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ObjectiveTo observe the effect of Scutellariae Radix-Coptidis Rhizoma on plaque stability in atherosclerotic (AS) mice and to explore its possible mechanism of action based on the Toll-like receptor 4 (TLR4)/myeloid differentiation factor 88 (MyD88)/nuclear factor kappa B (NF-κB) signaling pathway. MethodTen normal C57BL/6J mice were used as the normal group, and the same strain of ApoE knockout (ApoE-/-) mice were fed with a high-fat diet for 12 weeks to construct an atherosclerosis model. Mice were randomly divided into five groups, namely the model group, the atorvastatin group, and the Scutellariae Radix-Coptidis Rhizoma low-, medium-, and high-dose groups, with ten mice in each group. Then normal and model groups were given equal volume of saline gavage, and the low-, medium-, high-dose Scutellariae Radix-Coptidis Rhizoma groups were given 1.95, 3.9, 7.8 g·kg-1 of the drug by gavage for 8 weeks, respectively. The general state of mice was observed. Hematoxylin-eosin staining was utilized to observe the pathology of aortic root plaques and calculate the percentage of plaque area. Masson staining and oil red O staining combined with immunohistochemistry of F4/80 and α-SMA were used to detect the plaque components of aortic root plaques and calculate the plaque vulnerability index. Enzyme-linked immunosorbent assay was adopted to detect the expression levels of serum tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). Western blot was applied to detect the protein expression levels of matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9), TLR4, MyD88, NF-κB p65, and phosphorylation (p) -NF-κB p65 in the aortic tissues of mice in each group. Real-time fluorescence quantitative polymerase chain reaction (Real-time PCR) assay was employed to detect the expression levels of MMP-2, MMP-9, TLR4, and MyD88, NF-κB p65 mRNA. ResultCompared with the model group, the general state of the mice in each medication group was improved, and no obvious side effects were observed. Compared with the model group, the percentage of plaque area in the aortic root of AS mice was significantly reduced in the medium- and high-dose Scutellariae Radix-Coptidis Rhizoma groups (P<0.05). The content of collagen fibers and smooth muscle cells in the plaques of the high-dose Scutellariae Radix-Coptidis Rhizoma group was significantly increased (P<0.01), and the content of lipids and macrophages was significantly reduced (P<0.05), the plaque vulnerability index of each dose group of Scutellariae Radix-Coptidis Rhizoma was significantly reduced, with significant reduction of the medium- and high-dose groups (P<0.01). MMP-2 and MMP-9 protein and mRNA expression levels in aortic tissues were significantly reduced in medium- and high-dose Scutellariae Radix-Coptidis Rhizoma groups (P<0.05). The serum levels of TNF-α and IL-6 were significantly reduced in AS mice in medium- and high-dose Scutellariae Radix-Coptidis Rhizoma groups (P<0.05). In the medium- and high-dose Scutellariae Radix-Coptidis Rhizoma groups, the levels of TLR4, MyD88 protein, and mRNA expression in aortic tissues were significantly reduced (P<0.05), and the level of NF-κB p65 phosphorylation in aortic tissues was significantly reduced (P<0.05). ConclusionScutellariae Radix-Coptidis Rhizoma may play an anti-inflammatory and stabilizing role by inhibiting the TLR4/MyD88/NF-κB signaling pathway.
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ObjectiveTo investigate the molecular mechanism by which Si Junzitang in intervening in the development of hepatocellular carcinoma (HCC) by regulating the O-linked β-N-acetylglucosamine modification (O-GlcNAcylation) of nuclear factor kappa-B (NF-κB) p65 in the paracancerous tissues. MethodThe orthotopic liver cancer mouse model was established. Twenty-four C57BL/6 mice were randomly divided into four groups: Normal group, model group, Si Junzitang low-dose group (10 g·kg-1), and Si Junzitang high-dose group (25 g·kg-1), with 6 mice in each group. The O-GlcNAcylation level and phosphorylation modification level of p65 in the paracancerous tissues were detected using Western blot. The O-GlcNAcylation of p65 was assessed using immunoprecipitation (IP). The mRNA expression of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), transforming growth factor-β1 (TGF-β1), vascular endothelial growth factor A (VEGFA), matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9) in the paracancerous tissues was detected using real-time quantitative polymerase chain reaction (Real-time PCR). The tumor number, liver weight, locomotor activity, grip strength, and Qi status of the mice were observed and analyzed. ResultCompared with the normal group, the model group showed a significant decrease in O-GlcNAcylation in the paracancerous tissues (P<0.01), a significant decrease in p65 O-GlcNAcylation (P<0.01), a significant increase in p65 phosphorylation (P<0.01), significantly elevated mRNA levels of cytokines IL-6, TNF-α, TGF-β1, VEGFA, MMP-2, and MMP-9 (P<0.01), significantly increased liver weight (P<0.01), significantly declined grip strength, number of grid crossings, and number of vertical stand-ups (P<0.01), and significantly dwindled Qi status (P<0.01). Compared with model group, the Si Junzitang low-dose and high-dose groups showed significantly increased levels of O-GlcNAcylation in the paracancerous tissues (P<0.05, P<0.01), significantly upregulated p65 O-GlcNAcylation levels (P<0.05, P<0.01), and significantly decreased p65 phosphorylation levels (P<0.01). In the Si Junzitang low-dose group, the mRNA levels of IL-6, TGF-β1, and VEGFA significantly decreased (P<0.05, P<0.01). In the Si Junzitang high-dose group, the mRNA levels of IL-6, TNF-α, TGF-β1, VEGFA, MMP-2, and MMP-9 significantly decreased (P<0.01), the number of tumors larger than 3 mm in diameter significantly decreased (P<0.01), and liver weight significantly decreased (P<0.05). Additionally, grip strength, number of grid crossings, and number of vertical stand-ups significantly increased (P<0.05, P<0.01), along with a significant increase in qi status (P<0.01). ConclusionSi Junzitang can inhibit the progression of orthotopic HCC in mice, which may be achieved by increasing the O-GlcNAcylation level in the paracancerous tissues, enhancing the O-GlcNAcylation of p65, inhibiting the phosphorylation modification of p65, and ultimately suppressing the expression of downstream IL-6, TNF-α, TGF-β1, VEGFA, MMP-2, and MMP-9.
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ObjectiveTo explore the molecular mechanism of Sanhuang Xiexintang (SHXXT) in protecting stress gastric ulcer (SGU) in rats through network pharmacology, molecular docking, and animal experiments. MethodThe active ingredients and corresponding targets in SHXXT were collected and screened from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP), Traditional Chinese Medicine Information Database (TCMID), Bioinformation Analysis Tool for Molecular Mechanism of Traditional Chinese Medicine (BATMAN-TCM), and Swiss Target Prediction database. SGU-related targets were screened from the Online Mendelian Inheritance in Man (OMIM), Therapeutic Target Database (TTD), GeneCards database, and PharmGKB database. Herbal-ingredient-target (H-C-T) network was constructed by using Cytoscape 3.9.1 software. Protein-protein interaction (PPI) of drug and disease intersection targets was analyzed by using the Protein Interaction Platform (STRING) database. Gene ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were conducted through the Database for Annotation Visualization and Integrated Discovery (DAVID). The active ingredients and key targets were validated using AutodockVina 1.2.2 molecular docking software, and the experimental results were further validated through animal experiments. ResultThe 55 active ingredients were screened, and 255 potential target genes for SHXXT treatment of SGU were predicted. The PPI analysis showed that protein kinase B (Akt), phosphatase and tensin homolog deleted on chromosome ten (PTEN), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and cyclooxygenase-2 (COX-2) are the core targets of SHXXT for protecting SGU. GO and KEGG analyses showed that SHXXT may affect the development of SGU by regulating various biological processes such as the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway and inflammatory processes. The molecular docking results showed that both the active ingredients and key targets had good binding ability. Animal experiments showed that compared with the blank group, the ulcer index (UI) of the model group was significantly increased (P<0.01), and the serum levels of TNF-α and IL-1β significantly increased (P<0.01). The phosphorylation level of PTEN in gastric mucosal tissue was significantly down-regulated (P<0.05). The phosphorylation levels of PI3K, Akt, and nuclear factor kappa-B (NF-κB) were significantly up-regulated (P<0.05). Compared with the model group, the UI of the treatment group was significantly reduced (P<0.01), and the serum levels of TNF-α and IL-1β were significantly reduced (P<0.01). The phosphorylation level of PTEN in gastric mucosal tissue was significantly up-regulated (P<0.01), and the phosphorylation levels of PI3K, Akt, and NF-κB were significantly downregulated (P<0.01). ConclusionThe application of network pharmacology prediction, molecular docking simulation, and animal experimental validation confirms that SHXXT regulates the PI3K/Akt/NF-κB signaling pathway to regulate the inflammatory response of rats and thus protects the gastric mucosa of SGU rats.
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As people's living standards improve, the development trend of diabetes has gradually become severe. Diabetes is a chronic inflammatory disease associated with abnormal expression of nuclear factor-kappa B (NF-κB) in patients. NF-κB exists in various tissue cells and participates in the regulation of a variety of genes related to immune function and inflammation. Varieties of factors can activate NF-κB when the body is stimulated by external factors, so as to produce inflammation and other reactions. Previous studies on NF-κB mainly focus on cancer, and the pathological mechanism of the treatment of diabetes by related signaling pathways and the progress of traditional Chinese medicine (TCM) treatment have not been systematically elaborated on. By referring to the relevant literature in China and abroad, it was found that NF-κB is not isolated in the development and progression of diabetes but is associated with signal molecules related to inflammation, oxidative stress, and energy metabolism, and it is involved in mediating inflammation, pancreatic β cell apoptosis, insulin signal transduction, and other physiological functions. Therefore, blocking the transmission of NF-κB signaling pathway is beneficial to the treatment of diabetes. At present, Western medicine for the treatment of diabetes mainly includes oral hypoglycemic drugs and insulin injections, but the adverse reactions are obvious. TCM has been characterized by multi-target, extensive action, and excellent curative effects in the treatment of diabetes. TCM and its compounds with functions of tonifying Qi and promoting blood circulation, regulating qi and eliminating phlegm, clearing heat and detoxifying, and nourishing Yin and moistening dryness can effectively intervene in the abnormal expression of NF-κB signaling pathway in vivo through anti-inflammatory effects. In this paper, the association between NF-κB signaling pathway and diabetes was summarized, and the modern research progress of TCM intervention of NF-κB signaling pathway in the treatment of diabetes in the past five years was reviewed, so as to lay a laboratory foundation for the study of a new pathological mechanism of diabetes based on NF-κB signaling pathway and provide new targets and research direction for the prevention and treatment of diabetes and development of related TCM.
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Colorectal cancer (CRC) is one of the leading causes of cancer-related deaths worldwide, with increasing incidence and mortality rates. The disease often develops covertly and lacks specific symptoms in its early stages, leading to late-stage diagnoses in most patients. It has become a prominent research topic in the field of digestive system tumors. The exact mechanisms underlying CRC are not yet clear and involve factors such as genetics, gene mutations, inflammatory responses, and aberrant activation of tumor-related signaling pathways. Nuclear factor-kappa B (NF-κB) is a crucial transcription factor that participates in various biological processes, including inflammatory responses, immune responses, cell proliferation, and apoptosis. Research suggests that NF-κB, serving as a molecular link between inflammation and cancer, is highly expressed in CRC. It promotes the occurrence and development of CRC by regulating the activity of target genes such as cell pro-inflammatory factors, chemokines, angiogenic factors, metastasis factors, and anti-apoptotic proteins. Currently, common treatments for CRC include surgery, radiation therapy, and chemotherapy drugs like 5-fluorouracil. However, these treatments have limitations such as significant adverse reactions, high metastasis rates, and the development of drug resistance. Therefore, the search for effective, low-adverse-reaction drugs to replace or supplement current treatments is essential. In recent years, traditional Chinese medicine (TCM) has shown some effectiveness in preventing and treating CRC. TCM has been found to inhibit the growth of CRC cells by modulating the NF-κB signaling pathway, playing a positive role in the occurrence and progression of CRC. Based on the asthenia in origin and sthenia in superficiality and deficiency-excess in complexity in CRC, this article summarized and analyzed the mechanisms and effects of TCM interventions targeting the NF-κB signaling pathway in CRC, and reviewed advances of 10 Chinese medicinal compound formulas and 37 Chinese medicinal monomer components of different types, including flavonoids, phenols, alkaloids, glycosides, and terpenoids with the effects of dispelling pathogenic factors, reinforcing healthy qi, and removing toxins in the prevention and treatment of CRC by targeting the NF-κB pathway. It is found that Chinese medicine can inhibit CRC cell proliferation, invasion, metastasis, angiogenesis, and inflammation by modulating the NF-κB signaling pathway, induce cell apoptosis, restore drug and radiation sensitivity, and counteract CRC. This article is expected to provide insights and references for the in-depth exploration and treatment of CRC mechanisms.
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Objective:To observe the role of neuroinflammation in cognitive dysfunction induced by 1-bromopropane (1-BP) in rats.Methods:Male Wistar rats were randomly divided into control group, 1-BP group, pyrrolidine dithiocarbamate(PDTC)+ 1-BP group and PDTC group, with 15 rats in each group. Rats in 1-BP group and PDTC+ 1-BP group were given 800 mg / kg 1-BP by gavage, and rats in control group and PDTC group were given equal volume corn oil once a day for 12 days; rats in PDTC group and PDTC+ 1-BP group were intraperitoneally injected with 100 mg / kg PDTC 30 minutes after gavage, while rats in control group and 1-BP group were injected with equal volume of normal saline once a day for 12 days.From the 7th to 12th day of the experiment, ten rats in each group were randomly selected and subjected to Morris water maze test for detect the cognitive function. In the positioning navigation test, the learning ability of rats was evaluated by the escape latency and total swimming distance respectively. In the space exploration experiment, the memory ability of experimental animals was evaluated by the number of times crossing the target platform. After the experiment, ten rats were sacrificed, the cerebral prefrontal cortex was harvested. The cytosolic and nuclear NF-κB expression and phosphorylation were detected by Western blot, the mRNA levels of TNF-α and IL-1β were detected by qRT-PCR. After cardiac perfusion fixation, the brains of 5 rats were taken to make frozen sections for immunohistochemical staining and Nissl staining. SPSS 20.0 software was used for statistical analysis, repetitive measurement deviation analysis was used for the analysis of the swimming distance and the escape latency in positioning navigation test, One-way ANOVA was used for the analysis of the number of times crossed the target platform in spatial probe test and other data. Tukey's test was used for Post hoc comparison.Results:The results of Morris water maze showed that there was significant interaction between group and training time in the total swimming distance of rats in the four groups ( F=3.762, P<0.05). Simple effect analysis showed that the total swimming distance of 1-BP group in 1-4 days were longer than those of control group (all P<0.05), while the total swimming distance of PDTC+ 1-BP group in 1-4 days were shorter than those of 1-BP group (all P<0.05). There was significant interaction between group and training time in the escape latency among the four groups ( F=6.541, P<0.01). The escape latencies of 1-BP group in 1-4 days were longer than those of control group (all P<0.05), while the escape latencies of PDTC+ 1-BP group in 1-4 days were shorter than those of 1-BP group (all P<0.05). The results of space exploration experiment showed that there was significant difference in the number of crossing the platform among the four groups ( F=75.333, P<0.01). The number of crossing the platform (1.08±0.29) in 1-BP group was lower than that in the control (3.35±0.05) ( P<0.01). The number of crossing the platform (1.95±0.26) in PDTC+ 1-BP group was higher than that in 1-BP group ( P<0.01). It had significant difference both in the cytoplasm and in the nucleus of the NF-κB protein level in prefrontal cortex among rats of the four groups ( F=20.865, 23.877, both P<0.01). The levels of NF-κB in cytoplasm and nucleus of rats in 1-BP group were both higher than those in control group (cytoplasm: (177.3±32.1)%, (100.0±8.4)%, P<0.01; nucleus: ( 173.2±27.1)%, (100.0±8.4)%, P<0.01). While the levels of NF-κB in cytoplasm and nucleus of 1-BP+ PDTC group were both lower than those of 1-BP group (cytoplasm: (148.7±22.0)%, (177.3±32.1)%, P<0.01; nucleus: (149.7±18.8)%, (173.2±27.1)%, P<0.01). The results of qRT-PCR showed that there were significant differences in the mRNA levels of TNF-α and IL-1β in the prefrontal cortex among the four groups ( F=17.464, 17.382, both P<0.01). The levels of TNF-α and IL-1β mRNA in 1-BP group were higher than those in control group (both P<0.05), and the levels of TNF-α and IL-1β mRNA in PDTC+ 1-BP group were both lower than those in 1-BP group (both P<0.05). The results of immunohistochemistry showed that compared with the control group, the number of microglia and astrocytes in the 1-BP group increased (microglia: (158.30±9.68), (110.20±16.30), P<0.05; astrocytes: (122.76±4.35), (80.24±6.96), P<0.05), and the morphology was also activated, with light staining and reduced number of Nissl bodies in neurons.The number of microglia and astrocytes in PDTC + 1-BP group was lower than that in 1-BP group (microglia: (131.70±14.67), (158.30±9.68), P<0.05; astrocytes: (101.54±4.55), (122.76±4.35), P<0.05), and the Nissl body staining of neurons was significantly deepened. Conclusion:NF-κB signaling pathway might be the key mechanism of 1-BP neurotoxicity. PDTC intervention could significantly improve the neuroinflammatory response and behavioral disorders of experimental animals intoxicated with 1-BP.
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ObjectiveTo investigate the effect and mechanism of Biejiajian Wan on liver fibrosis by regulating the polarization of macrophages. MethodRaw264.7 cells were cultured in vitro by serum pharmacological method, and the hypoxia model of RAW264.7 cells was established by stimulating RAW264.7 cells with cobalt chloride (CoCl2). The cells were randomly divided into blank group, CoCl2 hypoxia model group (200 mmol·L-1), Biejiajian Wan low-dose group (200 mmol·L-1+0.55 g·kg-1 Fuzheng Quyu capsules), medium-dose group (200 mmol·L-1+1.1 g·kg-1 Biejiajian Wan), and high-dose group (200 mmol·L-1+2.2 g·kg-1 Biejiajian Wan) and Fuzheng Quyu capsule group (200 mmol·L-1+0.56 g·kg-1 Biejiajian Wan). Cell proliferation was detected by cell counting kit-8 (CCK-8), and the gene expression of hypoxia inducible factor-1α (HIF-1α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) in macrophages was detected by real time fluorescence quantitative polymerase chain reaction (Real-time PCR). The expression of macrophage polarization-related protein and HIF-1α/nuclear factor-kappa B (NF-κB) signaling pathway-related protein was tested by Western blot, and the distribution and expression of NF-κB signaling pathway-related protein and HIF-1α were determined by cell immunofluorescence. ResultCompared with the conditions in the blank group, the proliferation of RAW264.7 cells was inhibited after CoCl2 stimulation for 24 hours (P<0.05), the mRNA expression of HIF-1α, IL-1β and IL-6 in the model group were increased (P<0.05), the protein expression of HIF-1α and M1 macrophage phenotypic proteins IL-6 and tumor necrosis factor-α (TNF-α) was boosted while that of M2 macrophage phenotypic protein interleukin-10 (IL-10) was reduced (P<0.05), the protein expression of NF-κB p65, phosphorylation (p)-NF-κB p65, phosphorylated NF-κB inhibits protein kinase α/β (p-IKKα/β) and phosphorylated NF-κB inhibits protein α (p-IκBα) was elevated (P<0.05), the nuclear expression of HIF-1α and NF-κB p65 was promoted. Compared with the conditions in the model group, after 24 hours of treatment with corresponding drug-containing serum, each treatment group promoted the proliferation of RAW264.7 cells (P<0.05), the mRNA expression levels of HIF-1α, IL-1β and IL-6 in macrophages were reduced (P<0.05), the protein expression of HIF-1α, IL-6 and TNF-α was decreased, while that of CD163 and IL-10 was increased (P<0.05), the protein expression of NF-κB p65, p-NF-κB p65, p-IKKα/β and p-IκBα was lowered (P<0.05), the nuclear expression of HIF-1α and NF-κB p65 was inhibited. ConclusionBiejiajian Wan could modulate the polarization of macrophages, attenuate the injury of macrophage-associated inflammatory response under hypoxia, and thus delay the progression of liver fibrosis, which might be related to its regulation of HIF-1α/NF-κB signaling pathway.
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ObjectiveTo investigate the preventive and curative effect of Chaishao Liujunzi Tang (CSLJZT) on colonic mucosal injury induced by dextran sulfate sodium (DSS) in mice with ulcerative colitis (UC) and its mechanism. MethodFifty Balb/c male mice were randomly divided into normal group, model group, CSLJZT low-dose group, CSLJZT high-dose group, and sulfasalazine group. Except for the normal group, other groups were given 2.5% DSS freely for 7 d, and were given drug intervention after successful modeling for 7 d. Bodyweight, feces, and other general physiological statuses of mice were recorded every day, and disease activity index (DAI) scores were calculated.The colon length was measured, and stained by hematoxylin-eosin (HE) staining to observe the morphological changes of the colon.The enzyme-linked immunosorbent assay (ELISA) was used to determine the content of interleukin-1β (IL-1β), myeloperoxidase (MPO), and superoxide dismutase (SOD) in the serum. Western blot was used to determine the protein expression levels of Toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), nuclear factor kappa B (NF-κB), inhibitor-kappa binding protein (IκB), Caspase-1, and nucleotide-binding oligomerization domain-like receptor 3 (NLRP3) in the colon tissues. ResultAs compared with the normal group, mice in the model group had significantly decreased body weight (P<0.01), severe diarrhea and hematochezia, and significantly increased DAI score (P<0.01). As compared with the model group, the decreasing trend of body weight was significantly alleviated in the CSLJZT groups (P<0.01), diarrhea and hematochezia were significantly improved, DAI score was significantly decreased (P<0.01), and colon length increased (P<0.05). HE staining showed that the pathological damage of colon tissue was significantly improved and the inflammatory cell infiltration was reduced in the CSLJZT groups as compared with the model group. As compared with the normal group, the serum levels of IL-1β and MPO were significantly higher (P<0.01) and SOD levels were significantly lower (P<0.01) of mice in the model group.Compared with the model group, the treated group reduced the serum IL-1β and MPO levels (P<0.01), and raised the SOD level (P<0.01). The results of Western blot showed that as compared with the normal group, the expression levels of TLR4, MyD88, NF-κB, Ccaspase-1, and NLRP3 proteins were significantly increased (P<0.01), whereas the expression level of IκB protein was significantly decreased (P<0.01) in the colonic tissue of mice in the model group. As compared with the model group, the expression levels of TLR4, MyD88, NF-κB, Caspase-1, and NLRP3 proteins were decreased (P<0.01), whereas the expression level of IκB protein was increased (P<0.01) in the colonic tissue of mice in the CSLJZT groups. ConclusionCSLJZT improves the inflammatory injury of the colon tissue in DSS-induced UC mice through TLR4/MyD88/NF-κB signaling pathway.
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ObjectiveTo investigate the effect and mechanism of Biejiajian Wan on liver fibrosis by regulating the polarization of macrophages. MethodRaw264.7 cells were cultured in vitro by serum pharmacological method, and the hypoxia model of RAW264.7 cells was established by stimulating RAW264.7 cells with cobalt chloride (CoCl2). The cells were randomly divided into blank group, CoCl2 hypoxia model group (200 mmol·L-1), Biejiajian Wan low-dose group (200 mmol·L-1+0.55 g·kg-1 Fuzheng Quyu capsules), medium-dose group (200 mmol·L-1+1.1 g·kg-1 Biejiajian Wan), and high-dose group (200 mmol·L-1+2.2 g·kg-1 Biejiajian Wan) and Fuzheng Quyu capsule group (200 mmol·L-1+0.56 g·kg-1 Biejiajian Wan). Cell proliferation was detected by cell counting kit-8 (CCK-8), and the gene expression of hypoxia inducible factor-1α (HIF-1α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) in macrophages was detected by real time fluorescence quantitative polymerase chain reaction (Real-time PCR). The expression of macrophage polarization-related protein and HIF-1α/nuclear factor-kappa B (NF-κB) signaling pathway-related protein was tested by Western blot, and the distribution and expression of NF-κB signaling pathway-related protein and HIF-1α were determined by cell immunofluorescence. ResultCompared with the conditions in the blank group, the proliferation of RAW264.7 cells was inhibited after CoCl2 stimulation for 24 hours (P<0.05), the mRNA expression of HIF-1α, IL-1β and IL-6 in the model group were increased (P<0.05), the protein expression of HIF-1α and M1 macrophage phenotypic proteins IL-6 and tumor necrosis factor-α (TNF-α) was boosted while that of M2 macrophage phenotypic protein interleukin-10 (IL-10) was reduced (P<0.05), the protein expression of NF-κB p65, phosphorylation (p)-NF-κB p65, phosphorylated NF-κB inhibits protein kinase α/β (p-IKKα/β) and phosphorylated NF-κB inhibits protein α (p-IκBα) was elevated (P<0.05), the nuclear expression of HIF-1α and NF-κB p65 was promoted. Compared with the conditions in the model group, after 24 hours of treatment with corresponding drug-containing serum, each treatment group promoted the proliferation of RAW264.7 cells (P<0.05), the mRNA expression levels of HIF-1α, IL-1β and IL-6 in macrophages were reduced (P<0.05), the protein expression of HIF-1α, IL-6 and TNF-α was decreased, while that of CD163 and IL-10 was increased (P<0.05), the protein expression of NF-κB p65, p-NF-κB p65, p-IKKα/β and p-IκBα was lowered (P<0.05), the nuclear expression of HIF-1α and NF-κB p65 was inhibited. ConclusionBiejiajian Wan could modulate the polarization of macrophages, attenuate the injury of macrophage-associated inflammatory response under hypoxia, and thus delay the progression of liver fibrosis, which might be related to its regulation of HIF-1α/NF-κB signaling pathway.
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ObjectiveTo investigate the preventive and curative effect of Chaishao Liujunzi Tang (CSLJZT) on colonic mucosal injury induced by dextran sulfate sodium (DSS) in mice with ulcerative colitis (UC) and its mechanism. MethodFifty Balb/c male mice were randomly divided into normal group, model group, CSLJZT low-dose group, CSLJZT high-dose group, and sulfasalazine group. Except for the normal group, other groups were given 2.5% DSS freely for 7 d, and were given drug intervention after successful modeling for 7 d. Bodyweight, feces, and other general physiological statuses of mice were recorded every day, and disease activity index (DAI) scores were calculated.The colon length was measured, and stained by hematoxylin-eosin (HE) staining to observe the morphological changes of the colon.The enzyme-linked immunosorbent assay (ELISA) was used to determine the content of interleukin-1β (IL-1β), myeloperoxidase (MPO), and superoxide dismutase (SOD) in the serum. Western blot was used to determine the protein expression levels of Toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), nuclear factor kappa B (NF-κB), inhibitor-kappa binding protein (IκB), Caspase-1, and nucleotide-binding oligomerization domain-like receptor 3 (NLRP3) in the colon tissues. ResultAs compared with the normal group, mice in the model group had significantly decreased body weight (P<0.01), severe diarrhea and hematochezia, and significantly increased DAI score (P<0.01). As compared with the model group, the decreasing trend of body weight was significantly alleviated in the CSLJZT groups (P<0.01), diarrhea and hematochezia were significantly improved, DAI score was significantly decreased (P<0.01), and colon length increased (P<0.05). HE staining showed that the pathological damage of colon tissue was significantly improved and the inflammatory cell infiltration was reduced in the CSLJZT groups as compared with the model group. As compared with the normal group, the serum levels of IL-1β and MPO were significantly higher (P<0.01) and SOD levels were significantly lower (P<0.01) of mice in the model group.Compared with the model group, the treated group reduced the serum IL-1β and MPO levels (P<0.01), and raised the SOD level (P<0.01). The results of Western blot showed that as compared with the normal group, the expression levels of TLR4, MyD88, NF-κB, Ccaspase-1, and NLRP3 proteins were significantly increased (P<0.01), whereas the expression level of IκB protein was significantly decreased (P<0.01) in the colonic tissue of mice in the model group. As compared with the model group, the expression levels of TLR4, MyD88, NF-κB, Caspase-1, and NLRP3 proteins were decreased (P<0.01), whereas the expression level of IκB protein was increased (P<0.01) in the colonic tissue of mice in the CSLJZT groups. ConclusionCSLJZT improves the inflammatory injury of the colon tissue in DSS-induced UC mice through TLR4/MyD88/NF-κB signaling pathway.
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ObjectiveTo investigate the effect of Biejiajian Wan (BJJW) on transforming growth factor-β1 (TGF-β1)-induced epithelial-mesenchymal transition (EMT) of HepG2 cells, and explore its mechanism against EMT of hepatocellular carcinoma cells. MethodHepG2 cells were randomly divided into a blank group, a TGF-β1 model group (10 μg·L-1 TGF-β1), a low-dose BJJW group (10 μg·L-1 TGF-β1+0.55 g·kg-1 BJJW), a medium-dose BJJW group (10 μg·L-1 TGF-β1+1.1 g·kg-1 BJJW), a high-dose BJJW group (10 μg·L-1 TGF-β1+2.2 g·kg-1 BJJW), and a sorafenib group (10 μg·L-1 TGF-β1+0.03 g·kg-1 sorafenib). The EMT model was induced by 10 μg·L-1 TGF-β1 in HepG2 cells. After treatment with corresponding medicated serum, cell counting kit -8 (CCK-8) assay was used to detect cell proliferation. Cell migration ability was detected by the Transwell assay and wound healing assay. The protein expression related to EMT and nuclear factor-kappa B (NF-κB) signaling pathway was detected by cell immunofluorescence assay and Western blot. ResultCompared with the blank group 4 days later, the TGF-β1 model group showed fusiform and loose cells with widened gap and antennae reaching out, decreased protein expression of E-cadherin (P<0.05), and increased protein expression of N-cadherin and vimentin (P<0.05), which indicated that the EMT model was properly induced in HepG2 cells by TGF-β1 stimulation for 4 days. After 48 hours of treatment with the corresponding medicated serum, each medication group showed inhibited proliferation of HepG2 cells that had undergone EMT, especially the low- and high-dose BJJW groups (P<0.01), and the medium-dose BJJW group showed increased E-cadherin protein expression (P<0.05) and decreased p-p65, N-cadherin, and vimentin protein expression (P<0.05), as compared with the TGF-β1 model group. As revealed by the transwell assay and wound healing assay, TGF-β1 enhanced the migration ability of HepG2 cells (P<0.05, P<0.01) compared with the results in the blank group, compared with the TGF-β1 model group, the medication groups showed inhibited migration ability of HepG2 cells (P<0.05, P<0.01). Compared with the blank group, the TGF-β1 model group promoted the expression of p65 and Snail into the nucleus. Compared with the TGF-β1 model group, the medication groups inhibited the expression of p65 and Snail into the nucleus. ConclusionBJJW may inhibit the EMT, proliferation, and migration of HepG2 cells induced by TGF-β1 by suppressing the NF-κB signaling pathway to exert an anti-hepatocellular carcinoma effect.
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ObjectiveTo reveal the mechanism of action of Huangqi Guizhi Wuwutang in the treatment of rheumatoid arthritis by pharmacological research based on its clinical application. MethodThe collagen-induced arthritis (CIA) rat model was established by injecting bovine type Ⅱ collagen and Freund's adjuvant at the tail, and was treated with different concentrations of Huangqi Guizhi Wuwutang. The rats were randomly divided into blank group, model group, methotrexate (0.9 mg·kg-1) group, and Huangqi Guizhi Wuwutang low- and high-dose (5.13, 20.52 g·kg-1·d-1) groups, with continuous intragastric administration for 4 weeks. The degree of joint swelling, weight, degree of foot swelling and arthritis index score were determined and the pathological changes of ankle joints were detected by hematoxylin and eosin (HE) staining to observe the therapeutic effect of Huangqi Guizhi Wuwutang on rheumatoid arthritis. In addition, enzyme-linked immunosorbent assay (ELISA) and Western blot were used to measure the expression of interleukin 1β (IL-1β), interleukin 6 (IL-6), interleukin 10 (IL-10) and tumor necrosis factor-α (TNF-α) in serum and the expression of nuclear factor kappa-B (NF-κB) pathway related proteins in synovial tissue, respectively to clarify the molecular mechanism of Huangqi Guizhi Wuwutang in the treatment of rheumatoid arthritis. ResultCompared with the conditions in blank group, the body weight and IL-10 level were decreased (P<0.01), and the degree of foot swelling and arthritis index score, the levels of IL-1β, IL-6 and TNF-α, and the expression of NF-κB pathway related proteins were increased (P<0.01,) in the model group, with impaired morphology and function of the ankle joint. Additionally, compared with the model group, Huangqi Guizhi Wuwutang low- and high-dose groups had increased body weight of rats and IL-10 level (P<0.01), and reduced degree of foot swelling and arthritis index score (P<0.05, P<0.01), levels of IL-1β, IL-6 and TNF-α (P<0.01) and expression of NF-κB pathway related proteins (P<0.05, P<0.01), with improved function and morphology of the ankle joint. ConclusionHuangqi Guizhi Wuwutang can significantly alleviate joint inflammatory injury by down-regulating NF-κB pathway and reducing the inflammatory response in CIA rats.
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ObjectiveTo investigate the effect of combined therapy of lung and intestine (Mahuangtang + Da Chengqitang) on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in rats and its protective mechanism. MethodWistar rats were randomly divided into blank group, model group, low-, medium-, and high-dose groups with combined therapy of lung and intestine , and dexamethasone group. LPS (10 mg·kg-1) was given (ip) to induce ALI in rats. The general state of rats in each group was observed and recorded. The body temperature of rats in each group was recorded 0-8 h after modeling by means of anal temperature measurement. Serum and lung tissues were collected 24 h after modeling. Serum levels of interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), interleukin-10 (IL-10), and arginase-1 (Arg-1) were determined by enzyme-linked immunosorbent assay (ELISA). Western blot was used to detect the protein levels of nuclear factor kappa B p65 (NF-κB p65), phosphorylated NF-κB p65 (p-NF-κB p65), NF-κB inhibitor α (IκBα), and phosphorylated IκBα (p-IκBα) in lung tissues of rats. The levels of classically activated (M1) macrophage marker CD80 and IL-1β and macrophage markers F4/80 and IL-10 were detected by double immunofluorescence. ResultCompared with the blank group, the model group showed increased body temperature and thermal response index (TRI), elevated serum levels of pro-inflammatory factor TNF-α and IL-1β and anti-inflammatory factor IL-10 (P<0.01), up-regulated protein levels of p-NF-κB p65 and p-IκBα in lung tissues (P<0.01), and increased levels of F4/80, CD80, and IL-1β in lung tissues (P<0.01). Compared with the model group, the lung-intestine combined treatment groups and the dexamethasone group exhibited decreased body temperature and TRI in rats (P<0.01), declined serum levels of inflammatory factor TNF-α and IL-1β (P<0.05, P<0.01), elevated serum levels of anti-inflammatory factor IL-10 and Arg-1 (P<0.05, P<0.01), down-regulated protein levels of p-NF-κB p65 and p-IκBα in lung tissues (P<0.05, P<0.01), decreased levels of CD80 and IL-1β, and increased levels of IL-10 in lung tissues (P<0.01), while the level of F4/80 was not significantly changed. ConclusionThe combined therapy of lung and intestine can obviously alleviate the fever and inflammatory state of ALI rats, and the mechanism may be related to the inhibition of NF-κB inflammatory pathway and the polarization of lung tissue macrophages to anti-inflammatory phenotype.
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ObjectiveTo observe the intervention of modified Sanrentang on the lipopolysaccharide-induced proliferation of rat glomerular mesangial cells, the phosphatidylinositol-3 kinase(PI3K)/protein kinase B(PKB/Akt)/nuclear factor kappa B(NF-κB) signaling pathway, and to investigate its mechanism in improving kidney inflammation in rats with immunoglobulin A nephropathy(IgAN). MethodThe 18 rats were divided into 3 groups by serum pharmacology method: normal group, high-dose and low-dose (20.70,10.35 g·kg-1·d-1) groups with 6 rats in each group. Modified Sanrentang high- and low-dose groups were intragastric with the corresponding solution of modified Sanrentang, and normal group was intragastric with equal volume of distilled water. After 5 days of intragastric administration, blood samples were collected to prepare drug-containing serum. Rat mesangial (HBZY-1) were divided into five groups of normal group, LPS 10 mg·L-1 in the model group, benazepril(50 μmol·L-1), modified Sanrentang high- and low-dose group. Preclude the use of methyl thiazolyl tetrazolium(MTT) method detect the proliferation activity of HBZY-1 cells, enzyme-linked immunosorbent assay(ELISA) was used to determine the content of each group type Ⅳ collagen(ColⅣ),Western blot and Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR) were used to detect protein and mRNA expression levels of PI3K/Akt/NF-κB signaling pathway. ResultAs compared with the normal group, MTT assay showed that exposure to LPS significantly enhanced the proliferative activity, the ColⅣ was increased significantly of HBZY-1 cells(P<0.01), p-Akt, p-p65 was increased significantly (P<0.01). Compared with the model group, the proliferation and ColⅣ of rat chronic glomerulonephritis cells induced by LPS by inhibiting PI3K/Akt/NF-κB signaling pathway(P<0.01), and the phosphorylation of Akt was significantly inhibited(P<0.01), the expression levels of NF-κB p65 was reduced in modified Sanrentang high-dose group(P<0.01). ConclusionModified Sanrentang could inhibit cell proliferation and the content of ColⅣ in rat mesangial cells induced by LPS, and its mechanism might be related to suppression of PI3K/Akt signaling pathway.
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ObjectiveTo explore the effect and mechanism of Sangmei Zhike granule (SMZK) on airway inflammation in rats with cough variant asthma(CVA). MethodSix-week-old male SD rats were randomly divided into normal group, model group, and SMZK (2.48 g·kg-1) group. The rats in the model group and the SMZK group received intraperitoneal injection of a mixed solution containing 10% ovalbumin (OVA) and aluminium hydroxide on the 1st and 8th days and aerosol inhalation of 1% OVA solution from the 15th day for CVA model induction. The intervention lasted for two weeks from the 15th day. At the end of animal manipulation, the lung function was detected and inflammatory cells in the peripheral blood were counted. The serum interleukin-4 (IL-4), interleukin-5 (IL-5), and interleukin-10 (IL-10) levels were determined. Hematoxylin-eosin (HE) staining was performed on the lungs. Western blot was used to detect the protein expression of nuclear factor kappa-B (NF-κB) and its inhibitor α(IκBα) in lung tissues. ResultCompared with the normal group, the model group showed reduced forced expiratory volume in the first 0.1 second (FEV0.1),FEV0.1/forced vital capacity (FVC),and forced expiratory flow 50% (FEF50%) (P<0.05, P<0.01), increased white blood cells and eosinophils (P<0.01), and up-regulated serum IL-4, IL-5, and IL-10 (P<0.01). As revealed by HE staining, the model group displayed shed epithelial cells of the bronchus, airway stenosis, hyperplasia and expansion of mucous glands, disarrangement of layer structures, disorderly arranged cells, and extensive infiltration of inflammatory cells. The protein expression of NF-κB p65 was higher (P<0.01) and that of IκBα was lower (P<0.01) in the lung tissues of the model group than that in the normal group. Compared with the model group, the SMZK group showed increased FEV0.1,FEV0.1/FVC,and FEF50% (P<0.05), decreased white blood cells and eosinophils in the peripheral blood (P<0.01), and declining serum IL-4, IL-5, IL-10 (P<0.01). HE staining demonstrated mild bronchial mucosal injury and relieved inflammatory cell infiltration, gland hyperplasia, and epithelial degeneration and necrosis in the SMZK group. The protein expression of NF-κB p65 was decreased (P<0.05) and that of IκBα was increased (P<0.05) in lung tissues of the SMZK group than that in the model group. ConclusionSMZK can improve lung function and inhibit airway inflammation in rats with CVA. The underlying mechanism may be related to the regulation of IκBα/NF-κB protein expression in the lungs.
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ObjectiveTo investigate the mechanism by which Shenbai Jiedu prescription (SBJDF) inhibits the proliferation of colorectal cancer (CRC) HCT116 cells. MethodAfter 48 h treatment of HCT116 cells with SBJDF (0, 0.25, 0.5, 1, 2, 4 g·L-1), the viability of HCT116 cells were determined by methyl thiazolyl tetrazolium (MTT) colorimetry. Following the classification of cells into blank control group and SBJDF (1, 2, 4 g·L-1) groups, the effect of SBJDF on HCT116 cell morphology was observed under an inverted microscope. The effects of SBJDF on the proliferation of HCT116 cells and mitochondrial membrane potential (Δψm) were detected by colony formation assay and JC-1 probe, respectively. The flow cytometry was then performed for determining cell cycle distribution and apoptosis. The effects of SBJDF on cell cycle-, apoptosis-, and nuclear factor kappa-B (NF-κB) signaling pathway-related proteins were determined by Western blot. ResultSBJDF effectively inhibited the vitality of HCT116 cells and changed their morphology in a concentration-dependent manner. Compared with the blank control group, SBJDF at 1, 2, 4 g·L-1 significantly reduced cell colony formation (P<0.05, P<0.01),and SBJDF at 2 and 4 g·L-1 arrested the HCT116 cell cycle at G0/G1 phase (P<0.05, P<0.01). Compared with the blank control group, SBJDF at 1, 2, 4 g·L-1 remarkably down-regulated the protein expression of CyclinD1 (P<0.05, P<0.01). SBJDF at 2 and 4 g·L-1 lowered the CyclinA2 and cyclin-dependent kinase 4 (CDK4) (P<0.05, P<0.01). SBJDF at 4 g·L-1 reduced the cyclin-dependent kinase 1 (CDK1) (P<0.01). Compared with the blank control group, SBJDF at 2 and 4 g·L-1 induced HCT116 cell apoptosis, down-regulated the protein expression of anti-apoptosis-related proteins Bcl-2 and Bcl-xl as well as the NF-κB signaling pathway-related proteins IκB kinase α (IKKα),inhibitor α of NF-κB (IκBα),and phospho-NF-κB p65 (p-p65) (P<0.05, P<0.01), and diminished the mitochondrial membrane potential of HCT116 cells. ConclusionSBJDF inhibits the proliferation of HCT116 cells, which may be related to its inhibition of the activation of NF-κB signaling pathway and the induction of cell cycle arrest and apoptosis.
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ObjectiveTo explore the effects of phillygenin (PHI) on the inflammation in L02 cells induced by lipopolysaccharide (LPS) and adenosine triphosphate (ATP) and the expression of purinergic 2X7 receptor (P2X7R), NOD-like receptor family pyrin domain containing 3 (NLRP3), and nuclear factor kappa B (NF-κB) expression. MethodIn this study, the inflammation model was induced in L02 cells by 100 μg·L-1 LPS treatment for 24 h and 5 mmoL·L-1 ATP treatment for 5 h. The cells in the PHI groups were cultured with PHI (100, 50, 25 mg·L-1) for 6 h in the LPS treatment period, followed by LPS treatment for another 18 h. After ATP treatment for 5 h, the mRNA and protein expression of interleukin-1β (IL-1β), interleukin-18 (IL-18), P2X7R, NLRP3, Caspase-1 precursor (pro-Caspase-1), cleaved Caspase-1, NF-κB, and NF-κB inhibitor protein α (IκBα) in L02 cells was detected by real-time fluorescence quantitative polymerase chain reaction (Real-time PCR) and Western blot, respectively. Molecular docking was used to predict whether P2X7R could bind to PHI, and DCFH-DA was employed to detect the accumulation of reactive oxygen species (ROS) in cells. P2X7R was silenced by small interfering ribonucleic acid (siRNA), and then the mRNA expression of IL-1β, IL-18, P2X7R, NLRP3, Caspase-1, NF-κB, and IκBα was detected by Real-time PCR. ResultReal-time PCR and Western blot showed that compared with the normal group, the model group showed increased expression of IL-1β and IL-18 (P<0.05), and compared with the model group, the PHI groups showed down-regulated IL-1β, IL-18 mRNA and protein expression (P<0.05). Molecular docking suggested a good binding effect of PHI to P2X7R. Real-time PCR and Western blot analysis showed that the expression of P2X7R in the model group was significantly up-regulated compared with that in the normal group (P<0.05), and compared with the model group, the PHI groups showed down-regulated mRNA and protein expression of P2X7R (P<0.05). DCFH-DA results showed that compared with the normal group, the model group showed increased content of ROS (P<0.05), and compared with the model group, the PHI groups decreased the accumulation of ROS (P<0.05). As demonstrated by Real-time PCR and Western blot, compared with the normal group, the model group showed increased expression of NLRP3 inflammasome and NF-κB (P<0.05), and compared with the model group, the PHI groups significantly decreased the mRNA and protein expression of NLRP3 and cleaved Caspase-1, and up-regulated the mRNA and protein expression of NF-κB and IκBα (P<0.05). Real-time PCR analysis showed that compared with the results in the model group, after silencing P2X7R by siRNA, the mRNA expression of IL-1β, IL-18, P2X7R, NLRP3, Caspase-1, NF-κB, and IκBα was decreased (P<0.05). PHI exerted the same effect, and the mRNA expression was further reduced after the combination of them. ConclusionPHI is presumed to suppress the expression of the NLRP3/NF-κB signaling pathway by down-regulating upstream P2X7R to alleviate the LPS/ATP-induced inflammation in L02 cells, suggesting that P2X7R may be the target of PHI against inflammation.
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Objective:To explore the possible mechanism of Astragali Radix-Curcumae Rhizoma (AC) in inhibiting tumor growth in the orthotopic transplantation model of colon cancer in mice. Method:The molecular docking technology was used to predict the intermolecular interaction between the main active components of AC and the pathway target proteins, such as stromal cell-derived factor-1 (SDF-1), C-X-C motif chemokine receptor 4 (CXCR4), and nuclear factor kappa-B p65 (NF-<italic>κ</italic>B p65). The orthotopic transplantation model of CT26.WT colon cancer was established in mice for <italic>in vivo</italic> experimental verification. Sixty BALB/c male mice were randomly divided into a sham operation group, a model group, a 5-fluorouracil (5-Fu, 30 mg·kg<sup>-1</sup>) group,and low- (0.32 g·kg<sup>-1</sup>), medium- (0.64 g·kg<sup>-1</sup>), and high-dose (1.28 g·kg<sup>-1</sup>) AC groups, with 10 mice in each group. The sham operation group and the model group received normal saline by gavage. The corresponding drugs were administered by gavage in the 5-Fu group and by intraperitoneal injection in the AC groups. After intervention for 15 days, the tumor <italic>in situ</italic> was completely stripped, and the colon tissues 5-6 cm in length adjacent to the tumor were taken. The tumor volume was measured and calculated. The pathological changes of tumor tissues and colon tissues were observed by Hematoxylin-Eosin (HE) staining. Western blot was used to detect the protein expression of SDF-1, CXCR4, p-NF-<italic>κ</italic>B p65 in colon tissues. Western blot and Real-time quantitative polymerase chain reaction (Real-time PCR) were used to detect SDF-1, CXCR4, NF-<italic>κ</italic>B p65, Cyclin D<sub>1</sub>, oncogene c-Myc protein and mRNA expression in tumor tissues. Result:Compared with the model group, 5-Fu and AC groups showed reduced tumor volumes <italic>in situ</italic> (<italic>P</italic><0.05, <italic>P</italic><0.01), with the tumor inhibition rate in the 5-Fu group as high as (61.38±2.34)%. The tumor-inhibiting effect was optimal in the medium-dose AC group, with the tumor inhibition rate of (43.43±3.71)%. Compared with the model group, 5-Fu and AC groups showed relieved pathological changes of tumor and colon tissues. Specifically, AC down-regulated the protein expression levels of SDF-1, CXCR4, and p-NF-<italic>κ</italic>B p65 in colon tissues (<italic>P</italic><0.01), and down-regulated the protein and mRNA expression levels of SDF-1, CXCR4, NF-<italic>κ</italic>B p65, Cyclin D<sub>1</sub>, and c-Myc in tumor tissues (<italic>P</italic><0.05, <italic>P</italic><0.01). Conclusion:AC can inhibit the growth of orthotopic transplantation tumor of colon cancer, and its intervention mechanism may be related to the regulation of related protein and mRNA expression in the SDF-1/CXCR4/NF-<italic>κ</italic>B signaling pathway.
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Objective:To investigate the effects of ligustrazine combined with emodin on angiogenesis of ascites carcinoma Walker-256 cells by observing their inhibition against nuclear factor-<italic>κ</italic>B (NF-<italic>κ</italic>B), hypoxia-inducible factor-1<italic>α</italic> (HIF-1<italic>α</italic>), and vascular endothelial growth factor-C (VEGF-C) in HIF signaling pathway. Method:Fifty SD rats were randomly divided into sham operation group, model group, ligustrazine group, emodin group and ligustrazine combined with emodin group. Following the in situ injection of rat ascites carcinoma Walker-256 cells into the liver of normal rats, they were grouped and administered with ligustrazine (10 mg·kg<sup>-1</sup>), emodin (10 mg·kg<sup>-1</sup>), and ligustrazine (10 mg·kg<sup>-1</sup>) plus emodin (10 mg·kg<sup>-1</sup>) for seven days. Afterwards, the tumor-inoculated liver tissue was sampled from the experimental group and prepared into pathological sections for investigating tumor cell survival and VEGF expression. The <italic>in vitro</italic> hypoxia and hypoglycemia model (oxygen-glucose deprivation model), hypoxia model, and hypoglycemia model of Walker-256 cells were constructed respectively. In the ligustrazine group, emodin group, and ligustrazine combined with emodin group, three consecutive concentrations that did not affect the proliferation of Walker-256 cells were selected for investigation. The drugs were administered before modeling, and the model treatment lasted for 4 h. The levels of HIF-1<italic>α</italic>, VEGF-C, and NF-<italic>κ</italic>B in the cell culture supernatant of each group were tested. Result:After the rat liver was inoculated with Walker-256 cells, the total liver mass was significantly increased(<italic>P</italic><0.05), higher than that in the ligustrazine group, the emodin group, or the ligustrazine combined with emodin group(<italic>P</italic><0.05). Histopathological examination showed that the response of VEGF expression in the liver tissue of each administration group was lower than that of the model group. At the cellular level, the levels of HIF-1<italic>α</italic>, VEGF-C, and NF-<italic>κ</italic>B in oxygen-glucose deprivation model of the ligustrazine group and the ligustrazine combined with emodin group were significantly reduced(<italic>P</italic><0.05), exhibiting a certain dose-dependent response, followed by the reduction in the hypoxia model. The levels of HIF-1<italic>α</italic> and NF-<italic>κ</italic>B in the oxygen-glucose deprivation model and the hypoglycemia model of the emodin(1×10<sup>-2</sup>,1×10<sup>-3</sup> mol·L<sup>-1</sup>) group and the ligustrazine combined with emodin(1×10<sup>-2</sup>,1×10<sup>-3</sup> mol·L<sup>-1</sup>) group were significantly reduced, but there was no significant change in VEGF-C level of the hypoxia model of all the administration groups. Conclusion:Ligustrazine or emodin alone or their combination inhibits the abnormal increase in the weight of rat liver after inoculation with Walker-256 cells and the expression of VEGF in the liver tissue. Ligustrazine and emodin inhibit the protein expression of NF-<italic>κ</italic>B and HIF-1<italic>α</italic>, thereby reducing the gene and protein expression of metastasis-related target VEGF-A activated by HIF-1<italic>α</italic> transcription, restricting tumor cell neovascularization, and inhibiting the invasion and spread of ascites carcinoma cells. Among them, ligustrazine has the most significant effect against hypoxia. Glucose interferes with the effect of ligustrazine. The combination of ligustrazine with emodin is conducive to diminishing the intervention of glucose and stabilizing the inhibition against tumor cells.
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Objective:To explore the effect and mechanism of Xiangshenwan on ulcerative colitis (UC) induced by dextran sulfate sodium (DSS) in mice based on the classic Toll-like receptor (TLR)/nuclear factor kappa B (NF-<italic>κ</italic>B) signaling pathway. Method:The experimental mice were divided into a normal group, a model group, a Xiangshenwan group, and a mesalazine group. The mice, except for those in the normal group, received 3% DSS solution for 7 days to establish the acute UC model and were treated with Xiangshenwan (5 g·kg<sup>-1</sup>) and mesalazine (300 mg·kg<sup>-1</sup>) continuously from the 1st day to the 10th day of modeling. The body weight, disease activity index (DAI), colon weight, intestinal weight index, colon length, colon weight per unit length, and pathological changes of mice were evaluated respectively. The protein expression of TLR5, myeloid differentiation factor 88 (MyD88), interleukin-1 receptor-associated kinase 4 (IRAK4), tumor necrosis factor receptor-associated factor 6 (TRAF6), transforming growth factor <italic>β</italic>-activated kinase 1 (TAK1), p38 mitogen-activated protein kinase (MAPK), NF-<italic>κ</italic>B, IRAK1, TAK1-binding protein 1 (TAB1), TAB2, mitogen-activated protein kinase kinase 3 (MKK3), MKK6 and cyclic adenosine monophosphate response element-binding protein (CREB) in colon tissues of mice was detected by Western blot. Result:Compared with the normal group, the model group showed decreased body weight of mice, increased DAI scores, elevated colon weight, intestinal weight index, and colon weight per unit length, shortened colon length, severe colonic mucosal injury, and up-regulated protein expression of TLR5, MyD88, IRAK4, TRAF6, TAK1, p38 MAPK, NF-<italic>κ</italic>B, IRAK1, TAB1, TAB2, MKK3, MKK6, and CREB in colon tissues (<italic>P</italic><0.05,<italic> P</italic><0.01<bold>).</bold> Compared with the model group, the Xiangshenwan group and the mesalazine group displayed increased body weight of mice, decreased DAI scores, declining colon weight, intestinal weight index, and colon weight per unit length, increased colon length, improved colonic mucosal injury, and down-regulated protein expression of TLR5, MyD88, IRAK4, TRAF6, TAK1, p38 MAPK, NF-<italic>κ</italic>B, IRAK1, TAB1, TAB2, MKK3, MKK6, and CREB in colon tissues (<italic>P</italic><0.05,<italic> P</italic><0.01). Conclusion:Xiangshenwan can effectively treat DSS-induced UC presumedly by the inhibition of TLR/NF-<italic>κ</italic>B signaling pathway.