Relative mRNA Expression Levels Research Articles

Effects of in vivo targeted carboxylesterase 1f gene knockdown on the Kupffer cells polarization activity in mice with acute liver failure

Objective: To investigate the effect of targeted carboxylesterase 1f (Ces1f) gene knockdown on the polarization activity of Kupffer cells (KC) induced by lipopolysaccharide/D-galactosamine (LPS/D-GalN) in mice with acute liver failure. Methods: The complex siRNA-EndoPorter formed by combining the small RNA (siRNA) carrying the Ces1f-targeting interference sequence and the polypeptide transport carrier (Endoporter) was wrapped in β-1, 3-D glucan shell to form complex particles (GeRPs). Thirty male C57BL/6 mice were randomly divided into a normal control group, a model group (LPS/D-GalN), a pretreatment group (GeRPs), a pretreatment model group (GeRPs+LPS/D-GalN), and an empty vector group (EndoPorter). Real-time fluorescent quantitative PCR and western blot were used to detect Ces1f mRNA and protein expression levels in the liver tissues of each mouse group. Real-time PCR was used to detect the expression levels of KC M1 polarization phenotypic differentiation cluster 86(CD86) mRNA and KC M2 polarization phenotypic differentiation cluster 163 (CD163) mRNA in each group. Immunofluorescence double staining technique was used to detect the expression of Ces1f protein and M1/M2 polarization phenotype CD86/CD163 protein in KC. Hematoxylin-eosin staining was used to observe the pathological damage to liver tissue. A one-way analysis of variance was used to compare the means among multiple groups, or an independent sample nonparametric rank sum test was used when the variances were uneven. Results: The relative expression levels of Ces1f mRNA/protein in liver tissue of the normal control group, model group, pretreatment group, and pretreatment model group were 1.00 ± 0.00, 0.80 ± 0.03/0.80 ± 0.14, 0.56 ± 0.08/0.52 ± 0.13, and 0.26 ± 0.05/0.29 ± 0.13, respectively, and the differences among the groups were statistically significant (F = 9.171/3.957, 20.740/9.315, 34.530/13.830, P < 0.01). The percentages of Ces1f-positive Kupffer cells in the normal control group, model group, pretreatment group, and pretreatment model group were 91.42%, ± 3.79%, 73.85% ± 7.03%, 48.70% ± 5.30%, and 25.68% ± 4.55%, respectively, and the differences between the groups were statistically significant (F = 6.333, 15.400, 23.700, P < 0.01). The relative expression levels of CD86 mRNA in the normal control group, model group, and pretreatment model group were 1.00 ± 0.00, 2.01 ± 0.04, and 4.17 ± 0.14, respectively, and the differences between the groups were statistically significant (F = 33.800, 106.500, P < 0.01). The relative expression levels of CD163 mRNA in the normal control group, the model group, and the pretreatment model group were 1.00 ± 0.00, 0.85 ± 0.01, and 0.65 ± 0.01, respectively, and the differences between the groups were statistically significant (F = 23.360, 55.350, P < 0.01). The percentages of (F4/80(+)CD86(+)) and (F4/80(+)CD163(+)) in the normal control group and model group and pretreatment model group were 10.67% ± 0.91% and 12.60% ± 1.67%, 20.02% ± 1.29% and 8.04% ± 0.76%, and 43.67% ± 2.71% and 5.43% ± 0.47%, respectively, and the differences among the groups were statistically significant (F = 11.130/8.379, 39.250/13.190, P < 0.01). The liver injury scores of the normal control group, the model group, and the pretreatment model group were 0.22 ± 0.08, 1.32 ± 0.36, and 2.17 ± 0.26, respectively, and the differences among the groups were statistically significant (F = 12.520 and 22.190, P < 0.01). Conclusion: Ces1f may be a hepatic inflammatory inhibitory molecule, and its inhibitory effect production may come from the molecule's maintenance of KC polarization phenotypic homeostasis.

The inhibition of Wnt signaling attenuates RANKL-induced osteoclastogenic macrophage activation.

Abdominal aortic aneurysms (AAAs) have been linked to the activation of osteoclastogenic macrophages. Reports have suggested that Wnt signaling has a dual effect of proliferation and differentiation during osteoclastogenesis. The Wnt/β-Catenin pathway is a critical regulator of cell pluripotency, cell survival, and cell fate decisions. It regulates cell proliferation and differentiation through transcriptional co-activators, CBP, and p300, respectively. The inhibition of β-catenin suppresses proliferation but induces differentiation of osteoclast precursor cells. This study aimed to examine the effect of ICG-001, a β-catenin/CBP-specific Wnt signaling inhibitor, on osteoclastogenesis by inhibiting proliferation without inducing differentiation. To induce osteoclastogenesis, RAW 264.7 macrophages were stimulated with a soluble receptor activator of NF-κB ligand (RANKL). The effect of Wnt signaling inhibition was examined by treating macrophages with or without ICG-001 during RANKL stimulation. The activation and differentiation of macrophages were examined through western blotting, quantitative PCR, and tartrate-resistant acid phosphate (TRAP) staining in vitro. The relative expression level of the nuclear factor of activated T-cells cytoplasmic 1 protein was significantly suppressed by ICG-001 treatment. The relative expression levels of mRNA of TRAP, cathepsin K, and matrix metalloproteinase-9 were significantly lower in the ICG-001-treated group. The number of TRAP-positive cells decreased in the ICG-001-treated group relative to the non-treated group. The inhibition of Wnt signaling pathway via ICG-001 suppressed osteoclastogenic macrophage activation. Our previous studies have shown the importance of osteoclastogenic macrophage activation in AAA. Further research to examine the therapeutic potential of ICG-001 on AAA is warranted.

Association analysis of PMEL gene expression and single nucleotide polymorphism with plumage color in quail

To explore the relationship between PMEL gene and quail plumage color, to provide a reference for subsequent quail plumage color breeding. In this experiment, RT-qPCR technology was used to analyze the relative mRNA expression levels of Korean quail (maroon) and Beijing white quail embryos at different developmental stages. Two SNPs in PMEL gene were screened based on the RNA-Seq data of skin tissues of Korean quail and Beijing white quail during embryonic stage. The KASP technology was used for genotyping in the resource population and correlation analysis was carried out with the plumage color traits of quail. Finally, the bioinformatics technology was used to predict the effects of these two SNPs on the structure and function of the encoded protein. The results showed that the expression levels of PMEL gene during the embryonic development of Beijing white quail were extremely significantly higher than that of Korean quail (p < 0.01). The frequency distribution of the three genotypes (AA, AB, and BB) of the Beijing white quail at the c. 1030C > T and c. 1374A > G mutation sites were extremely significantly different from that of the Korean quail (p < 0.01). And there was a significant correlation between the c. 1374A > G mutation site with white plumage phenotype. Bioinformatics analysis showed that SNP1 (c. c1030t) located in exon 6 was a harmful mutation site, and SNP2 (c. a1374g) located in exon 7 was a neutral mutation site. Protein conservation prediction showed that the coding protein P344S site caused by SNP1 (c. c1030t) site and the coding protein I458M site caused by SNP2 (c. g2129a) site were non-conservative sites. The results of this experiment showed that the PMEL gene was associated with the plumage color traits of quail and could be used as a candidate gene for studying the plumage color of quail.

Multiple heteroresistance to tigecycline and colistin in Acinetobacter baumannii isolates and its implications for combined antibiotic treatment

BackgroundWe investigated the presence of heteroresistance against both tigecycline and colistin in Acinetobacter baumannii and then evaluated the effectiveness of combined antibiotic treatment given the existence of discrete tigecycline- and colistin-resistant subpopulations.MethodsWe performed population analysis profiling (PAP) to evaluate the degree of composite heteroresistance in A. baumannii isolates, with the extent of this resistance quantified using subsequent antibiotic susceptibility testing. We then evaluated the amino acid sequence of PmrBAC and the relative mRNA expression levels of pmrB. Finally, we investigated the combined antibiotic efficacy of tigecycline and colistin in multiple-heteroresistant isolates using dual PAP and in vitro time-killing assays.ResultsAll tigecycline-heteroresistant A. baumannii isolates, with the exception of one colistin-resistant isolate, were also heteroresistant to colistin. Evaluations of the colistin-resistant subpopulations revealed amino acid alterations in PmrA and PmrB and increased expression of pmrB. All tigecycline-resistant subpopulations were susceptible to colistin, and all colistin-resistant subpopulations were susceptible to tigecycline. Dual PAP analysis using tigecycline and colistin showed no heteroresistance, and in vitro time-killing assays revealed that a combination of these two antibiotics effectively eliminated the bacterial cells.ConclusionOur results suggest that multiple heteroresistance to tigecycline and colistin is highly prevalent among A. baumannii clinical isolates and that these resistant subpopulations exist independently in single multiple heteroresistant isolates. Therefore, our findings may explain the success of combined antibiotic therapies in these infections.

Increased METTL3 expression and m6A RNA methylation may contribute to the development of dry eye in primary Sjögren’s syndrome

BackgroundPrimary Sjögren’s syndrome (pSS) is a chronic autoimmune disorder defined by xerostomia and keratoconjunctivitis sicca, and its etiology remains unknown. N6-methyladenosine (m6A) is the predominant posttranscriptional modification in eukaryotic mRNAs and is dynamically regulated by m6A regulators. Dysregulation of m6A modification is closely associated with several autoimmune disorders, but the role of m6A modification in pSS remains unknown. This study investigated the potential role of m6A and m6A-related regulators in pSS patients with dry eye.MethodsThis cross-sectional study included forty-eight pSS patients with dry eye and forty healthy controls (HCs). Peripheral blood mononuclear cells (PBMCs) were isolated, and the level of m6A in total RNA was measured. The expression of m6A regulators was determined utilizing real-time PCR and western blotting. The serological indicators detected included autoantibodies, immunoglobulins (Igs), complement factors (Cs), and inflammatory indicators. Dry eye symptoms and signs were measured, including the ocular surface disease index, Schirmer’s test (ST), corneal fluorescein staining score (CFS), and tear break-up time. Spearman’s correlation coefficient was employed to assess the associations of m6A and m6A-related regulator expression with clinical characteristics.ResultsThe expression level of m6A was markedly increased in the PBMCs of pSS patients with dry eye compared to HCs (P value<0.001). The relative mRNA and protein expression levels of the m6A regulators methyltransferase-like 3 (METTL3) and YT521-B homology domains 1 were markedly elevated in pSS patients with dry eye (both P value<0.01). The m6A RNA level was found to be positively related to METTL3 expression in pSS patients (r = 0.793, P value<0.001). Both the m6A RNA level and METTL3 mRNA expression correlated with the anti-SSB antibody, IgG, ST, and CFS (all P values < 0.05). The m6A RNA level was associated with C4 (r = -0.432, P value = 0.002), while METTL3 mRNA expression was associated with C3 (r = -0.313, P value = 0.030).ConclusionsOur work revealed that the upregulation of m6A and METTL3 was associated with the performance of serological indicators and dry eye signs in pSS patients with dry eye. METTL3 may contribute to the pathogenesis of dry eye related to pSS.

Tibetan medicine Qi-Sai-Er-Sang-Dang-Song Decoction inhibits TNF-α-induced rheumatoid arthritis in human fibroblast-like synoviocytes via regulating NOTCH1/NF-κB/NLRP3 pathway.

Qi-Sai-Er-Sang-Dang-Song Decoction (QSD, ཆུ་སེར་སེང་ལྡེང་སུམ་ཐང་།), a Tibetan classical herbal formula, is commonly used in Tibetan hospital preparation for the treatment of rheumatoid arthritis (RA). Its efficacy is to relieve inflammation, dispel cold, remove dampness, and alleviate pain. However, its anti-RA mechanism is still unclear. This study aimed to investigate the effect of QSD on rheumatoid arthritis and explore its anti-inflammatory mechanism against human fibroblast-like synoviocytes (HFLSs) by regulating the notch family of receptors (NOTCH1)/Nuclear factor-κB (NF-κB)/nucleotide-binding (NLRP3) pathway. We used ultra-performance liquid chromatography coupled with Q-TOF mass spectrometry (UPLC-Q-TOF-MS) to identify the chemical composition of QSD. Then, HFLSs were exposed to drug-containing serum. The effect of QSD drug-containing serum on HFLS viability was detected using the cell counting kit-8 (CCK-8) assay. Next, we explored the anti-inflammatory effect of QSD using enzyme-linked immunosorbent assay (ELISA) for inflammatory factors, such as interleukin-18 (IL-18), interleukin-1β (IL-1β), and interleukin-6 (IL-6). The expression of NOTCH-related proteins, a member of the NOTCH1, Cleaved NOTCH1, hairy and enhancer of split-1 (HES-1), NF-κB p65, NF-κB pp65, NLRP3, and delta-like 1 (DLL-1), was examined using western blotting. Furthermore, the relative mRNA expression levels of NOTCH1, NF-κB p65, NLRP3, DLL-1, and HES-1 were detected using real-time quantitative (RT-qPCR). To explore the mechanism underlying the anti-RA effect of QSD, we the used the NOTCH signaling pathway inhibitor LY411575 and transfection with a NOTCH1 siRNA. In addition, we employed immunofluorescence to determine the expression of HES-1 and NF-κB p65 in vitro. Our results revealed that QSD ameliorated inflammation in HFLSs. Compared with the model group, the QSD drug-containing serum group had obviously down-regulated levels of IL-18, IL-1β, and IL-6. Consistently, the CCK-8 results showed that the QSD drug-containing serum had no obvious toxicity towards HFLSs. Moreover, both LY411575 and siNOTCH1, QSD could reduce NOTCH1, NLRP3, and HES-1 protein expression levels, and LY411575 could significantly inhibit the expression levels of NF-κB p65, NF-κB pp65, and Cleaved NOTCH1 (p<0.05). siNOTCH1 could also suppress the expression of DLL-1. The RT-qPCR results indicated that QSD could downregulate the relative mRNA expression levels of NOTCH1, NF-κB p65, NLRP3, DLL-1, and HES-1 in HFLSs (p<0.05). In the immunofluorescence experiment, the fluorescence intensities of HES-1 and NF-κB p65 in HFLSs were found to decrease after exposure to QSD drug-containing serum (p<0.05). Ultimately, 44 chemical components were detected in QSD using UPLC-Q-TOF-MS. This study reveals that the QSD can markedly ameliorate inflammation induced by TNF-α on HFLS. The effect of QSD on HFLS may be exerted by inhibition of the NOTCH1/NF-κB/NLRP3 signaling pathway.

Stress regulation of WFS1 and PERK-p-eIF2α-ATF4 signaling pathway in placental tissue cells of intrahepatic cholestasis of pregnancy

IntroductionThe placental tissue stress of intrahepatic cholestasis of pregnancy (ICP) is activated by ERS under hypoxia condition. PERK signaling pathway is the key pathway for UPR regulation, and is first to activated during ERS. WFS1, as an important regulatory gene of UPR pathway, participates in ERS regulation. The purpose of our study is to explore the expression level and mutual regulation mechanisms of WFS1 and PERK-mediated UPR pathway in ICP placental tissue cell under stress. MethodsBlood and placenta samples were obtained from the ICP patients and ethinylestradiol (EE)-induced intrahepatic cholestasis pregnant rats. IHC and WB were used to detect the expression of WFS1, key factors of PERK pathway (GRP78, PERK, eIF2a, P-eIF2α, ATF4) and placental stress peptides (CRH, UCN). Furthermore, qPCR was carried out to detect mRNA expression of above indicators. ResultsThe expression levels of WFS1 and key factors of PERK pathway were significantly increased in severe ICP placental tissues. Moreover, qPCR and WB showed that relative mRNA and protein expression levels of WFS1 and key factors of PERK pathways in placenta tissues of severe ICP and EE-induced intrahepatic cholestasis pregnant rats were higher than those in control group to varying degrees, while CRH and UCN were descended. Meanwhile, after WFS1-siRNA targeted silencing of the WFS1 gene, the protein expression levels of PERK, P-eIF2α, ATF4 were significantly increased, while CRH and UCN protein were significantly decreased. DiscussionOur study revealed that the activation of WFS1 and PERK-p-eIF2α-ATF4 signaling pathway may contribute to stress regulation in placental tissue cells of intrahepatic cholestasis of pregnancy, thereby avoiding adverse pregnancy outcomes.

TLRs expression in canine mammary gland neoplasms: a pathological and molecular study

TLRs are a class of PRRs that play a vital role in innate immunity. TLRs are expressed on immune cells and mammary epithelial cells. They can promote tumor growth, angiogenesis, invasion, and viability signaling. The current study aimed to test the correlation between histologic types and grades of neoplasms and TLRs gene expression levels. Twenty-one tissue samples of canine mammary neoplasms were stained with H&E. Then, it evaluated histologic type and grade according to the methods of Goldschmidt et al. and Peña, respectively. We established real-time PCR quantification assays to measure the mRNA abundances of TLRs in normal and neoplastic mammary glands. Profile pattern of TLR 1, 2, 3, 4, 5, 6, and 9 genes expression in canine mammary glands performed in 21 samples of mammary gland neoplasms and three non-neoplastic mammary gland samples from normal dogs. TLR 3, 4, and 9 mRNA overexpression were detected. In addition, tubulopapillary carcinoma grade II, SCC grade III, and carcinoma mixed type grade II demonstrated the highest relative TLR-3, and 9 mRNA expression levels. Complex carcinoma grade I, ductal carcinoma grade II, and anaplastic carcinoma grade II showed the highest relative TLR4 mRNA expression level. Although histopathological characteristics of tumors, including histologic type, grade, and inflammation, influenced TLRs mRNA expression level, such correlation was insignificant (P > 0.05).

Transcriptome analysis reveals ADAMTS15 is a potential inflammation-related gene in remote ischemic postconditioning.

Remote ischemic postconditioning (RIPostC) induced by brief episodes of the limb ischemia is a potential therapeutic strategy for myocardial ischemia/reperfusion injury, achieved by reducing cardiomyocyte death, inflammation and so on. The actual mechanisms underlying cardioprotection conferred by RIPostC remain unclear. Exploring gene expression profiles in myocardium at transcriptional level is helpful to deepen the understanding on the cardioprotective mechanisms of RIPostC. This study aims to investigate the effect of RIPostC on gene expressions in rat myocardium using transcriptome sequencing. Rat myocardium samples from the RIPostC group, the control group (myocardial ischemia/reperfusion group) and the sham group were performed transcriptome analysis using RNA sequencing. The levels of cardiac IL-1β, IL-6, IL-10 and TNFα were analyzed by Elisa. The expression levels of candidate genes were verified by qRT-PCR technique. Infarct size was measured by Evans blue and TTC staining. Apoptosis was assessed by TUNEL assays and caspase-3 levels were detected using western blotting. RIPostC can markedly decrease infarct size and reduce the levels of cardiac IL-1β, IL-6 and increase the level of cardiac IL-10. This transcriptome analysis showed that 2 genes were up-regulated (Prodh1 and ADAMTS15) and 5 genes (Caspase-6, Claudin-5, Sccpdh, Robo4 and AABR07011951.1) were down-regulated in the RIPostC group. Go annotation analysis showed that Go terms mainly included cellular process, metabolic process, cell part, organelle, catalytic activity and binding. The KEGG annotation analysis of DEGs found only one pathway, amino acid metabolism, was up-regulated. The relative mRNA expression levels of ADAMTS15, Caspase-6, Claudin-5 and Prodh1 were verified by qRT-PCR, which were consistent with the RNA-seq results. In addition, the relative expression of ADAMTS15 was negatively correlated with the level of cardiac IL-1β (r = -0.748, P = 0.005) and positively correlated with the level of cardiac IL-10 (r = 0.698, P = 0.012). A negative correlation statistical trend was found between the relative expression of ADAMTS15 and the level of cardiac IL-6 (r = -0.545, P = 0.067). ADAMTS15 may be a potential inflammation-related gene in regulation of cardioprotection conferred by remote ischemic postconditioning and a possible therapeutic target for myocardial ischemia reperfusion injury in the future.

Effect of Momordicacharantia polysaccharide on immunomodulatory activity in mice.

Momordica charantia polysaccharides (MCPs) have been reported to exert beneficial roles, such as disease healing, in medicine and pharmacy. However, little is known about their effects on immunomodulation. The present study aimed to explore the possible effects of Momordica charantia polysaccharide (MCP) on the immunomodulatory activity of mice lymphocytes. To this aim, male BALB/c mice aged 6-8 weeks were assigned to the following six experimental groups: i) Normal (NG); ii) model (MG); iii) positive (PG); iv) MCP low-dose (MLG); v) MCP medium-dose (MMG); and vi) MCP high-dose (MHG). An immunosuppressive model was established by the intraperitoneal injection of cyclophosphamide in all groups apart from NG. The NG and MG mice were fed with distilled water, whereas the PG mice were administered with levamisole and the MLG, MMG and MHG mice were fed on low, medium and high (100, 200 and 300 mg/kg, respectively) doses of MCP for 21 consecutive days. Subsequently, the mice underwent surgical procedure and were analysed using a range of procedures, including measurement of the thymus index (TI) and spleen index (SI), assessment of the lymphocyte proliferation rate and cell phagocytosis of peritoneal macrophages, lymphocyte proliferation, secretion and mRNA expression of cytokines IFN-γ, IL-6 and IL-12. The mice divided into six groups as mentioned above and treated for 7 days, in the first 6 days, except NG group, mice in each group were desiccated in the abdominal cavity and sensitized by 1% dinitrofluorobenzene (DNFB). On day 6, mice were sensitized with 20 µl DNFB/acetone/olive oil solution behind the right ear and in front of the right ear. Compared with those in the NG mice (not injected with 80 mg/kg cyclophosphamide), the TIs and SIs of the PG, MLG, MMG and MHG mice were increased. In addition, the inhibitory rate of ear swelling and the phagocytic activity of peritoneal macrophages in the PG, MLG, MMG and MHG mice were increased compared with those of MG. Furthermore, the lymphocyte proliferation rate, the secretion and relative mRNA expression levels of cytokines IFN-γ, IL-6 and IL-12 were significantly increased in the PG, MMG and MHG mice compared with those in the NG mice. The results from the present study suggest that treatment with MCP led to an upregulation of the organ indices of immunosuppressed mice, reduced their delayed allergic reaction indicated by the differential cytokine levels, improved the phagocytic activity of peritoneal macrophages, enhanced the proliferation rate of lymphocytes, increased the secretion and expression of IFN-γ, IL-6 and IL-12. Therefore, MCP may improve the immune function of the immunosuppressed mice.

Perilla, sunflower, and tea seed oils as potential dietary supplements with anti-obesity effects by modulating the gut microbiota composition in mice fed a high-fat diet.

Obesity has become a serious public health problem with its alarmingly increasing prevalence worldwide, prompting researchers to create and develop several anti-obesity drugs. Here, we aimed to investigate the protective effects of perilla seed oil (PSO), sunflower oil (SFO), and tea seed oil (TSO) against obesity through the modulation of the gut microbiota composition and related metabolic changes in mice fed a high-fat diet (HFD). Mice were divided into six equal groups: ND (normal diet); HFD; ORL (HFD supplemented with 20mg/kg body weight of orlistat); PSO, SFO, and TSO (HFD supplemented with 2g/kg body weight of PSO, SFO, and TSO, respectively). Our findings showed that PSO, SFO, and TSO supplementation significantly reduced body weight, organ weight, blood glucose, lipopolysaccharides (LPS), insulin resistance, and improved serum lipid levels (TG, TC, LDL-C, and HDL-C). Meanwhile, the three treatments alleviated oxidative stress and hepatic steatosis and reduced liver lipid accumulation. Relative mRNA expression levels of inflammatory cytokines (TNF-α, IL-1β, IL-6, and MCP-1) and lipid synthesis-related genes (PPAR-γ, FAS, and SREBP-1) were down-regulated, while β-oxidation-related genes (PPAR-α, CPT1a, and CPT1b) were up-regulated in the liver tissue of treated mice. Besides, dietary oil supplementation alleviated HFD-induced gut microbiota dysbiosis by promoting gut microbiota richness and diversity, decreasing the Firmicutes-to-Bacteroidetes ratio, and boosting the abundance of some healthy bacteria, like Akkermansia. PSO, SFO, and TSO supplementation could alleviate inflammation, oxidative stress, and hepatic steatosis, likely by modulating the gut microbiota composition in HFD-fed mice.

The Role of Thrombomodulin in Estrogen-Receptor-Positive Breast Cancer Progression, Metastasis, and Curcumin Sensitivity.

Estrogen and estrogen receptors (ER) play a key role in breast cancer progression, which can be treated with endocrine therapy. Nevertheless, resistance to endocrine therapies is developed over time. The tumor expression of thrombomodulin (TM) is correlated with favorable prognosis in several types of cancer. However, this correlation has not yet been confirmed in ER-positive (ER+) breast cancer. This study aims to evaluate the role of TM in ER+ breast cancer. Firstly, we found that lower TM expression correlates to poor overall survival (OS) and relapse-free survival (RFS) rates in ER+ breast cancer patients through Kaplan-Meier survival analysis (p < 0.05). Silencing TM in MCF7 cells (TM-KD) increased cell proliferation, migration, and invasion ability. Additionally, TM-KD MCF7 cells showed higher sensitivity (IC50 15 μM) to the anti-cancer agent curcumin than the scrambled control cells. Conversely, overexpression of TM (TM-over) in T47D cells leads to decreased cell proliferation, migration, and invasion ability. Furthermore, TM-over T47D cells showed more resistance (IC50 > 40 μM) to the curcumin treatment. The PI staining, DAPI, and tunnel assay also confirmed that the curcumin-induced apoptosis in TM-KD MCF7 cells was higher (90.34%) than in the scrambled control cells (48.54%). Finally, the expressions of drug-resistant genes (ABCC1, LRP1, MRP5, and MDR1) were determined by qPCR. We found that the relative mRNA expression levels of ABCC1, LRP1, and MDR1 genes after curcumin treatment were higher in scrambled control cells than in TM-KD cells. In conclusion, our results demonstrated that TM plays a suppressive role in the progression and metastasis of ER+ breast cancer, and it regulates curcumin sensitivity by interfering with ABCC1, LRP1, and MDR1 gene expression.

Changes in dopamine and octopamine levels caused disordered behaviour in red imported fire ants exposed to cinnamon essential oils

Some plant essential oils (EOs) are environmentally friendly insecticides because they can kill insects by manipulating the social behaviour of the red imported fire ants. In this study, we found that the main component of cinnamon oil, trans-cinnamaldehyde, which is extracted from the bark and leaves of Cinnamomum loureirii Nees and Cinnamomum cassia Presl, could cause leg-antennal and double-antennal grooming, as well as fighting behaviors in red imported fire ants. In particular, the bark oils not only produced strong fighting behaviour but also accelerated the death of the worker ants. Analysis of the contents of neurotransmitters (octopamine and dopamine) in red imported fire worker ants treated with trans-cinnamaldehyde, the main component of cinnamon oil, showed that trans-cinnamaldehyde destroyed the antennal receptor structure, causing a decrease in octopamine content and in the ants' ability to recognise their nest mates. Trans-cinnamaldehyde stimulated the synthesis of dopamine, thereby enhancing the aggression of the worker ants. Indeed, the relative mRNA expression levels of two genes needed for dopamine synthesis, tyrosine hydroxylase and DOPA decarboxylase, were significantly upregulated compared with those of the control group (CK) from the beginning of the fight to the peak of the fight. The ratio of octopamine to dopamine reached 1:86 during the peak of the fight. The findings revealed that trans-cinnamaldehyde acts on the nervous system of red imported fire ants to interfere with the behaviour of the workers, thus providing a strategy to control the spread of red imported fire ants using cinnamon EOs.

Cytotoxicity and anti-inflammatory activity of ethanol extract of Artocarpus altilis (Parkinson ex F.A.Zorn) Fosberg leaf in lipopolysaccharide-stimulated RAW 264.7 cells

Context: The flavonoid and phenolic compounds found in Artocarpus altilis have made their popularity. This plant has been used as a traditional medicine in Indonesia for various diseases. Aims: To evaluate the toxic concentration of the A. altilis extract and to investigate the capacity of the extract to inhibit inflammation against the macrophage cell line RAW 264.7, which has been induced with lipopolysaccharide (LPS). Methods: The MTT assay was used to determine the level of toxicity shown by the extract. The qRT-PCR method was used to examine the anti-inflammatory activity of the sample by comparing the relative expression levels of IL-1β, IL-6, and IL-10 mRNA in LPS-stimulated RAW 264.7 cells to those of dexamethasone, which served as the control. Results: The results of the test for toxicity indicated that the extract is safe. In RAW 264.7 cells that had been stimulated with LPS, we observed that the addition of A. altilis leaf extract drastically reduced the expression of IL-1β and IL-6 mRNA while simultaneously increasing the production of IL-10. Conclusions: The ethanol extract of A. altilis leaf was nontoxic and exhibited anti-inflammatory activity on LPS-induced RAW 264.7 cells.

Identification and validation of immune-related biomarkers and potential regulators and therapeutic targets for diabetic kidney disease

BackgroundDiabetic kidney disease (DKD) is a major complication of diabetes and the leading cause of end-stage renal disease worldwide. Renal inflammation and infiltration of immune cells contribute to the development and progression of DKD. Thus, the aim of the present study was to identify and validate immune-related biomarkers and analyze potential regulators including transcription factors (TFs), microRNAs (miRNAs), and drugs for DKD.MethodsImmune-related genes from the ImmPort database and glomeruli samples from GSE1009 and GSE30528 were used to identify differentially expressed immune-related genes (DEIRGs) of DKD. The expression level and clinical correlation analyses of DEIRGs were verified in the Nephroseq database. Murine podocytes were cultured to construct the high glucose-induced podocyte injury model. The reliability of the bioinformatics analysis was experimentally validated by RT-qPCR in podocytes. Networks among DEIRGs, regulators, and drugs were constructed to predict potential regulatory mechanisms for DKD.ResultsDKD-associated DEIRGs were identified. CCL19 and IL7R were significantly upregulated in the DKD group and negatively correlated with glomerular filtration rate (GFR). GHR, FGF1, FYN, VEGFA, F2R, TGFBR3, PTGDS, FGF9, and SEMA5A were significantly decreased in the DKD group and positively correlated with GFR. RT-qPCR showed that the relative mRNA expression levels of GHR, FGF1, FYN, TGFBR3, PTGDS, FGF9, and SEMA5A were significantly down-regulated in the high glucose-induced podocyte injury group. The enriched regulators for DEIRGs included 110 miRNAs and 8 TFs. The abnormal expression of DEIRGs could be regulated by 16 established drugs.ConclusionsThis study identified immune-related biomarkers, regulators, and drugs of DKD. The findings of the present study provide novel insights into immune-related diagnosis and treatment of DKD.

Differential expression of Tissue inhibitor of metalloproteinase 2 (TIMP2) gene in canine mammary tumour

Mammary tumour is the second most common cancer among dogs, just after skin cancer, accounting about fifty per cent of all tumours among dogs. Certain breeds like English Springer Spaniel, Boxer, Poodle, Bull Mastiff, German Shepherd, Cocker Spaniel, Dachshund and Fox Terrier dogs showed higher risk of incidence of mammary tumour, whereas certain other breeds like Collie, Shetland Sheep dog and Bernese Mountain Dog were considered to be at low risk. This breed predilection suggested that genetic factors play a role in canine mammary neoplasms. The role of numerous genes in canine mammary tumour (CMT) development and progression have been investigated, but the importance of the microenvironment is frequently disregarded as far as CMT is considered. Tissue inhibitor of metalloproteinase 2, commonly referred to as TIMP2, is one of several genes that influence the microenvironment of tumours. The interaction between Matrix Metalloproteinase 2 (MMP2) and TIMP2 is crucial for the invasion, development, and metastasis of tumours. The relative mRNA expression level of TIMP2 was examined in the current investigation in both CMT-affected and normal glands. A ten -fold reduced expression of TIMP2 was found in tumour samples than normal glands, which was statistically significant.

Targeted Delivery of Pennyroyal via Methotrexate Functionalized PEGylated Nanostructured Lipid Carriers into Breast Cancer Cells; A Multiple Pathways Apoptosis Activator.

Pennyroyal is a species of the Lamiaceae family with potent anti-cancer and antioxidant properties. Combining this antioxidant with chemotherapeutic agents enhances the effectiveness of these agents by inducing more apoptosis in cancerous cells. Here, methotrexate (MTX) combined with pennyroyal oil based on PEGylated nanostructured lipid carriers (NLCs) was assessed. These nanoparticles were physiochemically characterized, and their anti-cancer effects and targeting efficiency were investigated on the folate receptor-positive human breast cancer cell line (MCF-7) and negative human alveolar basal epithelial cells (A549). Results showed a mean size of 97.4 ± 12.1 nm for non-targeted PEGylated NLCs and 220.4 ± 11.4 nm for targeted PEGylated NLCs, with an almost small size distribution assessed by TEM imaging. Furthermore, in vitro molecular anti-cancer activity investigations showed that pennyroyal-NLCs and pennyroyal-NLCs/MTX activate the apoptosis and autophagy pathway by changing their related mRNA expression levels. Furthermore, in vitro cellular studies showed that these changes in the level of gene expression could lead to a rise in apoptosis rate from 15.6 ± 8.1 to 25.0 ± 3.2 (P<0.05) for the MCF-7 cells treated with pennyroyal-NLCs and pennyroyal-NLCs/MTX, respectively. Autophagy and reactive oxygen species (ROS) cellular evaluation indicated that treating the cells with pennyroyal-NLCs and pennyroyal-NLCs/MTX could significantly increase their intensity in these cells. Our results present a new NLCs-based approach to enhance the delivery of pennyroyal and MTX to cancerous breast tissues.

Carcase traits, meat quality, and lipogenic gene expression in muscle of lambs fed wheat bran feruloyl oligosaccharides

This study investigated the effects of dietary feruloyl oligosaccharides derived from fermented wheat bran (FOs-FWB) supplementation on carcase traits, meat quality, muscle fatty acid composition, and the expression of lipogenic genes in lambs. In a completely randomised design, 50 Dorper × thin-tailed Han lambs (20.21 ± 3.36 kg) were randomly divided into five treatments with ten replications. The lambs in the control group were fed a basal diet, while those in the experimental groups were fed the basal diet supplemented with 50, 100, 200, and 400 mg/kg FOs-FWB. The shear forces of longissimus dorsi (LD) in lambs fed 50 and 100 mg/kg FOs-FWB were significantly lower than that of the control group (p < .05). Lambs fed with 200 mg/kg FOs-FWB had higher crude protein and lower fat contents in LD muscle (p < .05), the ash content in LD muscle was increased by FOs-FWB supplementation (p < .05). Lambs fed 50 mg/kg FOs-FWB had significantly higher proportions of C18:1n − 9, C20:1, C18:3n − 3, C18:3n − 6 and total n − 3 (p < .05). The proportion of C18:3n − 6 was significantly increased by 100 mg/kg FOs-FWB supplementation (p < .05). Feeding 200 mg/kg FOs-FWB significantly decreased proportions of C14:0 and total saturated fatty acid (SFA), but increased proportions of C18:1n − 9, C18:2n − 6, total monounsaturated fatty acid (MUFA) and unsaturated fatty acid (UFA) to SFA ratio (p < .05). Moreover, increases in the proportion of C22:6n − 3 and total polyunsaturated fatty acid (PUFA) were observed in lambs fed FOs-FWB (p < .05). The relative expression levels of ACC, FAS, and SCD genes in the LD muscle of lambs fed 200 and 400 mg/kg FOs-FWB were higher than the control group (p < .05). Dietary supplementation with 100, 200, 400 mg/kg FOs-FWB significantly increased the relative mRNA expression level of LPL (p < .05). Furthermore, a decrease in the relative mRNA expression level of PPARγ was observed in lambs fed 50 mg/kg FOs-FWB (p < .05). In conclusion, dietary supplementation with FOs-FWB reduced shear force, enhanced intramuscular fat content and PUFAs proportion and stimulated lipogenic gene expression in LD muscle of lambs, especially at a level of 200 mg/kg addition. Highlights Dietary FOs-FWB improved the tenderness of the meat. Dietary supplementation with 200 mg/kg FOs-FWB increased the content of intramuscular fat and PUFAs, as well as the ratio of UFA to SFA. Dietary FOs-FWB affected the expression of genes related to lipid metabolism.

Clinical Significance of SFRP1 Gene Methylation in Patients with Childhood Acute Lymphoblastic Leukemia

To investigate the clinical significance of SFRP1 gene and its methylation in childhood acute lymphoblastic leukemia (ALL) . Methylation-specific PCR (MSP) was used to detect the methylation status of SFRP1 gene in bone marrow mononuclear cells of 43 children with newly diagnosed ALL before chemotherapy (primary group) and when the bone marrow reached complete remission d 46 after induction of remission chemotherapy (remission group), the expression of SFRP1 mRNA was detected by quantitative real-time polymerase chain reaction (qRT-PCR), the expression of SFRP1 protein was detected by Western blot, and clinical data of children were collected, the clinical significance of SFRP1 gene methylation in children with ALL was analyze. The positive rate of SFRP1 gene promoter methylation in the primary group (44.19%) was significantly higher than that in the remission group (11.63%) (χ2=11.328, P<0.05). The relative expression levels of SFRP1 mRNA and protein in bone marrow mononuclear cells of children in the primary group were significantly lower than those in the remission group (P<0.05). Promoter methylation of SFRP1 gene was associated with risk level (χ2=15.613, P=0.000) and survival of children (χ2=6.561, P=0.010) in the primary group, children with SFRP1 hypermethylation had significantly increased risk and shortened event-free survival time, but no significant difference in other clinical data. Hypermethylation of SFRP1 gene promoter may be involved in the development of childhood ALL, and its hypermethylation may be associated with poor prognosis.

Inhibitory Effect of Verapamil on the Growth of Human Airway Granulation Fibroblasts.

To explore the inhibitory effect of verapamil, a calcium channel blocker, on the growth of human airway granulation fibroblasts to provide an experimental basis for the clinical use of calcium channel blockers in preventing and treating benign airway stenosis. Primary human airway normal fibroblasts and human airway granulation fibroblasts were cultured by tissue block attachment culture method, and the experimental studies were carried out using 3-8 generation cells. Cell Counting Kit-8 (CCK-8) was used to test the proliferation of human normal airway fibroblasts and human airway granulation fibroblasts and the semi-inhibitory concentration of verapamil on normal airway fibroblasts and airway granulation fibroblasts. A scratch test detected the migration effect of verapamil on human airway granulation fibroblasts. The mRNA relative expression levels of related factors were detected by PCR to compare the differences between normal airway fibroblasts and airway granulation fibroblasts. Western blot was used to detect the relative amount of related proteins and compare the differences between normal airway fibroblasts and granulation airway fibroblasts. After 48 hours of treatment with half of the inhibitory concentration of Vera Pammy for granulation airway fibroblasts, the relative expression levels of related factors on mRNA and protein were observed. Human normal airway fibroblasts and human airway granulation fibroblasts with a purity of more than 95% could be obtained from primary culture by tissue block adherence method. CCK8 results showed that the proliferation rate of human airway granulation fibroblasts was faster than that of the normal human airway fibroblasts. The semi-inhibitory concentration of verapamil on human normal airway fibroblasts was 92.81 ug/ml, while the semi-inhibitory concentration on human airway granulation fibroblasts was 69.57 ug/ml. The scratch test indicated that the cell migration rate of human airway granulation fibroblasts treated with verapamil decreased significantly (P < 0.05). PCR results showed that the mRNA relative expression levels of TGFβ1, COL1A1, Smad2/3, VEGFA, IL6, and IL8 in human airway granulation fibroblasts were significantly higher than those in normal human airway fibroblasts (P < 0.05). The mRNA relative expressions of TGFβ1, smad2/3, and COL1A1 in human airway granulation fibroblasts treated with semi-inhibited verapamil for 48h were down-regulated (P < 0.05), while the mRNA relative expressions of VEGFA, IL6 and IL8 had no significant changes (P > 0.05). WB test showed that the relative protein expressions of TGFβ1, Smad2, and VEGFC in human airway granulation fibroblasts were upregulated (P < 0.05) but downregulated after verapamil treatment compared with before treatment (P < 0.05). Calcium channel blockers can inhibit the proliferation of human airway granulation fibroblasts through TGFβ1/ Smad pathway, which may be a method to prevent and treat benign airway stenosis.