Traditional Chinese medicine Zhusha Anshen Wan: protective effects on liver, kidney, and intestine of the individual drugs using 1H NMR metabolomics.

Introduction: Zhusha Anshen Wan (ZSASW) is a traditional Chinese medicine compound mainly composed of mineral drugs. In clinical practice, ZSASW did not show the toxicity of administering equal doses of cinnabar alone, suggesting that the four combination herbs in ZSASW can alleviate the damage of cinnabar. The effect of each herb on reducing the toxicity of cinnabar has not been fully explained. Methods: In our study, we utilized a metabonomics approach based on high-resolution 1H nuclear magnetic resonance spectroscopy to investigate the reduction of toxicity by each herb in ZSASW. Liver, kidney and intestinal histopathology examinations and biochemical analysis of the serum were also performed. Results: Partial least squares-discriminant analysis (PLS-DA) was conducted to distinct different metabolic profiles in the urine and serum from the rats. Liver and kidney histopathology examinations, as well as analysis of serum clinical chemistry analysis, were also carried out. The metabolic profiles of the urine and serum of the rats in the CGU (treated with cinnabar and Glycyrrhiza uralensis Fisch) and CCC (treated with cinnabar and Coptis chinensis French) groups were remarkably similar to those of the control group, while those of the CRG (treated with cinnabar and Rehmannia glutinosa Libosch) and CAS (treated with cinnabar and Angelica sinensis) groups were close to those of the cinnabar group. The metabolic profiles of the urine and serum of the rats in the CGU and CCC groups were remarkably similar to those of the control group, while those of the CRG and CAS groups were close to those of the cinnabar group. Changes in endogenous metabolites associated with toxicity were identified. Rehmannia glutinosa, Rhizoma Coptidis and Glycyrrhiza uralensis Fisch could maintain the dynamic balance of the intestinal flora. These results were also verified by liver, kidney and intestinal histopathology examinations and biochemical analysis of the serum. The results suggested that Discussion: The metabolic mechanism of single drug detoxification in compound prescriptions has been elucidated. Coptis chinensis and Glycyrrhiza uralensis serve as the primary detoxification agents within ZSASW for mitigating liver, kidney, and intestinal damage caused by cinnabar. Detoxification can be observed through changes in the levels of various endogenous metabolites and related metabolic pathways.

Adeno-Associated Virus-Mediated Knockdown of Agmatinase Attenuates Inflammation and Tumorigenesis in a Mouse Model of Colitis-Associated Colorectal Cancer.

Agmatinase (AGMAT) is an enzyme that hydrolyzes agmatine to putrescine and urea. In this study, we explored the functions of AGMAT in colorectal cancer (CRC). By performing gain-of-function and loss-of-function experiments, we investigated the roles of AGMAT in proliferation, cell cycle progression, and apoptosis of CRC cells. We also established a colitis-associated colorectal cancer model by challenging mice with azoxymethane (AOM) and dextran sodium sulfate (DSS), and we subsequently silenced AGMAT expression in mice by adeno-associated virus 9 (AAV9)-mediated delivery of short hairpin RNA (shRNA). In vitro experiments showed that overexpression of AGMAT accelerated the proliferation and inhibited the apoptosis of CRC cells, and AGMAT knockdown exhibited the opposite effects. Interestingly, the oncogenic transcription factor, c-Myc, could bind to the AGMAT promoter and transcriptionally increase AGMAT expression in CRC cells. Additionally, c-Myc and AGMAT were upregulated in the colon of AOM/DSS-treated mice, and AGMAT silencing significantly mitigated colitis in AOM/DSS-treated mice, as evidenced by the increased colon length, attenuated crypt damage, and reduced levels of inflammatory indicators (myeloperoxidase, interleukin-6, tumor necrosis factor-α, inducible nitric oxide synthase, and phosphorylated p65) in colon tissues. Notably, AGMAT silencing decreased both the number and size of tumors, reduced expression of proliferating cell nuclear antigen, and inhibited phosphorylation of protein kinase B (AKT) and extracellular signal-regulated kinase in the colon of AOM/DSS-treated mice. Overall, we determined that AGMAT facilitates tumor progression in CRC. Our findings will be helpful in the search for potential therapeutic targets for CRC.

Neuromedin U Induces Pulmonary ILC2 Activation via the NMUR1 Pathway during Acute Respiratory Syncytial Virus Infection.

Activated group 2 innate lymphoid cells (ILC2s) play a crucial role in respiratory syncytial virus (RSV)-induced airway inflammation and allergy-like symptoms because of their ability to secrete large quantities of type 2 cytokines. Cytokines such as IL-33, IL-25, and thymic stromal lymphopoietin are activators of ILC2s. Besides, a regulatory effect of neurotransmitters on ILC2 activation has been reported recently. However, whether and how RSV infection induces neurotransmitter production in the lungs and regulates pulmonary ILC2 activation remains unclear. In this study, using a murine model established by intranasal infection with RSV, we found that acute RSV infection induced the production of a neurotransmitter, neuromedin U (NMU), in the lungs of RSV-infected mice and upregulated the expression of NMUR1 (neuromedin U receptor 1) on ILC2s. Moreover, in vivo administration of NMU exacerbated RSV-induced airway inflammation by promoting the proliferation and activation of pulmonary ILC2s via the NMUR1 pathway, which involved PI3K, mitogen-activated protein kinase kinase, and NFAT signaling proteins. Furthermore, pulmonary neurons responded to the stimulation of RSV infection and secreted NMU in a Toll-like receptor 4- and Toll-like receptor 7-dependent manner. Collectively, our data suggest that NMU is a powerful neuropeptide to activate ILC2s, highlighting the critical regulatory effects of neurotransmitters on antiviral, inflammatory, and tissue homeostasis at the mucosal barrier during a viral respiratory infection.

Group 2 innate lymphoid cells mediate the activation of CD4+ T cells and aggravate Th1/Th2 imbalance via MHC II molecules during respiratory syncytial virus infection.

Respiratory syncytial virus (RSV) infection induces the activation of CD4+ T cells. However, the underlying mechanism of CD4+T-cell activation induced by RSV infection is not fully understood. In the present study, we found that depletion of CD4+ T cells can obviously reduce airway inflammation caused by RSV infection. Meanwhile, adoptive transfer of group 2 innate lymphocytes (ILC2s) significantly enhanced the number of CD4+ T cells and promoted their differentiation to Th2 in lung. In fact, RSV infection increased the expression of major histocompatibility complex-II (MHC II) molecules on the surface of pulmonary ILC2s. In vitro coculture experiments showed that ILC2s may act as promoters to promote the expansion and differentiation of RSV-infected CD4+ T cells. However, blocking the interaction between CD4+ T cells and ILC2s with anti-MHC-II mAbs significantly reduced CD4+T-cell expansion. These results suggest that pulmonary ILC2s may function as antigen-presenting cells to induce the activation of CD4+ T cells through the MHC II pathway during RSV infection.

Long Non-Coding RNA CCAT2 Activates RAB14 and Acts as an Oncogene in Colorectal Cancer.

Here, we investigated the clinicopathological and prognostic potential of the long noncoding RNA Colon Cancer-Associated Transcript 2 (CCAT2) in human colorectal cancer (CRC). We used qPCR to quantify CCAT2 levels in 44 pairs of CRC tissues and adjacent nontumor and healthy colon mucosa tissues, and in several CRC cell lines (SW620, SW480, HT-29, LOVO, HCT116 and DLD-1) and normal human colorectal epithelial cells (HFC). We assessed the effects of CCAT2 overexpression or knockdown on the proliferation, migration and invasion by SW620 and LOVO cells using CCK-8, transwell, and wound-healing assays, respectively. We also investigated the potential interaction between CCAT2 and TAF15 through RNA pull down and rescue experiments. Lastly, we evaluated the expression of the cell cycle progression markers and GSK3ß signaling pathway proteins using Western blotting. Our results showed that CCAT2 was upregulated in CRC tissues and cell lines as com-pared to controls. Ectopic expression of CCAT2 promoted CRC cell proliferation, migration and invasion, likely through direct interaction with TAF15, transcriptional activation of RAB14, and activation of the AKT/GSK3ß signaling pathway. In vivo, CCAT2 promoted CRC cell growth and metastasis in nude mice. Taken together, these results highlight the actions of CCAT2 as a CRC oncogene.

Oral administration of Clostridium butyricum rescues streptomycin-exacerbated respiratory syncytial virus-induced lung inflammation in mice.

Changes in the intestinal microbiota indirectly impact the health of mucosa distal to the intestine, particularly the respiratory tract. However, the effects of intestinal microbiota dysbiosis on the regulation of respiratory syncytial virus (RSV) infection are not clear. In this study, we examined the effects of altering the intestinal microbiota on the pulmonary immune response against RSV infection. BALB/c mice were treated with streptomycin before infection with RSV to study the altered immune response. The ingestion of streptomycin led to a marked alteration in the intestinal microbiota with a reduced abundance of Lactobacillus and Clostridium genera, followed by greatly aggravated pulmonary inflammation in response to RSV infection. This aggravated inflammation was associated with a dysregulated immune response against RSV infection, characterized by the increased expression of IFN-γ and IL-17 and increased pulmonary M1-like macrophage polarization, and decreased expression of IL-5. Supplementation of Clostridium butyricum (CB) prevented aggravated inflammation and the dysregulated immune response characterized by greater M2 polarization of pulmonary macrophages and decreased release of IFN-γ and IL-17 as well as increased IL-5 levels. Furthermore, in vitro and in vivo experiments identified that butyrate, the main metabolite produced by CB, promoted M2 polarization of macrophages in RSV-infected mice exposed to streptomycin. Together, these results demonstrate the mechanism by which intestinal microbiota modulate the pulmonary immune response to RSV infection.

A seven immune-related lncRNA signature predicts the survival of patients with colon adenocarcinoma.

This study aimed to explore immune-related lncRNAs for predicting the overall survival of patients with colon adenocarcinoma. RNA-sequencing data were downloaded from the TCGA data portal. The immune-related lncRNAs with differential expression were identified with Cox and LASSO regression analysis. With the stepwise regression analysis, a seven lncRNA signature was established for calculating the Risk Score with following formula: Risk Score = [Expression level of AC027307.2 * (0.156)] + [Expression level of AC074117.1 * (0.294)] + [Expression level of AC103702.2 * (-0.025)] + [Expression level of CYTOR * (0.205)] + [Expression level of LINC02381 * (0.251)] + [Expression level of MIR200CHG * (0.052)] + [Expression level of SNHG16 * (-0.101)]. The Risk Score was validated with survival analysis, achieving moderate area under the curve (AUC) of receiver operating characteristic (ROC) curve over 0.7. GSEA and immune-cell abundance analysis further supported the involved lncRNAs were immune-relevant. Finally, the prognosis prediction efficacy was verified with clinical samples with an AUC of 0.674 in ROC curve. Both the Risk Score and involved immune-related lncRNAs presented promising clinical significance.

Estimation of type i collagen structure dissolved in inorganical acids from circular dichroism spectra / Estimativa da estrutura de colágeno tipo i dissolvida em ácidos inorgânicos a partir de espectros de dicroísmo circular

Usually, weak inorganic acids have been used to disperse collagen as green solvents for fabricating kinds of biomaterials all the time. However, it is an open question how much the dissolving process preserves or alters the native structure of collagen till now. Herein, we have examined the effect of three different solvents (HAc, HCl, H3PO4) on the secondary structures of collagen, based on circular dichroism (CD) spectra of collagen from 185 to 260 nm together with CDNN programs. We have found that collagen almost completely preserved its triple helical structure in the three inorganic acids at pH=3.0 or so, which demonstrated that it was the concentration of free H+ in the above three solutions whose pH was fixed at 3.0 that can maintain an proper amount of surface charge on the collagen colloidal particles and appropriately loose the three-helix structure, which can not only lead to a better dispersion behavior, but also maximize the preservation of the integrity of the collagen structure. Although the fractions of kinds of secondary structures in collagen were different from all the three solvents based on CDNN data, which gave very similar results for each other. These results was tested for the first time in this work to estimate the secondary structures for collagen in the different common inorganic acids, which provides a new avenue for green collagen solvents to prepare collagen-based composite with well triple-helical structure for tissue engineering.

Habitualmente, os ácidos inorgânicos fracos têm sido usados para dispersar colágeno como solventes verdes para fabricar tipos de biomateriais o tempo todo. No entanto, é uma questão aberta quanto o processo de dissolução preserva ou altera a estrutura nativa do colágeno até agora. Aqui, examinamos o efeito de três solventes diferentes (HAc, HCl, H3PO4) nas estruturas secundárias de colágeno, com base em espectros de dicroísmo circular (CD) de colágeno de 185 a 260 nm em conjunto com programas CDNN. Descobrimos que o colágeno preservou quase completamente sua estrutura helicoidal tripla nos três ácidos inorgânicos a pH = 3,0 ou mais, o que demonstrou que foi a concentração de H+ livre nas três soluções acima cujo pH foi fixado em 3,0 que pode manter uma boa quantidade de carga superficial sobre as partículas coloidais de colágeno e destrói adequadamente a estrutura de três hélices, o que não só pode levar a um melhor comportamento de dispersão, mas também maximizar a preservação da integridade da estrutura de colágeno. Embora as frações de tipos de estruturas secundárias em colágeno fossem diferentes de todos os três solventes com base em dados CDNN, que deram resultados muito semelhantes entre si. Estes resultados foram testados pela primeira vez neste trabalho para estimar as estruturas secundárias para o colágeno nos diferentes ácidos inorgânicos comuns, o que fornece uma nova alternativa para solventes de colágeno verdes para preparar compósitos à base de colágeno com a estrutura helicoidal tripla para engenharia de tecidos.

Naringenin induces laxative effects by upregulating the expression levels of c-Kit and SCF, as well as those of aquaporin 3 in mice with loperamide-induced constipation.

Constipation is a common affliction which causes discomfort and affects the quality of life of affected individuals. Naringenin (NAR), a natural flavonoid widely found in citrus fruits and tomatoes, has been reported to exhibit various pharmacological effects, such as anti-inflammatory, anti-atherogenic, anti-mutagenic, hepatoprotective and anticancer effects. Increasing evidence has indicated that NAR has potential for use in the treatment of constipation. Thus, the aim of this study was to evaluate the laxative effects of NAR in mice with loperamide-induced (Lop-induced) constipation. The data indicated that NAR relieved Lop-induced constipation in mice based on the changes of fecal parameters (numbers, weight and water content), the intestinal charcoal transit ratio and the histological alteration. ELISA revealed that NAR regulated the production levels of gastrointestinal metabolic components, such as motilin (MTL), gastrin (Gas), endothelin (ET), substance P (SP), acetylcholinesterase (AChE) and vasoactive intestinal peptide (VIP) in serum. The expression levels of enteric nerve-related factors, glial cell line-derived neurotrophic factor (GDNF), transient receptor potential vanilloid 1 (TRPV1), nitric oxide synthase (NOS), c-Kit, stem cell factor (SCF) and aquaporin 3 (AQP3) were examined by western blot analysis and RT-PCR analysis. The results of this study suggest that NAR relieves Lop-induced constipation by increasing the levels of interstitial cells of Cajal markers (c-Kit and SCF), as well as AQP3. Thus, NAR may be effective as a candidate in patients suffering from lifestyle-induced constipation.

The function of BTG3 in colorectal cancer cells and its possible signaling pathway.

PURPOSE: B-cell translocation gene 3 (BTG3) has been identified as a candidate driver gene for various cancers, but its specific role in colorectal cancer (CRC) is poorly understood. We aimed to investigate the relationship between expression of BTG3 and clinicopathological features and prognosis, as well as to explore the effects and the role of a possible BTG3 molecular mechanism on aggressive colorectal cancer behavior. METHODS: BTG3 expression was assessed by immunohistochemistry (IHC) on specimens from 140 patients with CRC. The association of BTG3 expression with clinicopathological features was examined. To confirm the biological role of BTG3 in CRC, two CRC cell lines expressing BTG3 were used and BTG3 expression was knocked down by shRNA. CCK-8, cell cycle, apoptosis, migration, and invasion assays were performed. The influence of BTG3 knockdown was further investigated by genomic microarray to uncover the potential molecular mechanisms underlying BTG3-mediated CRC development and progression. RESULTS: BTG3 was downregulated in colorectal cancer tissues and positively correlated with pathological classification (p = 0.037), depth of invasion (p = 0.016), distant metastasis (p = 0.024), TNM stage (p = 0.007), and overall survival (OS) and disease-free survival (DFS). BTG3 knockdown promoted cell proliferation, migration, invasion, relieved G2 arrest, and inhibited apoptosis in HCT116 and LoVo cells. A genomic microarray analysis showed that numerous tumor-associated signaling pathways and oncogenes were altered by BTG3 knockdown. At the mRNA level, nine genes referred to the extracellular-regulated kinase/mitogen-activated protein kinase pathway were differentially expressed. Western blotting revealed that BTG3 knockdown upregulated PAK2, RPS6KA5, YWHAB, and signal transducer and activator of transcription (STAT)3 protein levels, but downregulated RAP1A, DUSP6, and STAT1 protein expression, which was consistent with the genomic microarray data. CONCLUSIONS: BTG3 expression might contribute to CRC carcinogenesis. BTG3 knockdown might strengthen the aggressive colorectal cancer behavior.

Overexpression of Livin promotes migration and invasion of colorectal cancer cells by induction of epithelial-mesenchymal transition via NF-κB activation.

Livin is a novel member of the inhibitors of apoptosis protein family and has been implicated in the development and progression of colorectal cancer (CRC). However, the underlying mechanisms of Livin in CRC remain not fully understood. In this study, we investigated the effects of Livin expression on the proliferation and metastasis of CRC cells and also addressed its related molecular mechanism to metastasis. The expression of Livin in CRC cells (HCT116, SW480, and HT-29 cell lines) was determined by Western blot analysis. Our results show that the overexpression of Livin significantly promotes the proliferation, migration, and invasion of SW480 cells. Concurrently, the inhibition of Livin reduces the proliferation, migration, and invasion of HCT116 cells. In addition, Livin overexpression promotes the epithelial-mesenchymal transition, as evidenced by a decrease in epithelial E-cadherin expression and an increase in mesenchymal markers, including vimentin, Slug, and Snail. Furthermore, adding the NF-κB inhibitor, BAY 11-7028, or transfecting with small interfering RNA against p65 notably restores the expression level of E-cadherin and attenuates the invasive ability of Livin-overexpressing cells. Taken together, these results indicate that Livin potentiates migration and invasion of CRC cells partially through the induction of epithelial-mesenchymal transition via NF-κB activation. Livin may be a potential therapeutic target for CRC.

Greener synthesis of electrospun collagen/hydroxyapatite composite fibers with an excellent microstructure for bone tissue engineering.

In bone tissue engineering, collagen/hydroxyapatite (HAP) fibrous composite obtained via electrospinning method has been demonstrated to support the cells' adhesion and bone regeneration. However, electrospinning of natural collagen often requires the use of cytotoxic organic solvents, and the HAP crystals were usually aggregated and randomly distributed within a fibrous matrix of collagen, limiting their clinical potential. Here, an effective and greener method for the preparation of collagen/HAP composite fibers was developed for the first time, and this green product not only had 40 times higher mechanical properties than that previously reported, but also had an excellent microstructure similar to that of natural bone. By dissolving type I collagen in environmentally friendly phosphate buffered saline/ethanol solution instead of the frequently-used cytotoxic organic solvents, followed with the key step of desalination, co-electrospinning the collagen solution with the HAP sol, generates a collagen/HAP composite with a uniform and continuous fibrous morphology. Interestingly, the nano-HAP needles were found to preferentially orient along the longitudinal direction of the collagen fibers, which mimicked the nanostructure of natural bones. Based on the characterization of the related products, the formation mechanism for this novel phenomenon was proposed. After cross-linking with 1-ethyl-3-(3-dimethyl-aminopropyl)-1-carbodiimide hydrochloride/N-hydroxysuccinimide, the obtained composite exhibited a significant enhancement in mechanical properties. In addition, the biocompatibility of the obtained composite fibers was evaluated by in vitro culture of the human myeloma cells (U2-OS). Taken together, the process outlined herein provides an effective, non-toxic approach for the fabrication of collagen/HAP composite nanofibers that could be good candidates for bone tissue engineering.