Salidroside regulates tumor microenvironment of non-small cell lung cancer via Hsp70/Stub1/Foxp3 pathway in Tregs.

BACKGROUND: The treatment of non-small cell lung cancer (NSCLC) is challenging due to immune tolerance and evasion. Salidroside (SAL) is an extract in traditional Chinese medicine and has a potential antitumor effect. However, the mechanism of SAL in regulating the immunological microenvironment of NSCLC is yet to be clarified. METHODS: The mouse model with Lewis lung cancer cell line (3LL) in C57BL/6 mice was established. And then, the percentage of tumor-infiltrating T cell subsets including Treg was detected in tumor-bearing mice with or without SAL treatment. In vitro, the effect of SAL on the expression of IL-10, Foxp3 and Stub1 and the function of Treg were detected by flow cytometry. Network pharmacology prediction and molecular docking software were used to predict the target of SAL and intermolecular interaction. Furthermore, the effect of SAL on the expression of Hsp70 and the co-localization of Stub1-Foxp3 in Treg was confirmed by flow cytometry and confocal laser microscopy. Finally, Hsp70 inhibitor was used to verify the above molecular expression. RESULTS: We discovered that SAL treatment inhibits the growth of tumor cells by decreasing the percentage of tumor-infiltrated CD4+Foxp3+T cells. SAL treatment downregulates the expression of Foxp3 in Tregs, but increases the expression of Stub1, an E3 ubiquitination ligase upstream of Foxp3, and the expression of Hsp70. Inhibiting the expression of Hsp70 reverses the inhibition of SAL on Foxp3 and disrupts the colocalization of Stub1 and Foxp3 in the nucleus of Tregs. CONCLUSIONS: SAL inhibits tumor growth by regulating the Hsp70/stub1/Foxp3 pathway in Treg to suppress the function of Treg. It is a new mechanism of SAL for antitumor therapy.

Dietary supplementation of botanical blends enhanced performance and disease resistance of weaned pigs experimentally infected with enterotoxigenic Escherichia coli F18.

Botanicals exhibit promising impacts on intestinal health, immune-regulation, and growth promotion in weaned pigs. However, these benefits may vary depending on major active components in the final feed additive products. Therefore, this study aimed to investigate two types of botanical blends (BB) that were comprised of 0.3% capsicum oleoresin and 12% garlic extracts from different sources on performance, diarrhea, and health of weaned piglets experimentally infected with a pathogenic Escherichia coli F18. Sixty weanling pigs (7.17 ± 0.97 kg body weight (BW)) blocked by weight and gender were assigned to one of five dietary treatments: negative control (NC), positive control (PC), or dietary supplementation with 100 mg/kg of BB1, 50 mg/kg or 100 mg/kg of BB2. This study lasted 28 d with 7 d before and 21 d after the first E. coli inoculation (day 0). All pigs, except negative control, were orally inoculated with 1010 cfu E. coli F18/3-mL dose for 3 consecutive days. Blood samples were collected periodically to analyze systemic immunity. Intestinal tissues and mucosa were collected on days 5 and 21 PI for analyzing histology and gene expression. All data, except for frequency of diarrhea, were analyzed by ANOVA using the PROC MIXED of SAS. The Chi-square test was used for analyzing frequency of diarrhea. Escherichia coli infection reduced (P < 0.05) growth rate and feed intake and increased (P < 0.05) frequency of diarrhea of weaned pigs throughout the experiment. Supplementation of 100 mg/kg BB1 or BB2 alleviated (P < 0.05) frequency of diarrhea of E. coli challenged pigs during the entire experiment. Escherichia coli infection also enhanced (P < 0.05) serum TNF-α and haptoglobin concentrations on day 4 post-inoculation (PI) but reduced (P < 0.05) duodenal villi height and area on day 5 PI, while pigs supplemented with 100 mg/kg BB1 or BB2 had lower (P < 0.05) serum TNF-α than pigs in PC on day 4 PI. Pigs fed with 100 mg/kg BB2 had higher (P < 0.05) jejunal villi height than pigs in PC on day 5 PI. Pigs fed with 100 mg/kg BB2 had reduced (P < 0.05) gene expression of IL1B, PTGS2, and TNFA in ileal mucosa than pigs in PC on day 21 PI. In conclusion, dietary supplementation of botanical blends at 100 mg/kg could enhance disease resistance of weaned pigs infected with E. coli F18 by enhancing intestinal morphology and regulating local and systemic immunity of pigs.

This experiment aimed to investigate two botanical blends consisting of 0.3% capsicum oleoresin and 12% garlic extracts on performance, diarrhea, and health of weaned piglets experimentally infected with a pathogenic Escherichia coli F18. The two botanical blends have the same formulation, except that different garlic oils were used. A total of 60 weaned pigs were randomly allotted to one of five experimental treatments: 1) a complex control diet without E. coli F18 challenge; 2) control diet with E. coli F18 challenge; 3) supplementing 100 mg/kg of botanical blend type 1 to pigs challenged with E. coli F18; 4) and 5) supplementing 50 or 100 mg/kg of botanical blend type 2 to pigs challenged with E. coli F18. The experiment lasted 28 d with 7 d adaptation and 21 d after the first F18 E. coli inoculation. Results of this experiment demonstrate that supplementation of 100 mg/kg of botanical blend enhanced disease resistance and tended to improve growth of weaned pigs, regardless of garlic oil variety. An improved intestinal morphology and reduced systemic inflammation was also observed in pigs supplemented with 100 mg/kg of botanical blends. In conclusion, supplementation of 100 mg/kg of botanical blends could reduce diarrhea of E. coli infected pigs and modify local or systemic immunity of pigs.

Genome-Wide Analysis for the Regulation of Gene Alternative Splicing by DNA Methylation Level in Glioma and its Prognostic Implications.

Glioma is a primary high malignant intracranial tumor with poorly understood molecular mechanisms. Previous studies found that both DNA methylation modification and gene alternative splicing (AS) play a key role in tumorigenesis of glioma, and there is an obvious regulatory relationship between them. However, to date, no comprehensive study has been performed to analyze the influence of DNA methylation level on gene AS in glioma on a genome-wide scale. Here, we performed this study by integrating DNA methylation, gene expression, AS, disease risk methylation at position, and clinical data from 537 low-grade glioma (LGG) and glioblastoma (GBM) individuals. We first conducted a differential analysis of AS events and DNA methylation positions between LGG and GBM subjects, respectively. Then, we evaluated the influence of differential methylation positions on differential AS events. Further, Fisher's exact test was used to verify our findings and identify potential key genes in glioma. Finally, we performed a series of analyses to investigate influence of these genes on the clinical prognosis of glioma. In total, we identified 130 glioma-related genes whose AS significantly affected by DNA methylation level. Eleven of them play an important role in glioma prognosis. In short, these results will help to better understand the pathogenesis of glioma.

Trace amounts of antibiotic exacerbated diarrhea and systemic inflammation of weaned pigs infected with a pathogenic Escherichia coli.

The experiment was conducted to investigate the effects of trace amounts of antibiotic on growth performance, diarrhea, systemic immunity, and intestinal health of weaned pigs experimentally infected with an enterotoxigenic Escherichia coli. Weaned pigs (n = 34, 6.88 ± 1.03 kg body weight [BW]) were individually housed in disease containment rooms and randomly allotted to one of the three dietary treatments: nursery basal diet (CON) and two additional diets supplemented with 0.5 or 50 mg/kg carbadox to the nursery basal diet (TRA or REC), respectively. The experiment lasted 18 d with 7 d before and 11 d after the first E. coli inoculation. The E. coli F18 inoculum was orally provided to all pigs with a dose of 1010 colony-forming unit (CFU)/3 mL for three consecutive days. Fecal and blood samples were collected on day 0 before inoculation and days 2, 5, 8, and 11 postinoculation (PI) to test the percentage of ß-hemolytic coliforms in total coliforms and complete blood cell count, respectively. Sixteen pigs were euthanized on day 5 PI, whereas the remaining pigs were euthanized at the end of the experiment to collect the jejunal and ileal mucosa and mesenteric lymph node for gene expression and bacterial translocation, respectively. Pigs in REC had greater (P < 0.05) final BW and lower (P < 0.05) overall frequency of diarrhea compared with pigs in the CON and TRA groups. Pigs in TRA had the lowest (P < 0.05) average daily gain and feed efficiency from day 0 to 5 PI, highest (P < 0.05) percentage of ß-hemolytic coliforms in fecal samples on days 2 and 5 PI, and greatest (P < 0.05) bacterial colonies in mesenteric lymph nodes on day 11 PI compared with pigs in the CON and REC groups. Pigs in TRA had the greatest (P < 0.05) neutrophils on day 5 PI and higher (P < 0.05) white blood cell counts and lymphocytes than other groups on day 11 PI. Pigs in TRA had the greatest (P < 0.05) serum C-reactive protein on days 2 and 5 PI and serum tumor necrosis factor-α on day 5 PI, compared with pigs in the CON and REC groups. Pigs fed REC had increased (P < 0.05) mRNA expression of zona occludens-1 (ZO-1) and occludin (OCDN) and reduced (P < 0.05) interleukin-1 beta (IL1B), interleukin-6 (IL6), and tumor necrosis factor-alpha (TNFA) in ileal mucosa on day 5 PI, compared with the CON, whereas TRA upregulated (P < 0.05) mRNA expression of IL1B, IL6, and cyclooxygenase-2 (COX2) in the ileal mucosa on day 11 PI, compared with the REC. In conclusion, trace amounts of antibiotic may exacerbate the detrimental effects of E. coli infection on pig performance by increasing diarrhea and systemic inflammation of weanling pigs.

Triad3A attenuates pathological cardiac hypertrophy involving the augmentation of ubiquitination-mediated degradation of TLR4 and TLR9.

Activation of TLRs mediated the NF-κB signaling pathway plays an important pathophysiological role in cardiac hypertrophy. Triad3A, a ubiquitin E3 ligase, has been reported to negatively regulate NF-κB activation pathway via promoting ubiquitination and degradation of TLR4 and TLR9 in innate immune cells. The role of Triad3A in cardiac hypertrophic development remains unknown. The present study investigated whether there is a link between Triad3A and TLR4 and TLR9 in pressure overload induced cardiac hypertrophy. We observed that Triad3A levels were markedly reduced following transverse aortic constriction (TAC) induced cardiac hypertrophy. Similarly, stimulation of neonatal rat cardiac myocytes (NRCMs) with angiotensin-II (Ang II) significantly decreased Triad3A expression. To determine the role of Triad3A in TAC-induced cardiac hypertrophy, we transduced the myocardium with adenovirus expressing Triad3A followed by induction of TAC. We observed that increased expression of Triad3A significantly attenuated cardiac hypertrophy and improved cardiac function. To investigate the mechanisms by which Triad3A attenuated cardiac hypertrophy, we examined the Triad3A E3 ubiquitination on TLR4 and TLR9. We found that Triad3A promoted TLR4 and TLR9 degradation through ubiquitination. Triad3A mediated TLR4 and TLR9 degradation resulted in suppression of NF-κB activation. Our data suggest that Triad3A plays a protective role in the development of cardiac hypertrophy, at least through catalyzing ubiquitination-mediated degradation of TLR4 and TLR9, thus negatively regulating NF-κB activation.

Genome-Wide Identification and Analysis of the Methylation of lncRNAs and Prognostic Implications in the Glioma.

Glioma is characterized by rapid cell proliferation and extensive infiltration among brain tissues, but the molecular pathology has been still poorly understood. Previous studies found that DNA methylation modifications play a key role in contributing to the pathogenesis of glioma. On the other hand, long noncoding RNAs (lncRNAs) has been discovered to be associated with some key tumorigenic processes of glioma. Moreover, genomic methylation can influence expression and functions of lncRNAs, which contributes to the pathogenesis of many complex diseases. However, to date, no systematic study has been performed to detect the methylation of lncRNAs and its influences in glioma on a genome-wide scale. Here, we selected the methylation data, clinical information, expression of lncRNAs, and DNA methylation regulatory proteins of 537 glioma patients from TCGA and TANRIC databases. Then, we performed a differential analysis of lncRNA expression and methylated regions between low-grade glioma (LGG) and glioblastoma multiform (GBM) subjects, respectively. Next, we further identified and verified potential key lncRNAs contributing the pathogenesis of glioma involved in methylation modifications by an annotation and correlation analysis, respectively. In total, 18 such lncRNAs were identified, and 7 of them have been demonstrated to be functionally linked to the pathogenesis of glioma by previous studies. Finally, by the univariate Cox regression, LASSO regression, clinical correlation, and survival analysis, we found that all these 18 lncRNAs are high-risk factors for clinical prognosis of glioma. In summary, this study provided a strategy to explore the influence of lncRNA methylation on glioma, and our findings will be benefit to improve understanding of its pathogenesis.

Effect of dietary soy oil, glucose, and glutamine on growth performance, amino acid profile, blood profile, immunity, and antioxidant capacity in weaned piglets.

Weaning stress results in gastrointestinal dysfunction and depressed performance in pigs. This study aimed to investigate the effect of soy oil, glucose, and glutamine on the growth and health of weaned piglets. Compared with those in the glutamine group, piglets in the glucose and soy oil groups had greater average daily gain, average daily feed intake, and gain: feed ratio from day 0 to 14, and gain: feed ratio for the overall period. There were no differences with regard to serum amino acids among the three groups on day 14, except glycine and threonine. The serum concentration of histidine, serine, threonine, proline, and cysteine was the highest in the glutamine group, while the content of glycine and lysine in the soy oil group on day 28 was the highest among all groups. Piglets fed with glutamine had greater serum glucose and creatinine on day 14, high-density lipoprotein on day 28, and serum IgG and IgM on day 28. Piglets in the glutamine group demonstrated lower serum total superoxide dismutase on day 14 and 28; however, they demonstrated higher total superoxide dismutase and total antioxidant capacity in the duodenum and ileum on day 14. Weaned pigs supplemented with glucose or soy oil demonstrate better growth performance possibly due to their enhanced feed intake, whereas those supplemented with glutamine may have improved immunity and intestinal oxidative capacity.

The TIR/BB-loop mimetic AS-1 attenuates mechanical stress-induced cardiac fibroblast activation and paracrine secretion via modulation of large tumor suppressor kinase 1.

The TIR/BB-loop mimetic AS-1 has been reported to prevent cardiac hypertrophy by inhibiting interleukin-1 receptor (IL-1R)-mediated myeloid differentiation primary response gene 88 (MyD88)-dependent signaling. To date, it remains unknown whether and if so how AS-1 contributes to mechanical stress (MS)-induced cardiac fibroblast activation, a key process in pressure overload-induced cardiac remodeling and heart failure. Here, we show that phosphorylation and expression of large tumor suppressor kinase 1 (LATS1), a key molecule in the Hippo-Yes associated protein (YAP) signaling pathway, were down-regulated in primary neonatal rat cardiac fibroblasts (NRCFs) in response to MS and in the hearts of mice subjected to transverse aortic constriction (TAC) procedure; AS-1 treatment was able to restore LATS1 phosphorylation and expression both in vitro and in vivo. AS-1 treatment suppressed the induction of proliferation, differentiation and collagen synthesis in response to MS in NRCFs. AS-1 also ameliorated cardiomyocyte hypertrophy and apoptosis through dampening paracrine secretion of stretched cardiac fibroblasts. In mice, AS-1 treatment could protect against TAC-induced cardiac hypertrophy, myocardial fibrosis and heart failure. Of note, LATS1 depletion using siRNA completely abrogated the inhibitory effects of AS-1 on NRCFs under MS including accelerated proliferation, differentiation, enhanced ability to produce collagen and augmented paracrine secretion of tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) to induce cardiomyocyte hypertrophy. Therefore, our results delineate a previously unrecognized role for LATS1 in cardiac fibroblast to mediate the beneficial effects of AS-1 in preventing pressure overload-induced cardiac remodeling and heart failure.

In vivo tracking of implanted stem cells using radio-labeled transferrin scintigraphy.

The possibility of monitoring stem cells in vivo with radionuclide imaging after transplantation was investigated. Based on the results of a radioligand receptors assay that human mesenchymal stem cells (hMSCs) express a high level of transferrin receptors, iodinated transferrin (131I-Tf(Fe)2) was chosen as the radiotracer for imaging the cells implanted into the spinal cords of rabbits. Accumulation of radioactivity at the cell transplanted sites was assessed 16 and 24 hours post-intrathecal injection of 131I-Tf(Fe)2. Transferrin receptors expression and Tf binding of the implanted cells were verified by immunofluorescence and ex vivo phosphor imaging. The specificity of Tf uptake of hMSCs was proved through control experiments, i.e., replacing 131I-Tf(Fe)2 with 131I labeled human serum albumin as the tracer or substituting hMSCs with phosphate buffered saline as the grafts. Despite some defects, such as the invasive administration of the tracer and the non-specificity of transferrin receptors as a marker of stem cells in this preliminary study, the technique of nuclear medicine imaging is considered to have great potential in tracking implanted cells in vivo.

Comparative analysis of patients not responding to a single dose of 153Sm-EDTMP palliative treatment for painful skeletal metastases.

OBJECTIVE: A certain fraction of patients failed palliative treatment of 153Sm- ethylenediaminetetramethy lenephosphate (153Sm-EDTMP) for painful skeletal metastases were reviewd. A comparative analysis was designed to identify the factors related to therapeutic response. METHODS: From a 3-year multi-center clinical trial, 51 cases were collected who did not respond to an intravenous injection of 153Sm-EDTMP at a dosage of 0.5-1.5 mCi/kg. The therapeutic efficacy was evaluated via changes of symptoms, general condition, consumption of analgesics, sum of effect product, and Karnofsky scores. The age, sex, history of treatment, tumor type, location of bony involvement, uptake ratio and number of metastases, and doses used by the patients were compared to those of the responders. RESULTS: In 51 non-responders, 43 were male, 34 suffered from lung cancer, 41 had bone lesions in the vertebrae, 39 in the pelvis, and 24 had metastases in the lower extremities. Sex distribution, tumor type and location of the lesion differed significantly between responders and non-responders. No other factor showed differences between the two groups. Though patients of younger age, and lesions with lower uptake of radiopharmaceutical seemed to fail the treatment more easily as observed clinically, this was not confirmed by statistical analysis. CONCLUSION: The sex of the patients, certain types of primary tumors and metastases to lower parts of the body were found to influence the patients' response to a single dose of 153Sm-EDTMP palliation. Further exploration of a better way to determine dosage and predict response for each individual case is needed.