of GABA transporters (GATs) is involved in tumor progression. Although antagonists of GABA metabolism and drugs that act on GABA receptors are considered therapeutic drugs for tumors, there have been few clinical studies conducted on them.
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Background/Aims@#This study aimed to explore the effect of gut microbiota-regulated Kupffer cells (KCs) on colorectal cancer (CRC) liver metastasis. @*Methods@#A series of in vivo and in vitro researches were showed to demonstrate the gut microbiota and its possible mechanism in CRC liver metastasis. @*Results@#Fewer liver metastases were identified in the ampicillin-streptomycin-colistin and colistin groups. Increased proportions of Parabacteroides goldsteinii, Bacteroides vulgatus, Bacteroides thetaiotaomicron, and Bacteroides uniforms were observed in the colistin group. The significant expansion of KCs was identified in the ampicillin-streptomycin-colistin and colistin groups. B.vulgatus levels were positively correlated with KC levels. More liver metastases were observed in the vancomycin group. An increased abundance of Parabacteroides distasonis and Proteus mirabilis and an obvious reduction of KCs were noted in the vancomycin group. P. mirabilis levels were negatively related to KC levels. The number of liver metastatic nodules was increased in the P. mirabilis group and decreased in the B. vulgatus group. The number of KCs decreased in the P. mirabilis group and increased in the B. vulgatus group. In vitro, as P. mirabilis or B. vulgatus doses increased, there was an opposite effect on KC proliferation in dose- and time-dependent manners. P. mirabilis induced CT26 cell migration by controlling KC proliferation, whereas B. vulgatus prevented this migration. @*Conclusions@#An increased abundance of P. mirabilis and decreased amount of B. vulgatus play key roles in CRC liver metastasis, which might be related to KC reductions in the liver.
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OBJECTIVE@#To investigate the effect and involved mechanism of RSL3 on ferroptosis action in acute leukemia cells MOLM13 and its drug-resistant cells.@*METHODS@#After MOLM13 treated with RSL3, CCK-8 assay was performed to detect cell viability, flow cytometry was used to detect the reactive oxygen species (ROS) level of the cells, RT-qPCR and Western blot were used to detect the expression of glutathione peroxidase 4 (GPX4). After MOLM13/IDA and MOLM13/Ara-C, the drug-resistant cell lines were constructed, the ferroptosis induced by RSL3 was observed. Bone marrow samples were collected from patients with acute monocytic leukemia. RT-qPCR and Western blot were performed to detect the expression of related genes and proteins involved in ferroptosis pathway.@*RESULTS@#RSL3 significantly inhibited the cell viability of MOLM13 and increased the intracellular ROS level, which were partially reversed by ferrostatin-1. The mRNA and protein expression of GPX4 decreased in MOLM13 treated with RSL3. RSL3 inhibited the viability of MOLM13/IDA and MOLM13/Ara-C cells more strongly than that of non-drug resistant cells, also increased the intracellular ROS level . The cytotoxic effects were partially reversed by ferrostatin-1. The mRNA and protein expressions of GPX4 in MOLM13/IDA and MOLM13/Ara-C cells were higher than those in non-drug resistant cells. The mRNA and protein levels of GPX4 in bone marrow of relapsed/refractory acute mononuclear leukemia patients were higher than those of ordinary acute mononuclear leukemia patients.@*CONCLUSION@#RSL3 can induce non-drug resistant cells MOLM13 ferroptosis by inhibiting GPX4 activity. MOLM13/IDA and MOLM13/Ara-C are more sensitive to RSL3 compared with non-drug resistant cells MOLM13, which may be caused by the differences in GPX4 expression. The expressions of GPX4 mRNA and protein in relapsed/refractory acute mononuclear leukemia are higher than those in ordinary acute mononuclear leukemia.
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Child , Humans , Carbolines , Cell Line , Ferroptosis , Leukemia, Myeloid, Acute , Pharmaceutical PreparationsABSTRACT
Objective: To evaluate the feasibility of immature permanent teeth pulp regeneration with a new method that utilizes the integration of concentrated growth factor (CGF) as a scaffold and stromal cell-derived factor-1 (SDF-1). Methods: Canine dental pulp cells (DPC) were isolated and cultured in vitro. Then the effects of SDF-1 and CGF were observed on DPC proliferation and differentiation. The pulpless model was established on the beagle’s immature incisors which were divided into four groups: natural pulp (A), empty cannel (B), CGF-filling (C) and SDF-1/CGF-filling (D). After 10 weeks, specimens were checked by imaging examination, RT-PCR and histological observation. Results: CGF extraction (CGFe) induced DPC proliferation while the combination of SDF-1 and CGFe enhanced this effect and also facilitated odontogenic and angiogenic differentiation of DPC. According to imaging examination, the apex growth of all four groups was in varying degrees. RT-PCR indicated the expressions of odontogenesis and angiogenesis related genes in group D were higher than those in group C. Besides, neonatal dentin and dental-pulp-like tissue were observed inside the canal of both group C and D, while only cementum-like tissue existed around root apex of group B. Conclusion:SDF-1 plays an important role in driving the process of pulp-like-tissue regeneration of immature permanent teeth by using CGF as an effective scaffold.
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Objective:To construct and identify the monoclonal antibody of ATP synthase beta subunit (ATP5B) with high purity, and to lay foundation for further study.Methods:The ATP5Bgene was amplified by PCR and cloned into the pET28avector and transformed into E.coli BL21 (DE3) .The protein expression was induced by IPTG and then the fusion protein was purified by nickel affinity chromatography column.The protein purity was detected by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) .Three female Balb/C mice were immunized with purified fusion protein and the tail vein blood was taken to detect the titer of ATP5Bantibody by indirect enzyme-linked immunosorbent assay (ELISA) .The spleen cells from the immunized mice with the highest serum titer were mixed with the SP2/0cells to establish the hybridoma cells and the fused cells were screened by indirect ELISA and monoclonally cultured.Karyotype analysis were performed in the positive cells.The hybridoma cells were intraperitoneally injected into 12weeks old BALB/C mice to estabilish the ascites models.The titer of ascites was detected by indirect ELISA.The purity of the antibody was detected by SDS-PAGE.The antibody subtype was detected by ELISA.Results:After PCR amplification, a specific band of 1 455bp was obtained, and the pET28aempty vector was ligated to obtain a recombinant pET28a/ATP5Bvector.The target protein was expressed in the IPTG-induced bacteria solution;the SDS-PAGE results showed that the protein band was found at51 000.The indirect ELISA results showed that the serum titer of the venous blood of immunized mice was up to1:64 000.In karyotype analysis, the total number of chromosomes in hybridoma cells was about the sum of myeloma cells and normal mouse spleen cells.The mouse ascites was prepared with the hybridoma cell line, and the highest titer of the antibody was 1:240 000.The subtype of the monoclonal antibody produced by the hybridoma cells was IgG1.Conclusion:The monoclonal antibody against ATP5Bprotein is successfully prepared by cloning, expressing and purifying the recombinant protein.
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Objective@#To study the correlation of lifestyle characteristics with thyroid nodules in a population-based sample of centenarians in Hainan.@*Methods@#The study was based on China Hainan Centenarian Cohort Study (CHCCS) conducted in 18 cities and counties in Hainan province from 2014 to 2016. A group of multidisciplinary team interviewed and examined local centenarians with structured questionnaires and ultrasonography procedures. A total of 918 centenarians were analyzed after excluding those who refused ultrasonographic examinations or had relevant missing data. Thyroids of centenarians were examined by 3-year experienced sonographer, details on lifestyle characteristics and dietary habits were collected by standard procedure.@*Results@#Of the 918 centenarians, 683 (74.4%) had thyroid nodules under the ultrasonography procedures. The prevalence of thyroid nodules in different group of areca nut consumption varied significantly (P<0.05). In multivariate logistic regression model, the results showed female and areca nut intake were independently associated with thyroid nodules in centenarians (P<0.05).@*Conclusions@#The modifiable variables including areca nut intake as well as non-modifiable such as gender are independent determinants of thyroid nodules in centenarians. Further studies are warranted to explore and verify these associations in populations with different ranges of age.
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Objective To explore the feasibility of establishing an acute pericardial effusion animal model guided by ultrasound. Methods Six experimental pigs were anesthetized. A PTC needle was injected and guided to the right ventricular anterior wall under real-time high frequency ultrasound,40 ml and 80 ml normal saline were respectively infused into the pericardial cavity within 5 minutes. Ultrasonography and pathologic examination were applied to confirm this porcine model. The amount of the fluid was estimated by ultrasound at 1 hour and 8 hours after infusion. Results With ultrasound guidance,the PTC needle smoothly entered the pericardial cavity and the saline was successfully injected. The fluid dispersed from local to the entire pericardial cavity. Pericardial effusion last within 8 hours and no significant change of the fluid amount was found (all P ＞0.05). This acute pericardial effusion model was successfully established with a rate of 100%. Conclusions It is feasible to establish an animal model of acute pericardial effusion under the guidance of ultrasound. This method is safe,rapid and effective. It can provide a suitable animal model for the study of acute pericardial effusion.
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Cyclophilin A was isolated from human colorectal carcinoma tissues by ultrafiltration,affinity chromatography,and two dimensional electrophoresis.By the aid of Western blot analysis,several isoforms of cyclophilin A were detected.,The tryptic digests from cyclophilin A were analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry as well as electrospray tandem mass spectrometry techniques.Several different modifications such as N-terminal acetylation (Ac-1Met and Ac-2Val) and phosphorylation of 5Thr were identified from these isoforms of cyclophilin A,which represented the majority of the proteoforms of cyclophilin A in this study.In addition,other kinds of modifications such as oxidation and deamidation were also found in these proteoforms.The results indicated that the developed method could be used to rapidly and correctly identify the several proteoforms of cyclophilin A isolated from human colorectal carcinoma tissues.
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<p><b>OBJECTIVE</b>To establish a MDS mouse model with iron overload and to study the effect of iron overload on MDS.</p><p><b>METHODS</b>The exogenous mutant gene RUNX1-S291fs was inserted into the mice bone marrow mononuclear cell's genome in mice by retrovirus and transplanted into C57BL/6 mice irradiated by Co γ-ray. After 8 weeks,intraperitoneal injection of iron was performed to establish an MDS mouse model with iron overload. After 24 weeks of transplantation, the peripheral blood, bone marrow, femur, liver and spleen of mice were taken, then the morphological characteristics of peripheral blood and bone marrow cells were observed by Wright's staining; the liver, spleen and bone marrow were stained with Prussian blue to observe the iron deposition. The surface antigens of bone marrow cells were detected by flow cytometry. Bone marrow mononuclear cells and spleen tissue proteins were detected by Western blot to confirm the transfection of RUNX1-S291fs gene and expression of protein. The blood routine and transplanted cell chimeric rate of mice were monitored periodically.</p><p><b>RESULTS</b>Compared with the empty plasmid control mice, levels of leukocyte and hemoglobin as well as platelet were decreased in RUNX1-S291fs mutant mice; the peripheral blood cells and bone marrow cells showed pathological hematopoiesis; the liver and spleen enlarged significantly; the tissue structure of femur, liver and spleen was abnormal; the expression of bone marrow cell surface antigens was abnormal. Bone marrow cells and spleen tissue expressed the RUNX1-S291fs protein. Compared with the controlled mice injected with normal saline, iron deposition occurred in the bone marrow, liver and spleen stained with Prussian blue in the mice injected with iron agent.</p><p><b>CONCLUSION</b>Mice engineered to carry exogenous mutant gene RUNX1-S291fs and injected with iron showed pathologic features of MDS and iron overload, resulting in establishing MDS iron overloaded mouse model successfully, which lays a foundation for studying the effect of iron overload on MDS.</p>
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Animals , Mice , Bone Marrow , Disease Models, Animal , Iron Overload , Mice, Inbred C57BL , SpleenABSTRACT
Objective To investigate the prevalence of thyroid nodules(TN)among centenarians in Hainan province and explore the association between thyroid nodules and major chronic diseases.Methods A mixed cross-sectional study of questionnaire survey,medical examination including ultrasonography and laboratory examination were conducted in elderly who resided in Hainan province and aged 100 and over,the subjects who have signed consent and complete data in both basic information and medical examination were included in this study.Eight hundred and four centenarians were finally enrolled and data of them was analyzed to investigate the prevalence of thyroid nodules and to explore its association with common chronic diseases by comparing the prevalence of chronic conditions between groups with and without thyroid nodules.Results The overall prevalence of TN was 73.5%(591、804)and the prevalence of TN was significantly higher in female when comparing with male[75.1%(505、672)vs 65.2%(86、132)](P <0.001).No significant correlation was found between the prevalence of TN and major chronic diseases (P>0.05),however,the factors associated with dyslipidemia and anemia such as TC[(4.6±1.2)mmol、L vs(4.3±1.5)mmol、L],LDL-C[(2.8±0.8)mmol、L vs(2.7±0.8)mmol、L],and Hb[(110.6±22.5)g、L vs(105.5±31.7)g、L]were significant higher in TN group than those in Non-TN group(P <0.05). Conclusions The thyroid nodule is common in centenarian population and its prevalence was higher in female than male.The associations between TN and biomarkers of dyslipidemia and anemia are found in the present study.
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Objective To evaluate the value of ultrasonic elastography for diagnosis of chronic nonspecific low back pain. Methods From March to September, 2016, 32 patients diagnosed as chronic nonspecific low back pain and other 32 healthy people (controls) were measured lumbar erector spinae with ultrasonic elastography, and calculated Yang's modulus. The correlation between Yang's modulus and Visual Analogue Scale (VAS) or Japanese Orthopaedic Association (JOA) score in the patients were investigated with Pearson's analysis. Re-sults There was no significant difference in Yang's modulus between bilateral lumbar erector spinae in both the patients and the controls (t0.05), but it was more in the patients than in the controls (t=9.931, P<0.001). The Yang's modulus was positively correlated with VAS score in the patients (r=0.614, P<0.001), but not with the JOA score (r=-0.243, P=0.180). Conclusion Ultrasonic elastography can be applied to differentiate low back pain from the healthy, and measure the intensity of pain.
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The purpose of this study was to investigate the potential cardioprotection roles of Rapamycin in anoxia/reoxygenation (A/R) injury of cardiomyocytes through inducing autophagy, and the involvement of PI3k/Akt pathway. We employed simulated A/R of neonatal rat ventricular myocytes (NRVM) as an in vitro model of ischemial/reperfusion (I/R) injury to the heart. NRVM were pretreated with four different concentrations of Rapamycin (20, 50, 100, 150 μmol/L), and pretreated with 10 mmol/L 3-methyladenine (3MA) for inhibiting autophagy during A/R. Then, Western blot analysis was used to examine variation in the expression of LC3-II, LC3-I, Bim, caspase-3, p-PI3KI, PI3KI, p-Akt and Akt. In our model, Rapamycin had a preferential action on autophagy, increasing the expression of LC3-II/LC3-I, whereas decreasing the expression of Bim and caspase-3. Moreover, our results also demonstrated that Rapamycin inhibited the activation of p-PI3KI and enhanced the activation of p-Akt. It is concluded that Rapamycin has a cardioprotection effect by inducing autophagy in a concentration-dependent manner against apopotosis through PI3K/Akt signaling pathway during A/R in NRVM.
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The purpose of this study was to investigate the potential cardioprotection roles of Rapamycin in anoxia/reoxygenation (A/R) injury of cardiomyocytes through inducing autophagy, and the involvement of PI3k/Akt pathway. We employed simulated A/R of neonatal rat ventricular myocytes (NRVM) as an in vitro model of ischemial/reperfusion (I/R) injury to the heart. NRVM were pretreated with four different concentrations of Rapamycin (20, 50, 100, 150 μmol/L), and pretreated with 10 mmol/L 3-methyladenine (3MA) for inhibiting autophagy during A/R. Then, Western blot analysis was used to examine variation in the expression of LC3-II, LC3-I, Bim, caspase-3, p-PI3KI, PI3KI, p-Akt and Akt. In our model, Rapamycin had a preferential action on autophagy, increasing the expression of LC3-II/LC3-I, whereas decreasing the expression of Bim and caspase-3. Moreover, our results also demonstrated that Rapamycin inhibited the activation of p-PI3KI and enhanced the activation of p-Akt. It is concluded that Rapamycin has a cardioprotection effect by inducing autophagy in a concentration-dependent manner against apopotosis through PI3K/Akt signaling pathway during A/R in NRVM.
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Animals , Rats , Autophagy , Base Sequence , Cells, Cultured , DNA Primers , Myocytes, Cardiac , Phosphatidylinositol 3-Kinases , Metabolism , Proto-Oncogene Proteins c-akt , Metabolism , Real-Time Polymerase Chain Reaction , Reperfusion Injury , Sirolimus , PharmacologyABSTRACT
Objective To investigate the change of HepG2 liver tumor perfusion after microbubble enhanced ultrasound cavitation treatment and observe the related pathological injury.Methods Twenty eight Balb/c(nu/nu) nude mice transplanted subcutaneous HepG2 tumor were divided into three groups randomly,including the microbubble enhanced ultrasound cavitation group,the ultrasound group and the sham group.Microbubble enhanced ultrasound cavitation treatment was performed by 0.1 ml microbubbles intravenous injection combined with pulse ultrasound emission in experimental group,while in control groups only ultrasound exposure or microbubble injection were applied.The perfusion of tumors was imaged using contrast-enhanced ultrasonography before and after treatments.Time-intensity curve and peak intensity were analyzed.The tumors were then harvested for histological examination.Results The perfusion of HepG2 tumors almost vanished immediately after treatment in experimental group,with the peak intensity reduced from (26.9 ± 10.9)％ to(8.2 ± 5.8)％ (P ＜0.05).There was no significant changes before and after treatments (P ＞ 0.05) in the two control groups.Histological findings were disruption of the endothelia,significant hemorrhage and increased intercellular fluid.Conclusions Microbubble-enhanced ultrasound cavitation can significantly reduce tumor blood perfusion and disrupt tumor vascularture.This new ultrasound therapy can potentially become a new physical anti-angiogenetic therapy for liver tumor.
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Objective To design Wound Area Measurement System based on the digital image processing.Methods A reference object whose area is known to the wound is set and the digital image processing is utilized to measure the wound area.Results The system was used in the experiment of animal model,and the anticipated results were achieved.Conclusion The system is fit for measuring the wound area in the studies of wound repair.
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Objective To study the biological function of Topical Oxygen Therapy(TOT) and Infrared Light-Emitting Diode(LED) therapy and mechanism of combination of both therapy in wound healing.To develop a device combined with TOT and LED therapy.Methods Wound models were made using rabbits which were grouped in four groups such as control group,TOT group,LED group and combined group by random.Except control group,the other groups were carried on TOT,LED and TOT+LED therapy respectively.Wound healing situations were observed.Results TOT+LED therapy could cut the wound healing time whose mechanism was to form composition force in the processing of wound healing by prompting inside growth factors and outside factors.Conclusion The therapy combined with TOT and LED can facilitate the wound healing.The theory of the wound healing device is feasible according to the results of the experiments.