WSB1/2 target chromatin-bound lysine-methylated RelA for proteasomal degradation and NF-κB termination.

Proteasome-mediated degradation of chromatin-bound NF-κB is critical in terminating the transcription of pro-inflammatory genes and can be triggered by Set9-mediated lysine methylation of the RelA subunit. However, the E3 ligase targeting methylated RelA remains unknown. Here, we find that two structurally similar substrate-recognizing components of Cullin-RING E3 ligases, WSB1 and WSB2, can recognize chromatin-bound methylated RelA for polyubiquitination and proteasomal degradation. We showed that WSB1/2 negatively regulated a subset of NF-κB target genes via associating with chromatin where they targeted methylated RelA for ubiquitination, facilitating the termination of NF-κB-dependent transcription. WSB1/2 specifically interacted with methylated lysines (K) 314 and 315 of RelA via their N-terminal WD-40 repeat (WDR) domains, thereby promoting ubiquitination of RelA. Computational modeling further revealed that a conserved aspartic acid (D) at position 158 within the WDR domain of WSB2 coordinates K314/K315 of RelA, with a higher affinity when either of the lysines is methylated. Mutation of D158 abolished WSB2's ability to bind to and promote ubiquitination of methylated RelA. Together, our study identifies a novel function and the underlying mechanism for WSB1/2 in degrading chromatin-bound methylated RelA and preventing sustained NF-κB activation, providing potential new targets for therapeutic intervention of NF-κB-mediated inflammatory diseases.

[Establishment and evaluation of pre-metastatic niche mouse model labelled with luciferase in lewis lung cancer cells].

Objective This study aimed to establish a pre-metastatic niche mouse model utilizing luciferase-labeled Lewis (Luc-Lewis) lung cancer cells and to assess the efficacy of this model employing both qualitative and quantitative methods. Methods C57BL/6 mice were categorized into two groups: a normal control group and a model group, each containing 15 individual mice. The pre-metastatic niche model was established via tail vein injection of Luc-Lewis lung cancer cells. Body mass were measured daily for all groups. Tumor fluorescence signals within the mice were detected using a high-throughput enzyme marker instrument. Lung tissue specimens were harvested to evaluate metastatic progression. HE staining was used to assess histopathological changes. Real-time quantitative PCR and Western blot analysis were used to detect the mRNA and protein expression of lysyl oxidase (LOX), matrix metalloproteinase 9 (MMP9), versican (VCAN), and fibronectin (FN), which are the specific markers for the formation of the microenvironment of lung tissues before metastasis. Results Significant declines in body mass and observable lethargy were noted in the model group when compared to the control group. Distinct fluorescence signals were observed in the lung tissue of the model group, demonstrating a positive correlation with the duration of model establishment. By day 14, elevated mRNA and protein expression levels of LOX, MMP9, VCAN, and FN were significantly evident. In addition, histopathological evaluations revealed augmented interstitial thickness, alveolar atrophy and significant inflammatory cell infiltration within the lung tissues of the model group. By the 21st day, metastatic lesions manifested in the lung tissues of the model group, suggesting an approximate pre-metastatic niche maturation timeline of 14 days. Conclusion A pre-metastatic niche mouse model for Lewis lung cancer is successfully established.

Inhibitory Effect of Astragalus Polysaccharide on Premetastatic Niche of Lung Cancer through the S1PR1-STAT3 Signaling Pathway.

As a common malignant tumor, the morbidity and mortality of lung cancer have been rising in recent years. The concept of "premetastatic niche" may lead to a revolutionary change in antitumor metastasis therapeutic strategies. Traditional Chinese medicine with multitargets and lower poisonous agents may be a potentially effective means to intervene in the "premetastatic niche (PMN)" to prevent and treat tumor metastasis. Astragalus polysaccharide (APS) is a substance with strong immune activity in Astragalus membranaceus that has excellent biological activities such as immunomodulation, anti-inflammatory, and antitumor. In this study, we constructed a tumor lung metastasis animal model to explore the intervention mechanism of APS on the premetastatic niche. We found that APS inhibited the formation of the lung premetastatic niche and inhibited the recruitment of myeloid-derived suppressor cells (MDSCs) in the lung. Mechanistically, we showed that the proteins and gene expression of S1PR1, STAT3, and p-STAT3 in the S1PR1/STAT3 signaling pathway were suppressed by APS. In line with the above findings, our results confirmed that APS may inhibit the accumulation of MDSCs in the premetastatic niche through the intervention of the S1PR1-STAT3 signaling pathway to achieve the antitumor effect.

Total flavonoids of Oxytropis falcata Bunge have a positive effect on idiopathic pulmonary fibrosis by inhibiting the TGF-ß1/Smad signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease with unknown etiology. Oxytropis falcata Bunge (O. falcata) is a 1-35 cm high perennial clustered herb, also known as edaxia, has viscosity and a special smell, and is mainly distributed in the western areas of China. The root of O. falcata has a diameter of 6 mm, is straight and deep, dark red and its stems are shortened, woody and multibranched. O. falcata has heat-clearing, detoxification, analgesic, anti-inflammatory, antibacterial, hemostatic and antitumor activities. Furthermore, O. falcata has excellent anti-inflammatory and analgesic effects, and it is one of the three major anti-inflammatory drugs in Tibetan medicine, known as "the king of herbs". Total flavonoids of Oxytropis falcata Bunge (FOFB) were previously extracted, and their pharmacological activities are consistent with those of the whole herb. In this study, FOFB was extracted from O. falcata by ethanol extraction, and the mechanism of FOFB on IPF was verified by in vivo and in vitro experiments. AIM OF THE STUDY: In this study, we aimed to observe the effects of FOFB on idiopathic pulmonary fibrosis. MATERIALS AND METHODS: In in vivo experiments, an IPF rat model was established by bleomycin induction. The rats were treated with FOFB (100, 200, 400 mg kg-1·d-1) for 4 weeks. Masson staining and the expression of TGF-ß, p-Smad2, p-Smad3 and Smad7 in the lung tissue of rats were detected. In in vitro experiments, we perfused normal rats with FOFB (100, 200, 400 mg kg-1·d-1) and obtained the corresponding drug-containing serum. The HFL-1 cell model induced by TGF-ß1 was used to detect the corresponding indices through intervention with drug-containing serum. The best intervention time for drug-containing serum was detected by the CCK-8 method. Changes in apoptosis, cytoskeleton and rough endoplasmic reticulum structure were detected. Finally, the expression of TGF-ß, p-Smad2, p-Smad3 and Smad7 in cells was examined. RESULTS: In vivo, Masson staining indicated that the degree of pulmonary fibrosis increased significantly, the expression of TGF-ß, p-smad2 and p-Smad3 increased significantly, and the expression of Smad7 decreased in the model group. We found that the degree of pulmonary fibrosis gradually decreased and that the inhibition of the TGF-ß/Smad signaling pathway became more obvious with increasing FOFB dose. FOFB (400 mg kg-1·d-1) significantly improved the degree of pulmonary fibrosis in rats. In in vitro experiments, the CCK-8 results showed that 120 h was the best intervention time for drug-containing serum. In the model group, there was no obvious apoptosis or changes in microfilaments and microtubules, the number of rough endoplasmic reticulum increased, and the expression of TGF-ß, p-Smad2 and p-Smad3 increased significantly, while the expression of Smad7 decreased significantly. We found that with the increase in drug-containing serum concentration, the apoptosis, cytoskeleton and degree of destruction of the rough endoplasmic reticulum in the HFL-1 cell model also increased, and the inhibition of the TGF-ß/Smad signaling pathway became more pronounced; the effect of the drug-containing serum administered with FOFB (400 mg kg-1·d-1) was the most significant. CONCLUSIONS: The results suggest that FOFB can improve the occurrence and development of IPF. The effect of FOFB on IPF may be mediated by inhibition of the TGF-ß1/Smad signaling pathway.

Baicalein suppresses the growth of the human thyroid cancer cells by inducing mitotic catastrophe, apoptosis and autophagy via NF-kB signalling pathway

The Editors of JBUON issue an Expression of Concern to 'Baicalein suppresses the growth of the human thyroid cancer cells by inducing mitotic catastrophe, apoptosis and autophagy via NF-kB signalling pathway', by Shijian Yi, Guowen Liu, Yang Wu, Qiankun Liang, Lanlan Li; JBUON 2020;25(1):389-394; PMID: 32277659. Following the publication of the above article, readers drew to our attention that part of the data was possibly unreliable. We sent emails to the authors with a request to provide the raw data to prove the originality, but received no reply. Therefore, as we continue to work through the issues raised, we advise readers to interpret the information presented in the article with due caution. We thank the readers for bringing this matter to our attention. We apologize for any inconvenience it may cause.

Molecular characterization of colorectal cancer: A five-gene prognostic signature based on RNA-binding proteins.

Background: Colorectal cancer (CRC) is one of the most common cancers worldwide. RNA-binding proteins (RBPs) regulate essential biological processes and play essential roles in a variety of cancers. The present study screened differentially expressed RBPs, analyzed their function and constructed a prognostic model to predict the overall survival of patients with CRC. Methods: We downloaded CRC RNA-sequencing data from the Cancer Genome Atlas (TCGA) portal and screened differentially expressed RBPs. Then, functional analyses of these genes were performed, and a risk model was established by multivariate Cox regression. Results: We obtained 132 differentially expressed RBPs, including 66 upregulated and 66 downregulated RBPs. Functional analysis revealed that these genes were significantly enriched in RNA processing, modification and binding, ribosome biogenesis, post-transcriptional regulation, ribonuclease and nuclease activity. Additionally, some RBPs were significantly related to interferon (IFN)-alpha and IFN-beta biosynthetic processes and the Toll-like receptor signaling pathway. A prognostic model was constructed and included insulin like growth factor 2 messenger ribonucleic acid binding protein 3 (IGF2BP3), poly (A) binding protein cytoplasmic 1 like (PABPC1L), peroxisome proliferator activated receptor gamma coactivator 1 alpha (PPARGC1A), peptidyl- transfer ribonucleic acid hydrolase 1 homolog (PTRH1) and tudor domain containing 7 (TDRD7). The model is an independent risk factor for clinicopathological characteristics. Conclusion: Our study provided novel insights into the pathogenesis of CRC and constructed a prognostic gene model, which may be helpful for determining the prognosis of CRC.

Compound amino acid combined with high-dose vitamin B6 attenuate traumatic coagulopathy via inhibiting inflammation by HMGB1/TLR4/NF-κB pathway.

BACKGROUND: Traumatic coagulopathy (TC) arises primarily from coagulation system failure to maintain adequate hemostasis after serious blood loss or trauma. Circulatory homeostasis restoration is the mainstay of the therapeutic approach to TC, but the effects are significantly inhibited by coagulopathy. OBJECTIVE: To identify the therapeutic effects and underlying mechanism of compound amino acid (CAA) combined with high-dosage of vitamin B6 (VB6) on TC. METHODS: Rabbit traumatic model and cellular model were used to evaluate the effect of CAA combined with high-dosage of VB6 in TC. Blood concentrations of AST and ALT were measured using the Vitros 250 device while blood APTT, PT and TT concentrations were measured using commercial diagnostics kits. Furthermore, qRT-PCR, ELISA and Western blotting were used to determine the expression of clotting factor (II, VII, IX, X and XI), inflammatory factors (TNF-α, IL-6 and IL-1ß) and HMGB1/TLR4/NF-κB signaling-related proteins, respectively. RESULTS: In the rabbit traumatic model, CAA combined with high-dosage of VB6 therapy inhibited the high expression of AST and ALT, but increased the expression of coagulation factors. Additionally, in both the rabbit trauma model and cellular injury model, CAA combined with high-dosage of VB6 inhibited the expression of inflammatory factors (IL-6, TNF-α and IL-1ß) and proteins (HMGB1, TLR4 and p-p65) in HMGB1/TLR4/NF-κB pathway. Most importantly, over-expression of HMGB1 reversed the effect of CAA and VB6 in HUVECs and EA.hy926 cells injury model. CONCLUSION: CAA combined with high-dosage of VB6 alleviated TC and inhibited the expression and secretion of inflammatory factors by inhibiting HMGB1-mediated TLR4/NF-κB pathway.

Baicalein suppresses the growth of the human thyroid cancer cells by inducing mitotic catastrophe, apoptosis and autophagy via NF-kB signalling pathway.

PURPOSE: The current study aimed at investigating the anticancer effects of plant-based flavone Baicalein against the thyroid cancer. METHODS: Cell viability was assessed using MTT assay. Flow cytometric-based estimation of cell cycle analysis was done for determining the cancer cell phase distribution. DAPI staining method followed by fluorescent microscope examination was used for inferring the cancer cell apoptosis. Transmission electron microscopy (TEM) was used for detection of autophagosomes. Western blotting was done for protein concentration estimation. RESULTS: Baicalein induced dose-dependent decline in proliferation of MDA-T68 thyroid cancer cells, while the reduction of cell proliferation was surprisingly lower for normal thyrocytes. IC50 of 10µM was estimated against cancer cells. Baicalein induced cell apoptosis in a concentration-dependent manner. Induction of apoptosis was attributed to increase in apoptotic protein concentration and signal was mediated through change Bax/Bcl-2 ratio. The autophagic cell death occurred in cancer cells when treated with Baicalein. The mechanism of cell death was inferred as modulation of NF-kB signaling pathway. Baicalein was also seen to induce mitotic cell cycle arrest in thyroid cancer cells by reducing the concentration of Cyclin B1 mitotic protein. CONCLUSION: The results of current study suggest Baicalein as an important anticancer agent against thyroid cancer. Future research to further investigate and enhance the effects of Baicalein against thyroid cancer is needed.

Pharmacological and non-pharmacological treatments for opioid-induced constipation: A protocol for systematic review and network meta-analysis.

BACKGROUND: Pain is very common and its management with a huge burden for patients and the healthcare system. And the network meta-analysis was designed to provide reference for the clinical practice. METHODS: PubMed, EMBASE, Cochrane library, CNKI, VIP, Wan Fang, and CBM will be systematically searched their inception to November 2018. Randomized controlled trials that compared the effect of differently pharmacological or non-pharmacological treatments for opioid-induced constipation will be included. The primary outcome is the efficacy of therapeutic regimens. Risk of bias assessment of the included studies will be performed using the Cochrane risk of bias tool. A network meta-analysis will be performed using STATA 13.0 software with WinBUGS 1.4.3 software. Grading of Recommendations Assessment, Development, and Evaluation will be used to assess the overall quality of evidence. RESULTS: This study is ongoing and will be submitted to a peer-reviewed journal for publication. CONCLUSION: This study will provide a comprehensive evidence on the effectiveness and safety of pharmacological and non-pharmacological treatments for opioid-induced constipation. PROSPERO REGISTRATION NUMBER: CRD42018116533.

Preventative effects of resveratrol and estradiol on streptozotocin-induced diabetes in ovariectomized mice and the related mechanisms.

Resveratrol, a non-flavonoid polyphenolic compound, is structurally and functionally similar to estrogen and has drawn great attention for its potentially beneficial effects on diabetes. However, it is not known whether it shares the same protective effect against diabetes as estrogen and the underlying mechanisms. The aim of the present study was to investigate the protective effects of phytoestrogen resveratrol and exogenous 17ß-estradiol against streptozotocin (STZ)-induced type 1 diabetes. Female mice were ovariectomized (OVX) and chronically injected with different concentrations of resveratrol (0.1, 1 or 10 mg/kg) and 17ß-estradiol (0.01, 0.1 or 1 mg/kg) subcutaneously for 4 weeks, and the levels of blood glucose, plasma insulin, plasma antioxidant capacity, the changes of pancreatic islet cells and the expressions of glucose transporter 4 (GLUT4), insulin receptor substrate 1 (IRS-1) and phosphorylation of extracellular signal-regulated kinase (p-ERK) were detected. Resveratrol and 17ß-estradiol significantly inhibited the increase of the blood glucose level and the rise of plasma malondialdehyde in STZ-induced diabetic mice, improved the levels of plasma antioxidant capacity and plasma insulin, protected the pancreatic islet cells, and increased the expressions of GLUT4 and IRS-1, but decreased p-ERK expression in skeletal muscle and myocardial tissue. The results suggest that resveratrol or 17ß-estradiol shows obvious protection against STZ-induced diabetes in OVX mice, the mechanisms probably involve their ameliorating antioxidant activities and islet function, promoting muscle glucose uptake and inhibiting the expression of p-ERK.

Evaluation of a modified rat model for functional dyspepsia.

Background/Aim: The purpose of this study was to establish a modified rat model with functional dyspepsia (FD) and analyze the changes in gastrointestinal motility and brain-gut peptide levels in serum and brain-gut axis. Materials and Methods: Male Wistar rats were divided into control group (Con) and FD model group. FD model was established by stimulating semi-starvation rats via tail damping, provocation, and forced exercise fatigue until gastrointestinal motility disorder appeared, and then levels of motilin, leptin, cholecystokinin (CCK), and vasoactive intestinal peptide (VIP) were detected in serum by enzyme linked immunosorbent assay and in duodenum, antrum, and hypothalamus by immunohistochemistry, reverse transcriptase-polymerase chain reaction, and Western blot. Results: The results showed rates of intestinal propulsion and gastric emptying slowed down markedly compared to Con (P < 0.05), the gastrointestinal electric activity attenuated, and migrating motor complex (MMC) interrupted in the model group. The levels of leptin and VIP markedly increased, but motilin decreased as compared to the Con (P < 0.05) in serum and in the above tissues. It is interesting that the level of CCK decreased in the antrum and duodenum but increased in the hypothalamus as compared to Con (P < 0.05). Conclusions: The modified rat model meets the diagnostic criteria of FD and can be used as a method for studying FD in animals.

Pingwei capsules improve gastrointestinal motility in rats with functional dyspepsia.

OBJECTIVE: To investigate the mechanism of Pingwei capsules (PWC) in improving gastrointestinal motility in rats with functional dyspepsia (FD). METHODS: We established an FD model by stimulating semi-starvation rats via tail damping, provocation, and forced exercise fatigue. The FD model group was further divided into five groups according to the treatment received: normal saline, domperidone, low-dose PWC, mid-dose PWC, or high- dose PWC. The effect of PWC on FD rat was evaluated by measuring gastrointestinal motility. Changes of leptin and cholecystokinin (CCK) were detected through enzyme-linked immunosorbent assay, reverse transcription-polymerase chain reaction, and immunohistochemistry. RESULTS: PWC significantly increased gastrointestinal motility in FD rats. Furthermore, PWC significantly increased CCK mRNA and protein concentrations in the duodenum and antrum, decreased leptin protein concentrations in the duodenum, antrum, and hypothalamus, and decreased CCK protein concentration in the hypothalamus. CONCLUSION: PWC improve gastrointestinal motor function in FD rats by decreasing the leptin concentration in serum and the brain-gut axis, and by increasing the CCK concentration in gastrointestinal tissue. Our findings help to elucidate the mechanism of FD and provide further insight into the pharmacokinetics of PWC.