Experimental Biology and Medicine

Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by chronic destructive joint disease. To date, the etiology and pathogenesis of RA have not been fully elucidated, but a large number of studies have indicated that hypoxia is an important feature of RA. Our study was designed to probe how hypoxia-induced exosome (exo) derived from synovial fibroblasts (SFs) affect RA. In this study, we found that hypoxic environment existed in synovial tissue of RA, and miR-424 expression was increased in RA, and exosome derived from synovial fibroblasts (SFs-exo) could significantly induce T cells differentiation, which Th17 cells increased and Treg cells decreased. Besides, SFs-exo affected the expression of related inflammatory cytokines. And, we also found that FOXP3 was a target gene of miR-424 and exo-miR-424 KD inhibited RA worsening. These results suggested that SFs-exo in hypoxia aggravates rheumatoid arthritis by regulating Treg/Th17 balance and thus may be a potential therapeutic target for RA.

Many countries are currently in a state of lockdown due to the SARS-CoV-2 pandemic. One key requirement to safely transition out of lockdown is the continuous testing of the population to identify infected subjects. Currently, detection is performed at points of care using quantitative reverse-transcription PCR, thus requiring dedicated professionals and equipment. Here, we developed a protocol based on reverse transcribed loop-mediated isothermal amplification for the detection of SARS-CoV-2. This protocol is applied directly to SARS-CoV-2 nose and throat swabs, with no RNA purification step required. We tested this protocol on over 180 suspected patients, and compared the results to those obtained using the standard method. We further succeeded in applying the protocol to self-collected saliva samples from confirmed cases. Since the proposed protocol can detect SARS-CoV-2 from saliva and provides on-the-spot results, it allows simple and continuous surveillance of the community.

Maintenance of the highly plastic intestinal epithelium relies upon stem cells localized to intestinal crypts. Recent evidence suggests muscarinic acetylcholine signaling impacts epithelial barrier function, proliferation, and apoptosis. We hypothesized that the intestinal crypt base would express specific muscarinic acetylcholine receptors that drive proliferation in this critical region. Intestinal segments spanning the small bowel were procured from wild-type C57Bl/6 mice to determine muscarinic acetylcholine receptor mRNA expression and create sections on laser capture microdissection slides for analysis of crypt base cells. RT-PCR was performed using primers targeting the five muscarinic acetylcholine receptor subtypes (M1–M5), LGR5, BIII-tubulin, and GAPDH. To determine the effects of muscarinic agonism in vivo, osmotic pumps delivering the M1 muscarinic acetylcholine receptor agonist McN-A-343 were implanted into wild type mice for one week. Segments were harvested, histologic sections created, and morphometric and proliferative parameters measured. In full-thickness intestinal samples, muscarinic acetylcholine receptor subtypes M1–M4 were found in all regions, while M5 was localized to the proximal jejunum. In crypt-base cells, the M1 muscarinic acetylcholine receptor subtype was the only subtype found and was present in all regions. LGR5 was present in all laser capture microdissection samples, indicating the capture of intestinal stem cells. In vivo experiments conducted with McN-A-343 revealed significantly increased villus height, crypt depth, and crypt-cell proliferation. The presence of M1 muscarinic acetylcholine receptor mRNA within the stem cell niche in the intestinal crypt base coupled with increased mucosal growth with M1 receptor stimulation in vivo suggests that the cholinergic system, via the M1 muscarinic acetylcholine receptor, is a critical mediator of intestinal mucosal homeostasis.

Ultrasound and microbubbles can induce apoptosis of tumor cells. However, the effects of ultrasound and microbubbles (USMB) combined with chemotherapy on in vivo tumors have rarely been studied. This research is to evaluate the efficacy of targeted USMB and chemotherapeutic drugs on apoptosis and inhibition of hepatic carcinoma cells in nude mice. We also summarize case reports of its clinical application. It was divided into three parts: First, the subcutaneous hepatic tumors of nude mice were irradiated with ultrasound (20 kHz, 2 W/cm², 40% duty cycle) and SonoVue microbubbles injected to the tail vein of the mice followed by chemotherapy comprising cisplatin, mitomycin, and 5-fluorouracil. The apoptosis of hepatoma cells was examined by TUNEL staining, and the expression of the Bax and Bcl-2 proteins in the tumor tissues was detected by immunohistochemistry. The microvasculature of the tumor tissues was observed by transmission electron microscope. Second, orthotopic hepatic tumors in nude mice were irradiated by USMB and chemotherapy. The therapeutic effects were evaluated by magnetic resonance imaging. Third, the malignant tumors in three patients were treated with USMB and chemotherapy. Our results showed that microbubbles focused by low-frequency US ruptured microvessel walls, through which more chemotherapy drugs entered into the carcinoma cells, thus enhancing cell apoptosis, increasing Bax protein expression, and decreasing Bcl-2 expression. The survival period of the nude mice in the USMB and chemotherapy group was the longest, compared with the control, USMB, and chemotherapy (χ²=29.37, P < 0.0001). Magnetic resonance imaging examinations revealed the tumor volumes in nude mice decreased after USMB and chemotherapy (t = 3.91, P = 0.0173). In clinical cases, after USMB and chemotherapy, the diameters of tumors decreased and the symptoms of the patients were relieved. Targeted low-frequency ultrasound combined with chemotherapy can promote tumor cell apoptosis, inhibit tumor growth, and relieve clinical symptoms of patients.

A quantitative analytical method to discriminate among the various types of cancerous esophagus tissue is essential for accurate cancer staging. This paper reports on the use of ratiometric nonlinear optical microscopy to reveal the ratio of two-photon excited fluorescence (TPEF) to second harmonic generation (SHG) and forward to backward (F/B) SHG from single collagen fibers only in submucosa of esophageal squamous cell carcinoma. This makes it possible to accurately differentiate among the four stages of esophageal cancer, providing results that are in good agreement with histopathology. Furthermore, it is confirmed by polarization-dependent SHG that the varied SHG response in esophageal cancer tissues is mainly from the shrinkage in diameter of collagen fibers instead of the collagen triple helixes altered by cancer cells. Based on the results of TPEF/SHG and F/B SHG ratio, they can cooperatively improve the precision of diagnostics on esophageal cancer and could be transferred to other types of cancer diseases with changed collagen fibers.

MicroRNA (miR) plays a critical role in the progression of multiple malignancies. Nevertheless, knowledge of the role it plays in laryngeal cancer is limited. In this study, we explored the role of miR-892a in laryngeal cancer cell proliferation and apoptosis. miR-892a expression was increased in 17 laryngeal cancer samples and cells compared with that in healthy tissues, laryngeal cancer normal surrounding tissues, and the NP69 human nasopharyngeal epithelial cell line. Conversely, Dicer expression was downregulated in human laryngeal cancer samples as well as in the laryngeal cancer cell lines. CCK-8 assays and colony formation assay confirmed that depleted miR-892a expression damaged the proliferation and growth of TU212 and M4E cells. Annexin V/PI flow cytometry displayed that miR-892a inhibition led to increased apoptosis of TU212 and M4E cells. By conducting bioinformatic analysis and dual-luciferase reporter assay, it was revealed that that miR-892a targets Dicer 3′-UTR for silencing. Dicer expression inhibition offsets the effect of miR-892a on the growth and apoptosis of laryngeal cancer cells. Dicer overexpression displayed similar phenotype with miR-892a inhibition on the properties of laryngeal cancer cells. Results of in vivo experiments further confirmed that miR-892a silencing suppressed tumor growth in a mouse model. Hence, the results of this study provide new ideas about the biological and molecular mechanisms behind laryngeal cancer progression, thereby obtaining novel laryngeal cancer treatments.

Recent studies have displayed that circular RNA plays a key regulatory role in tumorigenesis and development. However, evidence supporting the critical role of circular RNA in the prognosis of colorectal cancer (CRC) is still limited. This study was designed to screen and identify novel circular RNA biomarkers in CRC. The microarray analysis of circular RNA expression profile was performed in three matched CRC and normal tissues. Compared with normal mucosa, a total of 208 differentially expressed circular RNAs were found in CRC, of which 95 were upregulated and 113 were downregulated. Then, the top 10 circular RNAs with the most significant differential expression were selected for verification by quantitative polymerase chain reaction (qPCR) in the above samples. The results of qPCR basically coincided with the findings of microarray analysis. The hsa_circ_022382 expression was the most significant upregulation in CRC among the 10 circular RNAs validated by qPCR. Because hsa_circ_022382 is derived from the human FADS2 gene, we named it circFADS2. Next, the increased expression of circFADS2 was further confirmed by qPCR, and its correlation with clinicopathologic parameters was analyzed in 200 CRC tissues. Herein, the expression of circFADS2 was shown to increase in 187 cases and decrease in 13 cases of CRC, and the elevated circFADS2 expression was closely related to the size, differentiation, infiltration depth, lymphatic and distant metastasis, and tumor/node/metastasis (TNM) stage of CRC. The survival analyses by Kaplan–Meier method demonstrated that patients with higher circFADS2 expression levels had shorter overall survival time and vice versa. Cox regression and area under ROC curve analyses revealed that the circFADS2 expression may be a promised biomarker for prognosis of CRC patients and had better prediction value when combined with TNM stage. In conclusion, our study identifies circFADS2 as an oncogenic biomarker and a potential prognostic factor in CRC.

Immune-related genes have great potential as prognostic markers in many types of cancer. Therefore, we have attempted to develop immune-related gene markers to enhance the prognosis of breast cancer; 1159 samples of breast cancer gene expression data and clinical follow-up messages were downloaded from TCGA and GEO, which were classified into training set, test set, and validation set. In the training set, the gene pairs are established according to the relative expression levels between 320 immune genes, in which the prognosis-related gene pairs are screened, and Lasso is used for feature selection to screen the robust biomarkers. A prognostic model of immune gene correlation was set up and verified. Sixty-six IRGPs were obtained, and 17-IRGPs signature was established. 17-IRGPs signature is an independent prognostic indicator for BC patients, which can stratify the risk in the training set and testing series, and AUC of five years survival was greater than 0.7; 17-IRGPs signature had better classification performance in patients with advanced BC. In addition, we compared the prognostic characteristics of 17-IRGPs with four reported breast cancers and clinical stages; 17-IRGPs achieved the highest average C index (0.7, P < 0.05), and functional analysis found that the dysregulated immune environment may be the cause of the observed difference in survival between patient groups defined by our characteristics. 17-IRGPs signature was constructed as a newly developed prognostic indicator to calculate the survival of BC patients.

While neutrophil production in emergency states has been extensively studied, regulation of neutrophil homeostasis in the steady-state remained incompletely understood. We have shown that innate immune receptor toll-like receptor (TLR)4 and downstream TIR-domain-containing adapter-inducing interferon-β (TRIF) are indispensable for the generation of a granulocyte-colony stimulating factor (G-CSF)-dependent regulatory feedback loop upon antibody-induced neutropenia. These findings demonstrated that steady-state granulopoiesis is a demand-driven process, which may rely on differential triggering of innate immune receptors by microbial cell wall constituents such as lipopolysaccharide. Herein, we present further evidence on underlying mechanisms: oral intake of highly endotoxic lipopolysaccharide, but not TLR-antagonistic lipopolysaccharide derived from Rhodobacter sphaeroides, induces hematopoietic stem and progenitor cell fate decisions toward the neutrophil lineage independent of G-CSF. TLR4 has been identified as the indispensable sensor for oral lipopolysaccharide-modulated steady-state granulopoiesis. These results have important implications: food lipopolysaccharide content or the composition of the gastrointestinal microbiome may be strongly underrated as determinants of peripheral blood neutrophil levels. Both neutrophilia and neutropenia are associated with drastically worse outcomes in epidemiological studies of the general population as well as in diseased states.

Insulin resistance (IR) may be associated with oxidative stress and leads to cardiovascular disorders. Current research focuses on interplay between insulin-resistance indices and oxidant-antioxidant markers in elderly individuals with or without insulin-resistance. The assessment involved anthropometric data (weight, height, BMI, percentage of body fat (FAT)) and biochemical tests (glucose, lipids, serum insulin and plasma oxidant-antioxidant markers: Thiobarbituric Acid-Reacting Substances (TBARS), Cu,Zn-superoxide dismutase (SOD-1) and total antioxidant status). Insulin resistance index (IR) assuming a cut-off point of 0.3 allows to divides groups into: insulin sensitive group (InsS) IR < 0,3 (n = 35, median age 69.0 years) and insulin-resistant group (InsR) IR ≥ 0.3 (n = 51, median age 71.0 years). Lipids and antioxidant defense system markers did not differentiate the investigated groups. In the InsR elderly group, the FAT was increased (P < 0.000003) and TBARS (P = 0.008) concentration decreased in comparison with InsS group. A positive correlation for SOD-1 and total antioxidant status (P < 0.05; r =  0.434) and a negative correlation for TBARS and age (P < 0.05 with r = −0.421) were calculated in InsR individuals. In elderly individuals, oxidative stress persists irrespective of insulin-resistance status. We suggest that increased oxidative stress may be consequence of old age. An insulin action identifies those at high risk for atherosclerosis, via congruent associations with oxidative stress and extra- and intra-cellular antioxidant defense systems. Thus, we maintain that insulin-resistance is not the cause of aging.

Lipotoxicity, defined as the cell death and/or cellular dysfunction induced by ectopic lipid deposition, plays a pathological role in the development of many metabolic diseases. Although endoplasmic reticulum stress is a well-documented mechanism behind, how endoplasmic reticulum stress is initiated during lipotoxicity remains obscure. In this study, using palmitate exposure (a 16-C saturated fatty acid) of AML12 hepatocytes, a non-transformed murine hepatocyte cell line, as an experimental model, we identified mammalian target of rapamycin complex 1 (mTORC1) to be a critical contributor to palmitate-elicited lipotoxicity, manifested by incremental triglycerides secretion and cell death. Unlike oleate (an 18-C monounsaturated fatty acid), palmitate strongly induced mTORC1 activation in hepatocytes. Importantly, mTOR inhibitors, torin-1, and rapamycin prevented hepatocytes from palmitate-induced triglyceride overproduction and cell death. We further showed that the intracellular metabolism of palmitate is required for its stimulatory effect on mTORC1. Whereas the inhibition of long-chain acyl-CoA synthetase, which converts palmitate to palmitoyl-CoA, attenuated mTORC1 activation and protected against cell death, the inhibition of stearoyl-CoA desaturase-1, the enzyme desaturating palmitate to palmitoleate, strengthened mTORC1 activation and aggravated triglyceride overproduction and cell death. Our further investigations revealed that the palmitate-induced mTORC1 activation was required for its endoplasmic reticulum stress-inducing action as mTORC1 inhibition ablated palmitate-induced activation of IRE1α, one of the three canonical pathways activated during unfolded protein response. Finally, our data demonstrated that IRE1α inhibition ameliorated triglyceride overproduction and cell death in response to palmitate exposure. Collectively, our data identify that mTORC1-IRE1α pathway is coordinately implicated in the development of lipotoxicity in hepatocyte.

Diabetes mellitus has been shown to impair respiratory function. The diaphragm is an important skeletal muscle involved in respiration. Hydrogen sulfide (H₂S) is one of the three endogenous gas messengers in mammals, which exhibits anti-fibrotic activity in some types of diabetes-related complications. However, whether and how H₂S exerts its anti-fibrotic activity on the diabetic diaphragmatic muscle remains unclear. In this study, we explored the anti-fibrotic activity of exogenous H₂S on the diaphragm using a streptozotocin (STZ)-induced diabetic rat model. The results showed that diaphragmatic biomechanical parameters were decreased, whereas the levels of inflammatory cytokines, collagen, and nucleotide-binding oligomerization domain-like receptor protein (NLRP) 3 inflammasome-related protein expression were increased in diabetic diaphragms. This implies that diabetes causes fibrosis of the diaphragm muscle through activation of NLRP3 inflammasome. After supplementation with exogenous H₂S, the diaphragmatic biomechanical and pathological alterations were ameliorated and activation of NLRP3 inflammasome was inhibited, followed by a decline in diaphragm muscle inflammation and fibrosis. These results demonstrate for the first time that exogenous H₂S effectively attenuates STZ-induced diabetic diaphragm muscle fibrosis, and that the underlying mechanism may be associated with suppression of the NLRP3 inflammasome-mediated inflammatory reaction.

Chronic obstructive pulmonary disease (COPD) is a heterogeneous inflammatory disease and eosinophils (EOS) participate in inflammation process. Acute exacerbation of COPD (AECOPD) is an inevitable trend in the development of the disease and has attracted widespread attention. In the present study, 108 hospitalized patients with AECOPD were collected and the levels of interleukin-13 and eosinophil cationic protein in the serum and sputum were measured to explore their clinical value in eosinophilic AECOPD patients. The patients were divided into an eosinophilic group (52 cases, 48.15%) and a noneosinophilic group (56 cases, 51.85%). The eosinophilic group had fewer acute exacerbations in the past year, shorter average hospitalization days, lower respiratory failure rate, mechanical ventilation utilization rate, and lower CAT and mMRC scores (P < 0.05). The levels of interleukin-13 and eosinophil cationic protein in sputum in the eosinophilic group were higher than those in the noneosinophilic group (P < 0.05), and there was no significant difference in the serum between the two groups (P > 0.05). The receiver operating characteristic (ROC) curves of sputum interleukin-13 and eosinophil cationic protein predicting peripheral blood EOS% ≥2% of AECOPD patients were statistically significant (P < 0.05). The noneosinophilic group had a higher rate of rehospitalization due to acute exacerbation during the one-year follow-up, and there was no significant difference in mortality between the two groups. The results show that eosinophils in peripheral blood are a simple, convenient, and inexpensive index for assessing the condition and prognosis of AECOPD patients. Interleukin-13 and eosinophil cationic protein are involved in the pathogenesis of eosinophilic AECOPD and may be the new targeted anti-inflammatory therapies in the future.