Glyuranolide-Dependent Inhibition of NF-κB/Akt Pathways by Sini Decoction Enhances Caspase-3-Mediated Apoptosis in Colorectal Cancer

Sini Decoction (SND), as a classic prescription of Traditional Chinese Medicine (TCM), has been proved to be clinically useful in cardiomyopathy and inflammatory bowel diseases. However, the role and mechanism of SND in colitis-associated cancer remains unclear. This study aims to evaluate the effect of SND on colorectal cancer(CRC) symptoms and further explore the changes of gut microbes mediated by SND extract in azoxymethane (AOM)/dextran sulfate sodium (DSS)-induced CRC mice through 16 S rRNA sequencing. Our results indicated that treatment with SND extract could ameliorate the tumors' malignant degree by decreasing tumor number and size. Also, the expression levels of Cyclooxygenase 2 and Mucin-2, which are typical CRC biomarkers, were reduced compared to the CRC group. In the meantime, SND extract can upregulate CD8+ T lymphocytes' expression and Occludin in the colonic mucosal layer. Besides, SND inhibited the expression of CD4+ T cells and inflammatory cytokines in CRC tissue. According to bioinformatics analysis, SND extract was also suggested could modulate the gut microbial community. After the SND treatment, compared with the CRC mice model, the number of pathogenic bacteria showed a significant reduction, including Bacteroides fragilis and Sulphate-reducing bacteria; and SND increased the relative contents of the beneficial bacteria, including Lactobacillus, Bacillus coagulans, Akkermansia muciniphila, and Bifidobacterium. In summary, SND can effectively intervene in colorectal cancer development by regulating intestinal immunity, protecting the colonic mucosal barrier, and SND can change the intestinal microbiota composition in mice.

Sini decoction (SND) is a widely used Traditional Chinese Medicine (TCM). The reports of SND application in colorectal cancer (CRC) is limited. The objective of this study is to investigate the anti-tumor activity of SND in the treatmeant of CRC. SND was analyzed using high-performance liquid chromatography. A CRC metastasis model was established using murine CT-26 cells. Whole-body fluorescence imaging was used to observe CRC liver metastasis. Liver morphology was determined using hematoxylin-eosin staining. Cytokine mRNA expression (interleukin-2 (IL-2), interleukin-10 (IL-10), interferon-gamma (IFN-γ), and tumor necrosis factor beta (TNF-β)) were determined using real-time reverse transcription polymerase chain reaction. Spectral flow cytometry was used to detect mouse tumor immune subgroups. Databases were used to find potential target genes of SND. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes were used to identify potential signaling pathways of target genes. SND suppressed CRC liver metastasis and alleviated liver injury in vivo. After SND treatment, IL-2 and IFN-γ were upregulated, whereas IL-10 and TGF-β were downregulated. Moreover, CD3+, CD8+T cells, natural killer T cells, and macrophages increased significantly after SND treatment, while CD4+CD25+T cells decreased significantly. Importantly, increasing the aconite concentration had a better anti-tumor effect. Fifty-50 compounds in SND were screened, and 611 potential target genes were identified. Functional analyses showed that the genes were associated with the PI3K-Akt signaling pathway, EGFR tyrosine kinase inhibitor resistance, and HIF-1 signaling pathway. SND exerts anti-tumor activity by inhibiting tumor progression and enhancing antitumor immunity in mice, suggesting its application to prevent and treat CRC.

Sepsis, as life-threatening organ dysfunction caused by a dysregulated host response to infection, is characterized by the extensive release of cytokines and other mediators. Sini decoction (SND), a traditional Chinese prescription medicine, has been used clinically for the treatment of sepsis. But its explicit mechanism of action is still unclear.The present study aims to evaluate the potential protective effects of SND on sepsis-induced acute lung injury (ALI). After SND intervention, the lung tissues of each experimental group were collected. H&E sections were used to observe the pathological changes of lung tissue, and alveolar lavage fluid was collected to detect the infiltration of inflammatory cells. Level of inflammatory factors in lung tissue were analyzed by qRT-PCR. The change of Renin angiotensin system (RAS), as well as downstream MAPK/NF-κB signaling pathways were measured by Western blot. For in vitro experiments, human umbilical vein endothelial cells (HUVECs) were pretreated with lipopolysaccharide (LPS) and treated with SND. Subsequently, the expression levels of RAS and MAPK/NF-κB signaling pathways were measured by Western blot.In vivo, we found that SND significantly attenuated sepsis-induced pathological injury in the lung. SND also inhibited LPS-mediated inflammatory cell infiltration, the expression of pro-apoptotic proteins and the production of IL-6, IL-1β, TNF-α and MCP-1. In vitro, experiments using a co-culture of HUVECs with SND showed that there was a decrease in pro-apoptotic protein and pro-inflammatory mediator. In this research, we also found that SND protective action could be attributed to the regulation of renin-angiotensin system (RAS). MAPKs and NF-κB pathways.To conclude, our study demonstrated that SND ameliorates sepsis-induced-ALI via regulating ACE2-Ang (1-7)-Mas axis and inhibiting the MAPK signaling pathway.

The cell adhesion protein cadherin 19 (CDH19) has been reported to be involved in various types of cancer, but its role in cervical carcinoma remains unknown. We collected and analyzed the patients' data using the GEPIA Kaplan-Meier plotter databases. CDH19 was overexpressed in cervical carcinoma cells to assess its effect on cell proliferation and activation of AKT and NF-κB signaling pathways. A xenograft mouse model was established to study the function of CDH19 in vivo. We found that CDH19 expression was significantly downregulated in cervical carcinoma tissues compared to adjacent normal tissues. Patients with high expression of CDH19 had a significantly better overall survival rate than those with low CDH19 expression. CDH19 expression was negatively correlated with the expression of the proliferation marker Ki-67, and overexpression of CDH19 significantly inhibited cervical carcinoma cell proliferation. Furthermore, overexpression of CDH19 suppressed the activation of the AKT and NF-κB signaling pathways, and CDH19-overexpressing cervical carcinoma tumors exhibited significantly slower growth in vivo. CDH19 plays an important role in cervical carcinoma by suppressing both cell proliferation and the activation of AKT and NF-κB signaling pathways. Therefore, CDH19 may be a potential therapeutic target for cervical carcinoma.

Sini decoction alleviates E. coli induced acute lung injury in mice via equilibrating ACE-AngII-AT1R and ACE2-Ang-(1-7)-Mas axis

The paper aimed to investigate the effect of Pinoresinol diglucoside on the differentiation and bone resorption of osteoclast RAW264.7, and to evaluate the effect of Pinoresinol diglucoside on osteoclasts. In order to study the effect of Pinoresinol diglucoside on osteoclast differentiation, RANKL was used to induce RAW264.7 cells to differentiate into osteoclasts, and different concentrations of Pinoresinol diglucoside was added to intervene. CCK-8 method was applied to detect cell viability. TRAP staining was employed to observe cell morphology. Annexin V/PI flow cytometry was used to detect cell apoptosis. Phalloidin was used to detect F-actin formation of osteoclasts and to observe the effect on bone resorption. WB was employed to detect the effects of differentiation-related proteins and RANKL/RANK signaling pathways, and immunofluorescence detection technology was used to measure the distribution and nuclear translocation of p65. Pinoresinol diglucoside can effectively inhibit RANKL-induced osteoclast differentiation, especially in the early stage. The drug can inhibit the formation of F-actin of osteoclasts and inhibit bone resorption. Through inhibiting the ubiquitination and degradation of the homologous phosphatase tensin (PTEN), the drug up-regulated the viability of PTEN. The up-regulated PTEN viability then inhibited the NF-κB and AKT signaling pathways, resulting in a decrease in the expressions of nuclear factor c1 (NFATc1) in activated T cells. Pinoresinol diglucoside effectively inhibited the formation of F-actin and bone resorption in mature osteoclasts. The mechanism is through inhibiting the expression levels of osteoclast-related proteins NFATc1, c-Fos, CSTK and TRAP and RNAKL/RANK signaling pathways, and also inhibiting the activation of NF-κB and AKT signaling pathways.

The influence of compatibility of Si-Ni decoction with metabolism in intestinal bacteria on transports of toxic diterpenoid alkaloids from processed aconite root across Caco-2 monolayers

Sini decoction (SND), a classical traditional Chinese medicine emergency formula recorded in Shanghan Lun (Treatise on Febrile Diseases), which is composed of Aconiti Lateralis Preparata Radix, Glycyrrhizae Preparata Radix and Zingiberis Rhizoma. Modern clinical and pharmacological researches have shown that SND can protect the myocardium effectively during myocardial ischemia reperfusion injury (MI-RI). A myocardial ischemia reperfusion injury model of H9c2 cardiomyocytes in vitro has been established. Four groups, control group, MI-RI Model group, SND group and SND without Glycyrrhizae Radix group, were arranged. The livability, the level of LDH and CK activity in H9c2 cardiomyocytes in different groups were tested. By combining with principal components analysis (PCA), partial least squares discriminant analysis (PLS-DA), orthogonal partial least squares projection of latent structures-discriminant analysis (OPLS-DA), 17 biomarkers in extracellular fluid were identified and 15 of them were related to the pathway of biological processes. The results showed that the attenuate and synergistic mechanism between Aconiti Lateralis Praeparata Radix and Glycyrrhizae Radix for toxicity reduction was related with the glycolysis, lipid metabolism, citrate cycle and nitrogen metabolism of amino acids metabolism. The study proved the effect on H9c2 cardiomyocyte treated by MI-RI injury both SND group and SND without Glycyrrhizae Radix group, and compared with the SND without Glycyrrhizae Radix, the protective effect of myocardial ischemia reperfusion injury model of H9c2 cardiomyocyte from SND was stronger.

Cryopreservation is the primary technique for in vitro preservation of allogeneic tissue. However, its success is often hindered by factors such as low temperature, ischemia, and hypoxia. This study investigated the potential of Sini Decoction, known for its antioxidant and anti-apoptotic properties, to reduce cryopreservation-induced injury in rats' sciatic nerves. Sini Decoction was prepared according to the Chinese Pharmacopoeia, and its cytotoxicity on Rsc96 cells was assessed by using the CCK-8 method. Sini Decoction at concentrations of 4, 8, and 16 mg·mL~(-1), termed as low-(SL), medium-(SM), and high-(SH) doses group, was used for cryopreservation of rats' sciatic nerves. A normal control(NC) group and a fresh nerve control(fresh) group were set. Flow cytometry and TUNEL staining were used to detect the apoptosis of neural tissue cells after cryopreservation. Western blot was used to detect the expression of apoptosis-related proteins(Bcl-2, Bax, caspase-3, and caspase-8) and nerve regeneration proteins(NGF and BDNF) in vitro after cryopreservation. Oxidative damage of neural tissue after cryopreservation was evaluated by measuring levels of GSH, SOD, MDA, ROS, and ATP. Cryopreserved nerves were then used for allogeneic transplantation. One week after transplantation, CD4~+ and CD8~+ fluorescent double staining assessed inflammatory cell invasion in the transplanted nerve segment, and ELISA evaluated the expression of serum inflammatory factors(IL-1, IFN-γ, and TNF-α) in recipients. Twenty weeks after transplantation, electrophysiology and NF200 neurofilament staining were used to evaluate nerve regeneration. RESULTS:: showed that Sini Decoction at concentrations of below 32 mg·mL~(-1) exhibited no cytotoxicity to Rsc96 cells. During in vitro nerve cryopreservation, Sini Decoction significantly reduced cell apoptosis, ROS, and MDA production compared to the NC group. In the SH group, the protein expression of NGF and BDNF in vitro, as well as ATP, SOD, and GSH production, were significantly increased. In the rejection reaction one week after transplantation, compared to the fresh nerve transplantation group, the SL and SM groups showed reduced CD4~+ and CD8~+ T cell invasion in the transplanted nerve segment and down-regulated IL-1, IFN-γ, and TNF-α expression in recipient serum. Twenty weeks after transplantation, the electrophysiological test results of CMAP, NCV, and NF200 neurofilament protein fluorescent staining in the SM and SH groups were superior to those in the NC and fresh groups. These findings indicate that Sini Decoction offers protective benefits in the cryopreservation of rats' sciatic nerves and holds significant potential for the in vitro preservation of tissue and organs.

<div>Abstract<p>We have reported that a novel isoform of BTK (BTK-C) expressed in breast cancer protects these cells from apoptosis. In this study, we show that recently developed inhibitors of BTK, such as ibrutinib (PCI-32765), AVL-292, and CGI-1746, reduce breast cancer cell survival and prevent drug-resistant clones from arising. Ibrutinib treatment impacts HER2<sup>+</sup> breast cancer cell viability at lower concentrations than the established breast cancer therapeutic lapatinib. In addition to inhibiting BTK, ibrutinib, but not AVL-292 and CGI-1746, efficiently blocks the activation of EGFR, HER2, ErbB3, and ErbB4. Consequently, the activation of AKT and ERK signaling pathways are also blocked leading to a G<sub>1</sub>–S cell-cycle delay and increased apoptosis. Importantly, inhibition of BTK prevents activation of the AKT signaling pathway by NRG or EGF that has been shown to promote growth factor–driven lapatinib resistance in HER2<sup>+</sup> breast cancer cells. HER2<sup>+</sup> breast cancer cell proliferation is blocked by ibrutinib even in the presence of these factors. AVL-292, which has no effect on EGFR family activation, prevents NRG- and EGF-dependent growth factor–driven resistance to lapatinib in HER2<sup>+</sup> breast cancer cells. <i>In vivo</i>, ibrutinib inhibits HER2<sup>+</sup> xenograft tumor growth. Consistent with this, immunofluorescence analysis of xenograft tumors shows that ibrutinib reduces the phosphorylation of HER2, BTK, Akt, and Erk and histone H3 and increases cleaved caspase-3 signals. As BTK-C and HER2 are often coexpressed in human breast cancers, these observations indicate that BTK-C is a potential therapeutic target and that ibrutinib could be an effective drug especially for HER2<sup>+</sup> breast cancer. <i>Mol Cancer Ther; 15(9); 2198–208. ©2016 AACR</i>.</p></div>

<div>Abstract<p>We have reported that a novel isoform of BTK (BTK-C) expressed in breast cancer protects these cells from apoptosis. In this study, we show that recently developed inhibitors of BTK, such as ibrutinib (PCI-32765), AVL-292, and CGI-1746, reduce breast cancer cell survival and prevent drug-resistant clones from arising. Ibrutinib treatment impacts HER2<sup>+</sup> breast cancer cell viability at lower concentrations than the established breast cancer therapeutic lapatinib. In addition to inhibiting BTK, ibrutinib, but not AVL-292 and CGI-1746, efficiently blocks the activation of EGFR, HER2, ErbB3, and ErbB4. Consequently, the activation of AKT and ERK signaling pathways are also blocked leading to a G<sub>1</sub>–S cell-cycle delay and increased apoptosis. Importantly, inhibition of BTK prevents activation of the AKT signaling pathway by NRG or EGF that has been shown to promote growth factor–driven lapatinib resistance in HER2<sup>+</sup> breast cancer cells. HER2<sup>+</sup> breast cancer cell proliferation is blocked by ibrutinib even in the presence of these factors. AVL-292, which has no effect on EGFR family activation, prevents NRG- and EGF-dependent growth factor–driven resistance to lapatinib in HER2<sup>+</sup> breast cancer cells. <i>In vivo</i>, ibrutinib inhibits HER2<sup>+</sup> xenograft tumor growth. Consistent with this, immunofluorescence analysis of xenograft tumors shows that ibrutinib reduces the phosphorylation of HER2, BTK, Akt, and Erk and histone H3 and increases cleaved caspase-3 signals. As BTK-C and HER2 are often coexpressed in human breast cancers, these observations indicate that BTK-C is a potential therapeutic target and that ibrutinib could be an effective drug especially for HER2<sup>+</sup> breast cancer. <i>Mol Cancer Ther; 15(9); 2198–208. ©2016 AACR</i>.</p></div>

We have reported that a novel isoform of BTK (BTK-C) expressed in breast cancer protects these cells from apoptosis. In this study, we show that recently developed inhibitors of BTK, such as ibrutinib (PCI-32765), AVL-292, and CGI-1746, reduce breast cancer cell survival and prevent drug-resistant clones from arising. Ibrutinib treatment impacts HER2(+) breast cancer cell viability at lower concentrations than the established breast cancer therapeutic lapatinib. In addition to inhibiting BTK, ibrutinib, but not AVL-292 and CGI-1746, efficiently blocks the activation of EGFR, HER2, ErbB3, and ErbB4. Consequently, the activation of AKT and ERK signaling pathways are also blocked leading to a G1-S cell-cycle delay and increased apoptosis. Importantly, inhibition of BTK prevents activation of the AKT signaling pathway by NRG or EGF that has been shown to promote growth factor-driven lapatinib resistance in HER2(+) breast cancer cells. HER2(+) breast cancer cell proliferation is blocked by ibrutinib even in the presence of these factors. AVL-292, which has no effect on EGFR family activation, prevents NRG- and EGF-dependent growth factor-driven resistance to lapatinib in HER2(+) breast cancer cells. In vivo, ibrutinib inhibits HER2(+) xenograft tumor growth. Consistent with this, immunofluorescence analysis of xenograft tumors shows that ibrutinib reduces the phosphorylation of HER2, BTK, Akt, and Erk and histone H3 and increases cleaved caspase-3 signals. As BTK-C and HER2 are often coexpressed in human breast cancers, these observations indicate that BTK-C is a potential therapeutic target and that ibrutinib could be an effective drug especially for HER2(+) breast cancer. Mol Cancer Ther; 15(9); 2198-208. ©2016 AACR.

Homocysteine enhances MMP-9 production in murine macrophages via ERK and Akt signaling pathways

Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by complex symptoms. To treat AD without adverse effects, alternative therapeutic agents are required. The tubers of Helianthus tuberosus L. (Jerusalem artichoke) have been used in folk remedies for diabetes and rheumatism. However, its effect on AD development remains unknown. Therefore, this study examined the inhibitory effect of H. tuberosus (HT) on AD skin symptoms using an NC/Nga mouse model and HaCaT keratinocytes. The effect of HT and associated molecular mechanisms were evaluated in Dermatophagoides farina body (Dfb)-induced AD mice and tumor necrosis factor (TNF)-α/interferon (IFN)-γ-stimulated HaCaT keratinocytes by ELISA, western blot, and histological analysis. Topical HT administration attenuated AD skin symptoms in Dfb-induced AD mice, with a significant reduction in the dermatitis score and production of inflammatory mediators. HT also decreased epidermal thickness and mast cell infiltration. Moreover, HT restored filaggrin expression and inhibited adhesion molecules in the mice. These effects were confirmed in vitro. Furthermore, HT suppressed the activation of NF-κB, Akt, and mitogen-activated protein kinase (MAPK) signaling pathways induced by TNF-α/IFN-γ. These results suggest that HT is a potential therapeutic agent or supplement for skin allergic inflammatory diseases such as AD.

Vibrio harveyi infections induce production of proinflammatory cytokines in murine peritoneal macrophages via activation of p38 MAPK and NF-κB pathways, but reversed by PI3K/AKT pathways