Investigation of the mechanism of Prunella vulgaris in treatment of papillary thyroid carcinoma based on network pharmacology integrated molecular docking and experimental verification.

To analyze the molecular mechanism of Prunella vulgaris L. (PV) in the treatment of papillary thyroid carcinoma (PTC) by using network pharmacology combined with molecular docking verification. Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform database was used to predict the main active components of PV, Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform, PubChem, and Swiss Target Prediction databases were used to obtain the corresponding targets of all active components. Targets collected for PTC treatment through Gene Cards, Digest and Online Mendelian Inheritance in Man databases respectively. The Search Tool for the Retrieval of Interaction Gene/Protein database was used to obtain the interaction information between proteins, and the topology analysis and visualization were carried out through Cytoscape 3.7.2 software (https://cytoscape.org/). The R package cluster profiler was used for gene ontology and Kyoto encyclopedia of genes and genomes analysis. The "active ingredient-target-disease" network was constructed by using Cyto scape 3.7.2, and topological analysis was carried out to obtain the core compound. The molecular docking was processed by using Discovery Studio 2019 software, and the core target and active ingredient were verified. The inhibition rate was detected by CCK8 method. Western blot was used to detect the expression levels of kaempferol anti-PTC related pathway proteins. A total of 11 components and 83 corresponding targets in the component target network of PV, of which 6 were the core targets of PV in the treatment of PTC. It was showed that quercetin, luteolin, beta (ß)-sitosterol, kaempferol may be the core components of PV in the treatment of PTC. vascular endothelial growth factor A, tumor protein p53, transcription factor AP-1, prostaglandin endoperoxidase 2, interleukin 6, and IL-1B may be important targets for the treatment of PTC. The main biological processes mainly including response to nutrient levels, response to xenobiotic stimulus, response to extracellular stimulus, external side of plasma membrane, membrane raft, membrane microdomain, serine hydrolase activity, serine-type endopeptidase activity, antioxidant activity, etc IL-17 signaling pathway, and PI3K-Akt signaling pathway may affect the recurrence and metastasis of PTC. Kaempferol may significantly reduce the activity of Papillary cells of human thyroid carcinoma bcpap cell lines cells compared with quercetin, luteolin, ß-sitosterol. Kaempferol may reduce the protein expression levels of interleukin 6, vascular endothelial growth factor A, transcription factor AP-1, tumor protein p53, 1L-1B and prostaglandin endoperoxidase 2, respectively. PV has the characteristics of multi-components, multi-targets and multi- pathways in the treatment of PTC, which network pharmacology help to provides a theoretical basis for the screening of effective components of PV and further research.

Protective effect and mechanism of ginsenoside Rg1 on carbon tetrachloride­induced acute liver injury.

Liver injury is a common pathological state in various types of liver disease; severe or persistent liver damage is the basis of hepatic failure. Ginsenoside Rg1 (Rg1), one of the primary active ingredients of ginseng, has been reported to reduce concanalin A­induced hepatitis and protect against lipopolysaccharide­ and galactosamine­induced liver injury. However, the underlying protective mechanism of Rg1 in acute liver injury remains unclear. In the present study, a carbon tetrachloride (CCl4)­induced acute liver injury model was established, and the protective effect of Rg1 on CCl4­induced acute liver injury was demonstrated in cell culture and animal experimental systems. Further investigation of the mechanisms demonstrated that pretreatment with Rg1 reduced elevated levels of alanine aminotransferase and aspartate aminotransferase, enhanced the antioxidant activity of superoxide dismutase (SOD) and decreased malondialdehyde (MDA) content. Experiments in vitro demonstrated that Rg1 decreased p65 expression and inhibited nuclear factor (NF)­κB activity. In addition to the effect of Rg1, an NF­κB inhibitor promoted cell survival, enhanced SOD activity and reduced MDA level. It was observed through in vivo experiments that pretreatment with Rg1 inhibited NF­κB expression and activity in Kupffer cells and reduced the serum levels of tumor necrosis factor­α and interleukin­6. In conclusion, the results of the present study indicated that pretreatment with Rg1 may rescue CCl4­induced acute liver injury in vivo and in vitro through inhibition of NF­κB activity, to restore the anti­oxidative defense system and down­regulate pro­inflammatory signaling pathways. The present observations provide a theoretical foundation for the clinical application of Rg1 therapy in acute liver injury.

Formin-like2 regulates Rho/ROCK pathway to promote actin assembly and cell invasion of colorectal cancer.

Formin-like2 (FMNL2) is a member of the diaphanous-related formins family, which act as effectors and upstream modulators of Rho GTPases signaling and control the actin-dependent processes, such as cell motility or invasion. FMNL2 has been identified as promoting the motility and metastasis in colorectal carcinoma (CRC). However, whether FMNL2 regulates Rho signaling to promote cancer cell invasion remains unclear. In this study, we demonstrated an essential role for FMNL2 in the activations of Rho/ROCK pathway, SRF transcription or actin assembly, and subsequent CRC cell invasion. FMNL2 could activate Rho/ROCK pathway, and required ROCK to promote CRC cell invasion. Moreover, FMNL2 promoted the formation of filopodia and stress fiber, and activated the SRF transcription in a Rho-dependent manner. We also demonstrated that FMNL2 was necessary for LPA-induced invasion, RhoA/ROCK activation, actin assembly and SRF activation. FMNL2 was an essential component of LPA signal transduction toward RhoA by directly interacting with LARG. LARG silence inhibited RhoA/ROCK pathway and CRC cell invasion. Collectively, these data indicate that FMNL2, acting as upstream of RhoA by interacting with LARG, can promote actin assembly and CRC cell invasion through a Rho/ROCK-dependent mechanism.

FMNL2 is a positive regulator of cell motility and metastasis in colorectal carcinoma.

FMNL2 is a member of diaphanous-related formins which act as effectors of Rho family GTPases and control the actin-dependent processes such as cell motility or invasion. We previously found that FMNL2 overexpression in metastatic cell lines and tissues of colorectal carcinoma is associated with more aggressive tumour behaviour. Here we used gain-of-function and loss-of-function approaches to investigate the effects of FMNL2 on colorectal carcinoma in vitro and in vivo. Forced expression of FMNL2 caused a significant increase in tumour cell proliferation, motility, invasion in vitro, and metastasis in vivo, whereas FMNL2 depletion showed opposite effects. We examined gene expression profiles following knockdown of FMNL2 in SW480/M5 cells. Expression of 323 genes was up-regulated by more than two-fold, whereas 222 genes were down-regulated by more than two-fold in FMNL2-depleting SW480/M5 cells. Gene ontology analysis showed that most of genes belong to functional categories such as cell cycle, cytoskeleton, transcription factor, and G-protein modulator. Pathway analysis revealed that cytoskeletal regulation by the Rho GTPase pathway, the Wnt pathway, the G-protein pathway, and the P53 pathway were affected by FMNL2. Many of these genes are in functional networks associated with cell proliferation, metastasis, Wnt or the Rho signalling pathway involved in the regulation of FMNL2. The expression of five differentially expressed genes including CXXC4, CD200, VAV1, CSF1, and EPHA2 was validated by real-time PCR and western blot analysis. Thus, FMNL2 is a positive regulator of cell motility, invasion, and metastasis of colorectal carcinoma.

FMNL2 enhances invasion of colorectal carcinoma by inducing epithelial-mesenchymal transition.

FMNL2 is a member of diaphanous-related formins that control actin-dependent processes such as cell motility and invasion. Its overexpression in metastatic cell lines and tissues of colorectal carcinoma has been associated with aggressive tumor development in our previous study. But its specific role in cancer is largely unknown. Here we report that FMNL2 is involved in epithelial-mesenchymal transition (EMT) maintenance in human colorectal carcinoma cells. A positive correlation between FMNL2 and vimentin expression and an inverse correlation between FMNL2 and E-cadherin expression were found in colorectal carcinoma cell lines and cancer tissues. Specific knockdown of FMNL2 led to an epithelial-state transition, confirmed by the cobblestone-like phenotype, upregulation of E-cadherin, α-catenin, and γ-catenin, and downregulation of vimentin, snail, slug. Loss of FMNL2 expression lowered the ability of TGF-ß to induce cell invasion and EMT, as shown by morphology and the expression levels. Upregulation of vimentin, slug, snail, downregulation of E-cadherin and activation of receptor-Smad3 phosphorylation were observed in M5 and MDCK cells induced by TGF-ß, whereas altered expression of these markers was not obvious in FMNL2-depleting M5 cells. High levels of activation of p-MAPK and p-MEK, but not p-PI3K and p-AKT, were observed in SW480/FMNL2+ cells compared with control cells. Treatment with U0126 could abrogate the activation of p-MAPK and p-MEK, whereas LY294002 treatment had no effect on the PI3K/AKT pathway. In conclusion, these findings identify a novel EMT and tumor promoting function for FMNL2, which is involved in TGF-ß-induced EMT and colorectal carcinoma cell invasion via Smad3 effectors, or in collaboration with MAPK/MEK pathway.

[Expression of FMNL2 and its relation to the metastatic potential of human colorectal cancer cells].

OBJECTIVE: To explore the association of FMNL2 expression with the metastatic potential of colorectal cancer cells. METHODS: FMNL2 mRNA and protein expressions in 6 human colorectal cancer cell lines were detected by real-time RT-PCR and immunohistochemical method, respectively, and analyzed for their correlations to the in vitro invasiveness of the cell lines evaluated by Boyden assay. In SW620 and SW480/M5 cell lines, the expression of FMNL2 was repressed by FMNL2 short hairpin RNA (shRNA), and the changes in the invasiveness of the cells were observed. RESULTS: FMNL2 was highly expressed in SW480/M5, LoVo and SW620 cells derived from metastatic colorectal cancers in comparison with that in LS174T, SW480 and HT29 cells, which were derived from primary colorectal cancers. In vitro analysis of the cell invasiveness demonstrated that SW480/M5, LoVo and SW620 cells had higher invasiveness than LS174T, SW480 and HT29 in vitro. In SW480/M5 and SW620 cells, transfection with FMNL2 shRNA resulted in significantly lowered cell invasiveness. CONCLUSION: FMNL2 may play an important role in the invasion and metastasis of colorectal cancer.

Overexpression of FMNL2 is closely related to metastasis of colorectal cancer.

BACKGROUND AND AIMS: Formin-like 2 (FMNL2) is a member of diaphanous-related formins which can control the actin-dependent processes such as cell motility and invasion. In this study, we investigated the expression of FMNL2 in colorectal cancer (CRC) and its correlation with CRC metastasis. PATIENTS-METHODS: Paraffin-embedded specimens of CRC (including 75 primary CRC tumors and 45 corresponding metastatic lymph nodes) and normal colorectal mucosa (30 samples) were immunostained with a FMNL2 antibody. Thirty-two paired snap-frozen tumor tissues and adjacent normal colorectal mucosa were subjected to real-time reverse-transcription polymerase chain reaction (RT-PCR). Six CRC cell lines (SW480, SW620, SW480/M5, LoVo, LS174T, and HT29) were assayed for FMNL2 expression by Western blotting and real-time RT-PCR. Their invasive abilities in vitro were determined by Boyden chamber assay. RESULTS: The immunohistochemical analysis showed FMNL2 expression was considerably higher in CRC tumors and corresponding metastatic lymph nodes than in normal colorectal mucosa (P < 0.05, respectively). Elevated FMNL2 expression was significantly correlated with lymphatic metastasis of CRC (P < 0.05). Real-time RT-PCR analysis confirmed the results obtained by immunohistochemistry. At mRNA and protein levels, FMNL2 expression was substantially upregulated in cell lines derived from CRC metastases (SW620, SW480/M5, and LoVo) compared to ones derived from primary CRC (HT29, LS174T, and SW480; P < 0.05). In vitro cell invasive assay demonstrated that the former three cell lines had higher invasive ability than the latter cell lines. CONCLUSIONS: FMNL2 may play an important role in the metastasis of CRC and may be a useful marker for metastasis of CRC.

[Galectin-1 expression in human colorectal carcinoma and its clinical significance].

OBJECTIVE: To investigate the correlation between galectin-1 expression and the biological behaviors of human colorectal carcinoma. METHODS: SP immunohistochemistry was used to detect the expression of galectin-1 in 158 paraffin-embedded specimens including 30 normal mucosa, 25 adenoma, 65 colorectal carcinoma and 38 metastatic tumor specimens. Real-time RT-PCR was used to detect galectin-1 mRNA expression in 32 fresh specimens of colorectal carcinoma and normal mucosa. RESULTS: The positive expression level of galectin-1 was significantly different between normal mucosa, adenoma, colorectal carcinomas and metastatic tumors, with positivity rate of 0, 8%, 66% and 86%, respectively (P<0.05). Galectin-1 expression in moderately or well differentiated colorectal carcinomas was significantly lower than that in poorly differentiated ones (P=0.031), and its expression in invasive carcinomas was significantly higher than that in non-invasive carcinomas (P=0.000). Galectin-1 expression in colorectal carcinomas was significantly related with lymph node metastasis (P=0.004). In poorly differentiated colorectal carcinomas, the expression of galectin-1 mRNA was about 2.27 times that in moderately or well differentiated colorectal carcinomas (P=0.00); galectin-1 mRNA expression in invasive carcinoma was 1.98 times that in non-invasive carcinoma (P=0.002). In tumors with lymph node metastasis, galectin-1 mRNA expression was 1.42 times that in tumors without metastasis (P=0.018). CONCLUSION: Galectin-1 can be involved in the development and progression of colorectal carcinoma, and may relate to the infiltration, differentiation and lymph node metastasis of colorectal carcinoma.

[Effect of AcSDKP on the proliferation and collagen synthesis in cultured rat cardiac fibroblasts stimulated by PDGF].

AIM: To investigate whether AcSDKP can inhibit proliferation and collagen synthesis in cultured rat cardiac fibroblasts mediated by PDGF. METHODS: Neonatal rat cardiac fibroblasts were isolated. The cell proliferation was observed by 3H-proline incorporation assay. RESULTS: On the culture of 0.4% FBS, PDGF stimulated cardiac fibroblasts proliferation and collagen synthesis with a dose-dependent manner at the concentrations from 1 ng/ml to 20 ng/ml, in which 10 ng/ml PDGF reached its peak. AcSDKP at the concentration from 10(-10) mol/L to 10(-8) mol/L could inhibit cardiac fibroblasts proliferation and collagen synthesis mediated by PDGF. 10(-9) mol/L AcSDKP attained its peak on inhibiting cardiac fibroblasts proliferation and collagen synthesis. CONCLUSION: AcSDKP can inhibit proliferation and collagen synthesis in cultured rat cardiac fibroblasts mediated by PDGF.

[Role of AcSDKP on collagen synthesis and degradation in cultured rat cardiac fibroblast].

OBJECTIVE: To investigate the role of AcSDKP on collagen synthesis and degradation in cultured rat cardiac fibroblasts. METHODS: Neonatal rat cardiac fibroblasts were isolated and stimulated by PDGF. The cell proliferation was observed by (3)H-TdR incorporation assay. The synthesis of collagen was measured by (3)H-proline incorporation assay. The expression of type I and type III collagen and MMP-1 protein were measured by Western blot. The MMP-2 and MMP-9 activity was evaluated with zymography assay. RESULTS: PDGF stimulated cardiac fibroblasts proliferation with increased collagen synthesis and type I and type III collagen protein expressions as well as MMP-2 and MMP-9 activities and MMP-1 expression. AcSDKP inhibited cardiac fibroblasts proliferation induced by PDGF and reduced collagen synthesis and type I and type III collagen protein expression. AcSDKP also further up-regulated MMP-2 and MMP-9 activities and MMP-1 expression in cardiac fibroblasts induced by PDGF. CONCLUSION: AcSDKP inhibited proliferation and collagen synthesis and up-regulated matrix metalloproteinases activity or expression induced by PDGF, which was possibly related with the effect of AcSDKP anti-fibrosis.