MiR-744 Functions as an Oncogene Through Direct Binding to c-Fos Promoter and Facilitates Non-small Cell Lung Cancer Progression.

Metastasis is the leading cause of death in non-small cell lung cancer (NSCLC) patients. Previously, we reported that miR-744 exerted proto-oncogenic function in nasopharyngeal carcinoma, but the role of miR-744 during NSCLC development has not been established. We focused on the function and molecular mechanism of miR-744 in NSCLC. The clinical cohort data from TCGA were analyzed for the correlation of miR-744 and outcomes in NSCLC patients. Gain- and loss-of-function experiment was performed by transfection with miR-744 agomir or antagomir in NSCLC cell lines. The expression of mRNA and protein were analyzed by qPCR assays and Western blotting respectively. Cellular proliferation, migration, and invasion were analyzed by CCK8 assays, wound healing, and transwell assays, respectively. Promoter activities and gene transcription were analyzed by luciferase reporter assays. Xenograft model was applied for in vivo study. High miR-744 expression correlated with lymph node metastasis and poor prognosis in NSCLC patient. MiR-744 aggravated the growth, invasion, and metastasis of NSCLC cells eventually induced the malignant phenotype and promotes radio/chemoresistance in vitro. The -1195 to -1227 and -298 to -323 bp upstream of c-FOS gene was observed to bind with miR-744. Lastly, miR-744 acted as a tumor promoter in lung cancer growth and metastasis in vivo. Taken together, our results indicated that miR-744 up-regulated c-Fos by binding with its promoter contributed to development of NSCLC cells malignant phenotype. Our findings highlight the potential value of miR-744, which may serve as a possible therapeutic target for NSCLC.

Phytoestrogen arctigenin preserves the mucus barrier in inflammatory bowel diseases by inhibiting goblet cell apoptosis via the ERß/TRIM21/PHB1 pathway.

Intestinal mucus barrier dysfunction is closely involved in the pathogenesis of inflammatory bowel diseases (IBD). To investigate the protective effect and underlying mechanism of arctigenin, a phytoestrogen isolated from the fruits of Arctium lappa L., on the intestinal mucus barrier under colitis condition. The role of arctigenin on the intestinal mucus barrier and the apoptosis of goblet cells were examined by using both in vitro and in vivo assays. Arctigenin was demonstrated to promote the mucus secretion and maintain the integrity of mucus barrier, which might be achieved by an increase in the number of goblet cells via inhibiting apoptosis. Arctigenin selectively inhibited the mitochondrial pathway-mediated apoptosis. Moreover, arctigenin elevated the protein level of prohibitin 1 (PHB1) through blocking the ubiquitination via activation of estrogen receptor ß (ERß) to competitively interact with PHB1 and disrupt the binding of tripartite motif 21 (TRIM21) with PHB1. ERß knock down in the colons of mice with DSS-induced colitis resulted in significant reduction of the protection of arctigenin and DPN against the mucosal barrier. Arctigenin can maintain the integrity of the mucus barrier by inhibiting the apoptosis of goblet cells through the ERß/TRIM21/PHB1 pathway.

Pharmacological activation of ERß by arctigenin maintains the integrity of intestinal epithelial barrier in inflammatory bowel diseases.

Intestinal epithelial barrier dysfunction is deeply involved in the pathogenesis of inflammatory bowel diseases (IBD). Arctigenin, the main active constituent in Fructus Arctii (a traditional Chinese medicine), has previously been found to attenuate colitis induced by dextran sulfate sodium (DSS) in mice. The present study investigated whether and how arctigenin protects against the disruption of the intestinal epithelial barrier in IBD. Arctigenin maintained the intestinal epithelial barrier function of mice with DSS- and TNBS-induced colitis. In Caco-2 and HT-29 cells, arctigenin lowered the monolayer permeability, increased TEER, reversed the abnormal expression of tight junction proteins, and restored the altered localization of F-actin induced by TNF-α and IL-1ß. The specific antagonist PHTPP or shRNA of ERß largely weakened the protective effect of arctigenin on the epithelial barrier function of Caco-2 and HT-29 cells. Molecular docking demonstrated that arctigenin had high affinity for ERß mainly through hydrogen bonds as well as hydrophobic effects, and the protective effect of arctigenin on the intestinal barrier function was largely diminished in ERß-mutated (ARG346 and/or GLU305) Caco-2 cells. Moreover, arctigenin-blocked TNF-α induced increase of the monolayer permeability in Caco-2 and HT-29 cells and the activation of myosin light chain kinase (MLCK)/myosin light chain (MLC) pathway in an ERß-dependent manner. ERß deletion in colons of mice with DSS-induced colitis resulted in a significant attenuation of the protective effect of arctigenin on the barrier integrity and colon inflammation. Arctigenin maintained the integrity of the intestinal epithelial barrier under IBD by upregulating the expression of tight junction proteins through the ERß-MLCK/MLC pathway.

Oral administration of curcumin ameliorates pulmonary fibrosis in mice through 15d-PGJ2-mediated induction of hepatocyte growth factor in the colon.

Oral administration of curcumin has been shown to inhibit pulmonary fibrosis (PF) despite its extremely low bioavailability. In this study, we investigated the mechanisms underlying the anti-PF effect of curcumin in focus on intestinal endocrine. In bleomycin- and SiO2-treated mice, curcumin (75, 150 mg· kg-1 per day) exerted dose-dependent anti-PF effect when administered orally or rectally but not intravenously, implying an intestinal route was involved in the action of curcumin. We speculated that curcumin might promote the generation of gut-derived factors and the latter acted as a mediator subsequently entering the lungs to ameliorate fibrosis. We showed that oral administration of curcumin indeed significantly increased the expression of gut-derived hepatocyte growth factor (HGF) in colon tissues. Furthermore, in bleomycin-treated mice, the upregulated protein level of HGF in lungs by oral curcumin was highly correlated with its anti-PF effect, which was further confirmed by coadministration of c-Met inhibitor SU11274. Curcumin (5-40 µM) dose-dependently increased HGF expression in primary mouse fibroblasts, macrophages, CCD-18Co cells (fibroblast cell line), and RAW264.7 cells (monocyte-macrophage cell line), but not in primary colonic epithelial cells. In CCD-18Co cells and RAW264.7 cells, curcumin dose-dependently activated PPARγ and CREB, whereas PPARγ antagonist GW9662 (1 µM) or cAMP response element (CREB) inhibitor KG-501 (10 µM) significantly decreased the boosting effect of curcumin on HGF expression. Finally, we revealed that curcumin dose-dependently increased the production of 15-deoxy-Δ12, 14-prostaglandin J2 (15d-PGJ2) in CCD-18Co cells and RAW264.7 cells, which was a common upstream of the two transcription factors. Moreover, both the in vitro and in vivo effects of curcumin were diminished by coadministration of HPGDS-inhibitor-1, an inhibitor of 15d-PGJ2 generation. Together, curcumin promotes the expression of HGF in colonic fibroblasts and macrophages by activating PPARγ and CREB via an induction of 15d-PGJ2, and the HGF enters the lungs giving rise to an anti-PF effect.

Alpinetin improves intestinal barrier homeostasis via regulating AhR/suv39h1/TSC2/mTORC1/autophagy pathway.

The injury of intestinal epithelial barrier is considered as the key pathophysiological process in response to gastrointestinal infection and inflammation, and plays an important role in the initiation and development of colitis. Alpinetin has been shown to improve intestinal barrier homeostasis under colitis condition, but the mechanism is still unclear. Here, we showed that alpinetin significantly improved transepithelial electrical resistance (TEER) in TNF-α-stimulated Caco-2 cells, which was mainly mediated by inhibiting the apoptosis. Mechanistic studies demonstrated that alpinetin markedly increased the production of autophagosomes, along with obvious regulation of LC3B-II, beclin-1, p62, Atg7 and Atg5 expressions. In addition, it also markedly repressed the activation of mTORC1 signaling pathway, which was ascribed to TSC2 rather than p-AKT, p-ERK, p-AMPKα or PTEN expressions in Caco-2 and NCM460 cells. Furthermore, the enrichment of H3K9me3 at TSC2 promoter region was decreased and ubiquitin proteasome degradation of suv39h1 was increased. Additionally, alpinetin activated aryl hydrocarbon receptor (AhR) and promoted co-localization of AhR with suv39h1 in the cytoplasm. The relationship between alpinetin-regulated AhR/suv39h1/TSC2/mTORC1 signals, autophagy and apoptosis of Caco-2 and NCM460 cells was confirmed by using CH223191, siAhR, siTSC2 and chloroquine. Finally, CH223191 and leucine abolished alpinetin-mediated inhibition of intestinal epithelial cells apoptosis, improvement of intestinal epithelial barrier and amelioration of colitis.

Orally administered berberine ameliorates bleomycin-induced pulmonary fibrosis in mice through promoting activation of PPAR-γ and subsequent expression of HGF in colons.

Berberine has been demonstrated to alleviate renal interstitial, liver and myocardial fibrosis when administered orally despite its extremely low bioavailability. Here, we inspected effect of berberine on pulmonary fibrosis (PF) and explored underlying mechanisms on the basis of intestinal endocrine. The results showed that either oral or rectal administration of berberine exhibited marked alleviation of bleomycin-induced PF in mice. In contrast, anti-PF activity of berberine disappeared when given by an intravenous injection, implying that it functioned in a gut-dependent manner. Moreover, berberine promoted both mRNA and protein levels of HGF and PTEN in colons, but only their protein levels in lungs of PF mice. In addition, SU11274 but not BPV abolished the anti-PF effect of berberine. In vitro, berberine preferentially induced expression of HGF in fibroblast cells than epithelial, preadipocyte and endothelial cells. Similarly, rosiglitazone and 15dPGJ2 also enhanced expression of HGF in fibroblasts cells, and GW9662 and siPPAR-γ diminished induction of berberine on HGF expression. Berberine could enter into the cytoplasm, activate PPAR-γ directly and synergistically with 15dPGJ2, as shown by an up-regulation of CD36 and aP2 mRNA expression, nuclear translocation and DNA-binding activity of PPAR-γ both in vitro and in vivo. Additionally, GW9662 almost abolished anti-PF effect of berberine and induction of HGF expression in colons. In conclusion, oral administration of berberine displays anti-PF action probably in a colon-dependent manner, and mechanisms involve activation of PPAR-γ and resultant promotion of HGF expression in colonic fibroblasts. The up-regulated HGF arrives in lung tissues via blood circulation to palliate PF.

Combination of radiotherapy and Anlotinib enhances benefit from immunotherapy to liver metastasis and abscopal tumor from lung cancer.

Many studies have shown that liver metastasis can weaken the efficacy of immunotherapy. Immunotherapy combined with radiotherapy or anti-angiogenic therapy has been proven to have synergistic anti-tumor effects. So we devote to explore whether the combination of the three therapies can exert effective anti-tumor effects on liver metastasis. The clinical information of 118 patients with liver metastasis were collected to compare the intrahepatic progression-free survival between immunotherapy and immunotherapy combined with other treatments. We used Lewis lung cancer (LLC) cell to establish a mouse liver metastasis tumor model and record tumor burden and survival. Tumor-infiltrating immune cells detected by flow cytometry. RNA sequencing was performed and the proportion of immune cells were analyzed by TIMER2.0 database. Compared with immunotherapy group, the combination therapy group showed a trend for longer median intrahepatic progression-free survival. Radiotherapy combined with PD-1 inhibitor and Anlotinib can inhibit liver metastasis and subcutaneous tumor growth and prolong the survival compared with other groups in vivo. Compared with the anti-PD-1 treatment group, triple therapy can increase CD4+T, CD8+T, and IFN-γ+CD8+T cells and decrease infiltration of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) in tumors. PPAR signaling pathway were significantly activated and CD8+T and dendritic cells (DC) were increased in the triple therapy group compared to the PD-1 inhibitor combined with Anlotinib group. Radiotherapy combined with PD-1 inhibitor and Anlotinib can effectively exert anti-tumor efficacy and reshape the tumor immune microenvironment by increasing the infiltration of anti-tumor immune cells and reducing the infiltration of immunosuppressive immune cells.

Aryl Hydrocarbon Receptor Activation Limits the Fatty Acid Synthesis and Subsequent "miR-193a-3p-HDAC3-FASN" Signals to Alleviate Intestinal Fibrosis.

Intestinal fibrosis is a common complication of Crohn's disease and characterized by excessive extracellular matrix (ECM) deposition. The aryl hydrocarbon receptor (AhR) detects micronutrients and microbial metabolites in diet and can attenuate intestinal fibrosis with unclear mechanisms. In this study, AhR activation was demonstrated to downregulate the transcription of collagen I and fibronectin in a Sp1- but not Sp3- or AP-1-dependent manner. A suppressed fatty acid synthesis was highlighted using untargeted metabolomics analyses, and synthetic products, palmitic acid (PA), were used as the intermediary agent. After a screening study, fatty acid synthase (FASN) was identified as the main targeted protein, and AhR activation regulated "HDAC3-acetylation" signals but not glycosylation to enhance FASN degradation. Furthermore, results of bioinformatics analysis and others showed that after being activated, AhR targeted miR-193a-3p to control HDAC3 transcription. Collectively, AhR activation inhibited ECM deposition and alleviated intestinal fibrosis by limiting fatty acid synthesis subsequent to the inhibition of "miR-193a-3p-HDAC3-FASN" signals.

High-dose radiation-resistant lung cancer cells stored many functional lipid drops through JAK2/p-STAT3/FASN pathway.

BACKGROUND: The understanding of radiation resistance is still unclear. This study aims to explore the new mechanism of radiation resistance in lung cancer from the perspective of lipid metabolism. METHODS: Oil red O was used to detect the amount of lipid droplets in high-dose radiation-resistant lung cancer cells (HDRR-LCCs) and the primary lung cancer cells. Western blot analysis was used to determine the protein expression levels of key molecules related to de novo fatty acid synthesis and fatty acid transport. Orlistat was used to inhibit the de novo fatty acid synthesis. The prediction of the transcriptional regulators of fatty acid synthetase (FASN) was analyzed by bioinformatics. AZD-1480 was used to inhibit the JAK2/STAT3 pathway to observe its effects on FASN and intracellular lipid droplets. The regulation of the transcription factor p-STAT3 on the FASN gene was verified by Chip-qPCR. Finally, we used the public data of lung cancer patients to analyze the correlation between FASN and LPL gene expression with the prognosis. RESULTS: There were more lipid drops in the HDRR-LCCs than in the primary lung cancer cells. HDRR-LCCs preferred de novo synthesis of fatty acids, and high expression of LPL homodimers indicated a high intake of extracellular fatty acids. The expression of FASN was increased in HDRR-LCCs compared with the primary lung cancer cells in a radiation-dose-dependent way, while LPL homodimers did not show such a trend. The lipid droplets, cell proliferation, and radiation resistance were decreased in HDRR-LCCs after orlistat treatment. Lipid droplets were significantly reduced, and the protein expression of FASN also decreased when using AZD-1480 to inhibit the JAK2/STAT3 pathway. The Chip-qPCR showed that p-STAT3 was the upstream regulator which binds to the promoter region of FASN. Survival analysis showed that high expression of the FASN gene was associated with a poor prognosis in lung cancer patients who received radiotherapy. CONCLUSION: Our studies discovered that lipids deposited in HDRR-LCCs were due to endogenous de novo fatty acids synthesis and exogenous lipids uptake. JAK2/p-TAT3/FASN could be used as promising targets for radiotherapy sensitization. Our study provided a new theoretical basis for studying the mechanism of radiation resistance in lung cancer.

The kinesin light chain-2, a target of mRNA stabilizing protein HuR, inhibits p53 protein phosphorylation to promote radioresistance in NSCLC.

BACKGROUND: Radioresistance hinders radiotherapy for the treatment of lung cancer. Kinesin light chain-2 (KLC2) has been found to be upregulated in lung cancer and also to be associated with poor prognosis. This study aimed to investigate the effect of KLC2 on radiosensitivity in lung cancer. METHODS: The radioresistant role of KLC2 was determined by colony formation, neutral comet assay, and γH2AX immunofluorescent staining assay. We further verified the function of KLC2 in a xenograft tumor model. The downstream of KLC2 was identified through gene set enrichment analysis and validated by western blot. Finally, we analyzed clinical data from the TCGA database to reveal the upstream transcription factor of KLC2, which was validated by RNA binding protein immunoprecipitation assay. RESULTS: Here, we found that downregulation of KLC2 could significantly reduce colony formation, increase γH2AX level, and double-stranded DNA breaks in vitro. Meanwhile, overexpressed KLC2 significantly increased the proportion of the S phase in lung cancer cells. KLC2 knockdown could activate P53 pathway, and ultimately promoting radiosensitivity. The mRNA of KLC2 was observed to bind with Hu-antigen R (HuR). The mRNA and protein expression of KLC2 in lung cancer cells was significantly reduced when combined with siRNA-HuR. Interestingly, KLC2 overexpression significantly increased the expression of HuR in lung cancer cells. CONCLUSION: Taken together, these results indicated that HuR-KLC2 forms a positive feedback loop, which decreases the phosphorylation of p53 and thereby weaken the radiosensitivity of lung cancer cells. Our findings highlight the potential prognosis and therapeutic target value of KLC2 in lung cancer patients treated with radiotherapy.

Radiotherapy combined with PD-1/PD-L1 inhibitors in NSCLC brain metastases treatment: The mechanisms, advances, opportunities, and challenges.

At present, whole-brain radiation therapy/stereotactic radiosurgery is one of the main local treatments for brain metastasis of non-small-cell lung cancer (NSCLC). Currently, it has been proved that radiotherapy (RT) can regulate the immune response, and small-sample studies have shown that patients with NSCLC brain metastases (BMs) can benefit from RT combined with immunotherapy (IO). However, the efficacy and safety of the combination treatment have not been deeply elaborated. Notably, as a challenge that is still being explored, the timing of RT combined with IO is likely to be an important factor affecting efficacy and prognosis. This article reviews the current application and challenges of RT combined with IO from the perspectives of molecular mechanism, combination timing, safety, and efficacy. The purpose is to provide information on clinical evidence-based medicine of combination between RT with IO. For further investigation, we also discuss the major challenges and prospects of RT combined with IO in NSCLC BMs.

Radiotherapy in the preoperative neoadjuvant treatment of locally advanced rectal cancer.

Radiotherapy and chemotherapy are effective treatments for patients with locally advanced rectal cancer (LARC) and can significantly improve the likelihood of R0 resection. Radiotherapy can be used as a local treatment to reduce the size of the tumor, improve the success rate of surgery and reduce the residual cancer cells after surgery. Early chemotherapy can also downgrade the tumor and eliminate micrometastases throughout the body, reducing the risk of recurrence and metastasis. The advent of neoadjuvant concurrent radiotherapy (nCRT) and total neoadjuvant treatment (TNT) has brought substantial clinical benefits to patients with LARC. Even so, given increasing demand for organ preservation and quality of life and the disease becoming increasingly younger in its incidence profile, there is a need to further explore new neoadjuvant treatment options to further improve tumor remission rates and provide other opportunities for patients to choose watch-and-wait (W&W) strategies that avoid surgery. Targeted drugs and immunologic agents (ICIs) have shown good efficacy in patients with advanced rectal cancer but have not been commonly used in neoadjuvant therapy for patients with LARC. In this paper, we review several aspects of neoadjuvant therapy, including radiation therapy and chemotherapy drugs, immune drugs and targeted drugs used in combination with neoadjuvant therapy, with the aim of providing direction and thoughtful perspectives for LARC clinical treatment and research trials.

Prognostic value of cell-free DNA in cerebrospinal fluid from lung cancer patients with brain metastases during radiotherapy.

BACKGROUND: During the last decades, radiotherapy (RT) for non-small cell lung cancer (NSCLC) with brain metastases (BM) has been developed. However, the lack of predictive biomarkers for therapeutic responses has limited the precision treatment in NSCLC-BM. PATIENTS AND METHODS: In order to find the predictive biomarkers for RT, we investigated the influence of RT on the cell-free DNA (cfDNA) from cerebrospinal fluid (CSF) and the frequency of T cell subsets of NSCLC patients with BM. A total of 19 patients diagnosed as NSCLC with BM were enrolled. The CSF from 19 patients and matched plasma samples from 11 patients were collected before RT, during RT, and after RT. The cfDNA from CSF and plasma were extracted, and the cerebrospinal fluid tumor mutation burden (cTMB) was calculated after through next-generation sequencing. The frequency of T cell subsets in peripheral blood was using flow cytometry. RESULTS: The detection rate of cfDNA was higher in CSF compared to plasma in the matched samples. The mutation abundance of cfDNA in CSF was decreased after RT. However, no significant difference was observed in cTMB before and after RT. Although the median intracranial progression-free survival (iPFS) has not yet been reached in patients with decreased or undetectable cTMB, there was a trend that these patients possessed longer iPFS compared to those with stable or increased cTMB (HR 0.28, 95% CI 0.07-1.18, P = 0.067). The proportion of CD4+T cells in peripheral blood was decreased after RT. CONCLUSION: Our study indicates that cTMB can serve as a prognostic biomarker in NSCLC patients with BMs.

Cholinergic dysfunction-induced insufficient activation of alpha7 nicotinic acetylcholine receptor drives the development of rheumatoid arthritis through promoting protein citrullination via the SP3/PAD4 pathway.

Both cholinergic dysfunction and protein citrullination are the hallmarks of rheumatoid arthritis (RA), but the relationship between the two phenomena remains unclear. We explored whether and how cholinergic dysfunction accelerates protein citrullination and consequently drives the development of RA. Cholinergic function and protein citrullination levels in patients with RA and collagen-induced arthritis (CIA) mice were collected. In both neuron-macrophage coculture system and CIA mice, the effect of cholinergic dysfunction on protein citrullination and expression of peptidylarginine deiminases (PADs) was assessed by immunofluorescence. The key transcription factors for PAD4 expression were predicted and validated. Cholinergic dysfunction in the patients with RA and CIA mice negatively correlated with the degree of protein citrullination in synovial tissues. The cholinergic or alpha7 nicotinic acetylcholine receptor (α7nAChR) deactivation and activation resulted in the promotion and reduction of protein citrullination in vitro and in vivo, respectively. Especially, the activation deficiency of α7nAChR induced the earlier onset and aggravation of CIA. Furthermore, deactivation of α7nAChR increased the expression of PAD4 and specificity protein-3 (SP3) in vitro and in vivo. Our results suggest that cholinergic dysfunction-induced deficient α7nAChR activation, which induces the expression of SP3 and its downstream molecule PAD4, accelerating protein citrullination and the development of RA.

The neuropeptide cortistatin attenuates Th17 cell response through inhibition of glycolysis via GHSR1.

The neuropeptide cortistatin (CST) has been reported to attenuate Th17 cell response in multiple disease models, but the mechanism of action remains obscure. Here, we show that either overexpression or exogenous addition of CST obviously restricts Th17 cell differentiation. As metabolic reprogramming plays an important role in Th17 cell development, we explore whether CST inhibits Th17 cell differentiation by regulating glycolysis. The results show that CST substantially attenuates the glycolysis during Th17 differentiation and down-regulates the mRNA expression of myelocytomatosis oncogene (Myc) and hexokinase 2 (HK2), the glycolysis rate-limiting enzyme. Following the overexpression of Myc and HK2, the inhibitory effect of CST on Th17 differentiation nearly disappears, suggesting that Myc-HK2 pathway is deeply involved. Furthermore, growth hormone secretagogue receptor 1 (GHSR1) is identified as the key receptor subtype for CST attenuating glycolysis and Th17 cell differentiation by the combined uses of CST with various receptor subtype blockers. The knockdown of GHSR1 abrogates the inhibition of CST on Th17 differentiation and glycolysis. Finally, in the colitis mice induced by dextran sulfate sodium, an intraperitoneal injection of CST markedly inhibits Th17 cell response and down-regulates the expression of HK2 in the Th17 cells, which is reversed by the combined use of GHSR1 antagonist. These findings suggest that inhibition of glycolysis is the key pathway for CST attenuating Th17 cell response, and GHSR1, Myc and HK2 are potential therapeutic targets of Th17 cell-related diseases.

Changes in T Lymphocyte Subsets in Different Tumors Before and After Radiotherapy: A Meta-analysis.

Purpose: Radiation therapy (RT) induces an immune response, but the relationship of this response with tumor type is not fully understood. This meta-analysis further elucidated this relationship by analyzing the changes in T lymphocyte subsets in different tumors before and after radiotherapy. Methods: We searched English-language electronic databases including PubMed, EMBASE, and the Cochrane Library to collect studies on the changes in peripheral blood CD3+ T lymphocytes, CD4+ T lymphocytes, and CD8+ T lymphocytes before and after radiotherapy in tumor patients from January 2015 to April 2021. The quality of the included literature was evaluated using the NOS scale provided by the Cochrane Collaboration, and statistical software RevMan 5.4 was used to analyze the included literature. P<0.05 was considered to indicate statistical significance. Results: A total of 19 studies in 16 articles involving 877 tumor patients were included. All data were collected within 1 month before or after radiotherapy. Meta-analysis showed that numbers of CD3+ T lymphocytes (SMD: -0.40; 95% CI [-0.75, -0.04]; p = 0.03) and CD4+ T lymphocytes (SMD: -0.43; 95% CI: [-0.85, -0.02]; p = 0.04) were significantly reduced after radiotherapy compared with before treatment, but there was no statistically significant difference for CD8+ T lymphocytes (SMD: 0.33; 95% CI: [-0.88, 0.74]; p = 0.12). Subgroup analysis showed that peripheral blood T lymphocytes decreased in head and neck cancer. However, in prostate cancer and breast cancer, there was no significant change in peripheral blood. 1 month after radiotherapy, it has a potential proliferation and activation effect on lymphocytes in esophageal cancer and lung cancer. The results showed that CD8+T lymphocytes increased in peripheral blood after SBRT. Radiotherapy alone reduced CD3+ T lymphocyte numbers. Conclusions: Within 1 month of radiotherapy, patients have obvious immunological changes, which can cause apoptosis and reduction of T lymphocytes, and affect the balance of peripheral blood immune cells. The degree of immune response induced by radiotherapy differed between tumor types.

Circulating miRNAs in Serum as Biomarkers for Early Diagnosis of Non-small Cell Lung Cancer.

BACKGROUND: Non-small cell lung cancer (NSCLC) accounts for about 85% of lung cancers. This study aimed to discover the potential miRNA biomarkers for early detection of NSCLC. METHODS: Total circulating miRNAs were extracted from six patients and six volunteers and run on the miRNA chip. The differentially expressed miRNAs acquired by data mining were intersected with chip results, and qRT-PCR were carried out. Then the differentially miRNAs were validated by using a validation cohort (120 participants). ROC curves were established to evaluate the diagnostic efficacy of the differentially circulating miRNAs. The target genes of the differential miRNAs were identified using the miRTarBase database, and follow-up GO and KEGG enrichment analysis were conducted. RESULTS: We identified 577 miRNA which screened according to the criteria (fold change > 2 and p value < 0.05). Among them, seven circulating miRNAs passed additional filtering based on data mining. These miRNAs were further validated in the training and validation cohort. miR-492, miR-590-3p, and miR-631 were differentially expressed in the patients' serum, and the area under the ROC curve (AUC) values of these miRNAs were 0.789, 0.792, and 0.711, respectively. When using them as a combination to discriminate healthy volunteers from patients, the AUC reached 0.828 (95% CI, 0.750-0.905, p = 0.000) with a sensitivity of 86.7% and specificity of 71.7%. The follow-up enrichment analysis showed that target genes of three miRNA were associated with tumorigenesis and progression, such as cell cycle and P53 signaling pathway. CONCLUSIONS: The combination of miR-492, miR-590-3p, and miR-631 can be utilized to distinguish healthy individuals and early-stage NSCLC patients. IMPACT: The combination of miR-492, miR-590-3p, and miR-631 might be a promising serum biomarker in patients for the early diagnosis of NSCLC.

Inhibition of the activation of γδT17 cells through PPARγ-PTEN/Akt/GSK3ß/NFAT pathway contributes to the anti-colitis effect of madecassic acid.

Type-17 immune response, mediated mainly by IL-17, plays a critical role in ulcerative colitis. Previously, we showed that madecassic acid (MA), the main active ingredient of Centella asiatica herbs for anti-colitis effect, ameliorated dextran sulfate sodium (DSS)-induced mouse colitis through reducing the level of IL-17. Here, we explore the effect of MA on the activation of γδT17 cells, an alternative source of IL-17 in colitis. In DSS-induced colitis mice, oral administration of MA decreased the number of γδT17 cells and attenuated the inflammation in the colon, and the anti-colitis effect of MA was significantly counteracted by redundant γδT17 cells, suggesting that the decrease in γδT17 cells is important for the anti-colitis effect of MA. In vitro, MA could inhibit the activation but not the proliferation of γδT17 cells at concentrations without evident cytotoxicity. Antibody microarray profiling showed that the inhibition of MA on the activation of γδT17 cells involved PPARγ-PTEN/Akt/GSK3ß/NFAT signals. In γδT17 cells, MA could reduce the nuclear localization of NFATc1 through inhibiting Akt phosphorylation to promote GSK3ß activation. Moreover, it was confirmed that MA inhibited the Akt/GSK3ß/NFATc1 pathway and the activation of γδT17 cells through activating PPARγ to increase PTEN expression and phosphorylation. The correlation between activation of PPARγ, decrease in γδT17 cell number, and amelioration of colitis by MA was validated in mice with DSS-induced colitis. In summary, these findings reveal that MA inhibits the activation of γδT17 cells through PPARγ-PTEN/Akt/GSK3ß/NFAT pathway, which contributes to the amelioration of colitis.

Regulation of gut microbiota substantially contributes to the induction of intestinal Treg cells and consequent anti-arthritis effect of madecassoside.

Previously, we reported that oral administration of madecassoside, the main active triterpene in Centella asiatica L., exerted anti-arthritis effect by inducing the generation of regulatory T (Treg) cells in small intestine. This study investigates the action site and mechanism of madecassoside to induce Treg cells. In collagen-induced arthritis (CIA) of rats, oral administration of madecassoside significantly alleviated arthritis symptoms, but its main metabolite madecassic acid did not, suggesting that madecassoside functions in the parent form. Madecassoside was shown to increase the number of Treg cells and promote the expression of Foxp3 and IL-10 in rat ileum rather than duodenum and jejunum, as detected using the immunohistochemistry assay and quantitative PCR assay, respectively. Unexpectedly, madecassoside was absent of significant effect on in vitro Treg cell differentiation and the expression of Foxp3 and IL-10. A combined use of broad-spectrum antibiotics resulted in significant reduction of the anti-arthritis effect of madecassoside in CIA rats. The 16S rRNA gene sequence showed that madecassoside could reverse the changes of gut microbiota under arthritis condition, and enrich several bacteria such as Lachnospiraceae, Butyricicoccus, Faecalibacterium, Butyricicoccus pullicaecorum and so on. GC-MS assay showed that madecassoside elevated the levels of acetic acid and butyric acid, but not other short chain fatty acids (SCFAs) in the cecum contents of CIA rats. Butyric acid rather than acetic acid could induce the in vitro differentiation of Treg cells and the expression of Foxp3 and IL-10. Accordingly, when madecassoside was co-administered with heptanoyl CoA, the competitive inhibitor of butyrate synthase, its effect on butyric acid production, Treg cell proportion and arthritis nearly disappeared. These findings indicate that oral madecassoside induces the generation of Treg cells and therefore displays anti-arthritis effect in the parent form but not metabolites, and the ileum is the main action site. The mechanism of madecassoside can be summarized as: expansion of the richness of butyrate-producing bacteria-up-regulation of intestinal butyrate level-induction of Treg cell differentiation and IL-10 expression.

Arctigenin disrupts NLRP3 inflammasome assembly in colonic macrophages via downregulating fatty acid oxidation to prevent colitis-associated cancer.

Arctigenin, the major active constituent of Fructus Arctii, has been reported to inhibit the growth of various tumors and alleviate colitis. This study aimed to prove the protective effect of arctigenin on colitis-associated cancer (CAC) and explore its mechanisms. Orally administered arctigenin prevented the progression of colitis and protected against colon carcinogenesis in azoxymethane (AOM)/dextran sulfate sodium (DSS)-induced CAC mice. Arctigenin downregulated NLRP3 inflammasome activation and fatty acid oxidation (FAO) metabolism in macrophages, as determined by untargeted metabolomics. Arctigenin also inhibited the expression of carnitine palmitoyltransferase 1 (CPT1), reduced the acetylation of α-tubulin, and disrupted NLRP3 complex formation, which in turn inactivated the NLRP3 inflammasome. Downregulation of the CPT1-FAO-acetyl-coenzyme A (acetyl-CoA)-acetylated α-tubulin pathway was observed to inhibit the effect of arctigenin on NLRP3 inflammasome assembly, as confirmed by CPT1 overexpression. Lastly, arctigenin was shown to inhibit NLRP3 inflammasome activation and improve CAC in mice, and the effect was significantly diminished by the overexpression of adeno-associated virus (AAV)9-CPT1. Taken together, these results show that the inhibition of NLRP3 inflammasome assembly in macrophages due to FAO downregulation contributes to the preventative effect of arctigenin against CAC. Our findings highlight the potential value of arctigenin to reduce the risk of CAC in patients with colitis.