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Reflux esophagitis is an inflammatory disease of esophageal mucosa damage caused by the reflux of gastric contents into the esophagus. Its incidence is on the rise， and it has become an important precancerous disease of esophageal cancer. Studies have shown that the continuous inflammatory response stimulates the esophageal mucosa， causing abnormal proliferation of esophageal epithelial cells and damage to esophageal mucosal tissue， which eventually leads to the occurrence of heterogeneous hyperplasia and even carcinogenesis. The nuclear transcription factor-kappa B （NF-κB） signaling pathway is one of the most classical inflammatory and cancer signaling pathways. It has been found that abnormal activation of the NF-κB signaling pathway is crucial to the development and prognosis of reflux esophagitis and esophageal cancer. It is widely involved in the proliferation， autophagy， apoptosis， and inflammatory response of esophageal epithelial cells and tumor cells， accelerating the transformation of reflux esophagitis to esophageal cancer and making it a potential target for the treatment of reflux esophagitis and esophageal cancer. Currently， there is no specific treatment for reflux esophagitis and esophageal cancer， and large side effects often appear. Therefore， finding a promising and safe drug remains a top priority. In recent years， traditional Chinese medicine scholars have conducted a lot of research on NF-κB signaling pathway， and the results indicate that NF-κB signaling pathway is an important potential target for traditional Chinese medicine to prevent and treat reflux esophagitis and esophageal cancer， but there is a lack of comprehensive and systematic elaboration. Therefore， this paper summarized the relevant studies in recent years， analyzed the relationship among NF-κB signaling pathway， reflux esophagitis， esophageal cancer， and transformation from inflammation to cancer， and reviewed the research literature on the regulation of the NF-κB signaling pathway in traditional Chinese medicine to prevent and treat reflux esophagitis and esophageal cancer， so as to provide new ideas for the prevention and treatment of reflux esophagitis and esophageal cancer.
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ObjectiveTo explore the protective effect and mechanism of Zingiberis Rhizoma Recens alcohol extract on lipopolysaccharide （LPS）-induced acute lung injury in mice. MethodBalb/c mice were randomly divided into normal group， model group， dexamethasone group， and low- and high-dose Zingiberis Rhizoma Recens groups. Mice in the normal group were instilled with normal saline through the nose， and the other groups were instilled with normal saline containing LPS （50 μg）. After 30 minutes of modeling， the dexamethasone group was gavaged with 5 mg·kg-1 of dexamethasone acetate solution， the low- and high-dose Zingiberis Rhizoma Recens groups were gavaged with different doses of （7， 14 g·kg-1） of Zingiberis Rhizoma Recens alcohol extract， and the normal group and the model group were gavaged with the same volume of water. After 24 hours of modeling， the total number of white blood cells in bronchoalceolar lavage fluid （BALF） was detected by cell counter， and the levels of the inflammatory factors including tumor necrosis factor （TNF）-α， interleukin （IL）-1β， IL-6， and superoxide dismutase （SOD）， and myeloperoxidase （MPO） was detected by enzyme-linked immunosorbent assay （ELISA）. Haematoxylin-eosin （HE） staining method was used to observe the pathological changes of lung tissue in each group， and the Western blot was used to detect the protein expression of nuclear transcription factor （NF）-κB p65， phosphorylation （p）-NF-κB p65， and Toll-like receptor 4 （TLR4） in lung tissue. ResultCompared with the normal group， the white blood cell count in BALF and the levels of TNF-α， IL-1β， IL-6， and MPO in the model group was increased （P<0.01）， and the level of SOD was decreased （P<0.05）. Pathological damage of lung tissue was obvious， and the protein expression of NF-κB p65， p-NF-κB p65， and TLR4 in lung tissue was increased （P<0.01）. Compared with the model group， the white blood cell count in BALF and the levels of TNF-α， IL-1β， IL-6， and MPO in the treatment group was decreased （P<0.05，P<0.01）， and the level of SOD was increased （P<0.05，P<0.01）. Pathological damage of lung tissue was alleviated， and the protein expression of NF-κB p65， p-NF-κB p65， and TLR4 in lung tissue was decreased （P<0.01）. ConclusionZingiberis Rhizoma Recens alcohol extract may play a protective role in LPS-induced acute lung injury in mice by inhibiting the TLR4/NF-κB signaling pathway.
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ObjectiveTo investigate the effect and mechanism of echinacoside （ECH） in improving liver injury in rats with acute pancreatitis by establishing a rat model of acute pancreatitis and liver injury. MethodsA total of 24 Sprague-Dawley rats were randomly divided into blank group （Con group）， control group （Con+ECH group）， acute pancreatitis group （AP group）， and acute pancreatitis+ECH intervention （AP+ECH group）. The rats were given intraperitoneal injection of 10 mg/kg ECH on day 7 before the establishment of the model of acute pancreatitis； at 24 hours after the last administration of cerulein， blood samples were collected via the abdominal aorta， and serum was separated for biochemical analysis including alanine aminotransferase （ALT）， aspartate aminotransferase （AST）， lactate dehydrogenase （LDH）， gamma-glutamyl transpeptidase （GGT）， alkaline phosphatase （ALP）， albumin （Alb）， total bilirubin （TBil）， cholinesterase， blood amylase （Amy）， and lipase （LPS）. HE staining was used to observe the histopathological changes of the pancreas and the liver； transmission electron microscopy （TEM） was used to observe the microstructural changes of pancreas and liver tissue； ELISA was used to measure the levels of interleukin-1β （IL-1β）， interleukin-16 （IL-6）， tumor necrosis factor-α （TNF-α）， and interleukin-10 （IL-10） in liver tissue homogenate； immunohistochemistry was used to measure the levels of TNF-α and p-p65 NF-κB in pancreas and liver tissue； Western blot was used to measure the expression levels of NF-κB pathway proteins in liver tissue. A one-way analysis of variance was used for comparison of continuous data between multiple groups， and the SNK test or the Dunnett’s T3 method was used for further comparison between two groups. ResultsCompared with the Con group， the AP group had significant increases in ALT， AST， GGT， LDH， ALP， TBil， Amy， and LPS （all P<0.01）， as well as significant increases in the levels of IL-1β， IL-6， IL-10， and TNF-α in liver tissue homogenate （all P<0.01）. ECH intervention reduced the levels of ALT， AST， GGT， LDH， ALP， TBil， AMY， and LPS and inhibited the secretion of IL-1β， IL-6， and TNF-α in rats with acute pancreatitis. HE staining showed that ECH intervention alleviated the vacuolar degeneration of acinar cells， inflammatory cell infiltration in pancreatic tissue， and the necrosis of hepatocytes compared with the AP group. TEM showed that compared with the AP group， there was a reduction in the degree of mitochondrial swelling in liver and pancreatic cells after ECH intervention. ECH intervention partially reversed the elevated expression levels of p-p65 NF-κB and TNF-α in liver and pancreatic tissue. In addition， the expression levels of MyD88， p-IκBα， p-IKKα， and p-p65 were upregulated in liver tissue of rats with acute pancreatitis， which could be partially reversed after ECH intervention. ConclusionEchinacoside can alleviate liver and pancreatic injury induced by acute pancreatitis by inhibiting the TLR4/MyD88/NF-κB pathway.
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Parkinson's disease （PD） is a common neurological degenerative disease in the middle-aged and elderly， characterized by pathological changes of progressive degeneration of dopaminergic neurons in the substantia nigra and Lewy body formation， with high prevalence and long course of disease. The drug is mainly used to treat PD in western medicine， and the early curative effect is remarkable. However， with the progression of the disease and the long-term use of the drug， the efficacy will be significantly reduced， or there may be sports complications， and the long-term efficacy is not good. As a traditional medical system， traditional Chinese medicine has a unique understanding of PD. Traditional Chinese medicine plays an important role in the treatment of PD， which is natural， mild， safe， and effective， and it can cooperate with western medicine to enhance its efficacy and reduce the adverse reactions of western medicine. The pathogenesis of PD is complex， involving multiple levels such as mitochondrial dysfunction and apoptosis. Neuroinflammation is also involved in the progressive degeneration of dopaminergic neurons in PD. The Toll-like receptor 4 （TLR4）/nuclear factor-κB （NF-κB） signaling pathway is a classic inflammatory pathway， and its expression changes play an important role in the occurrence and development of inflammatory response in the body. In recent years， the research on this pathway in TCM is increasing. This paper summarized the literature of traditional Chinese and western medicine in the past 10 years and reviewed the relevant mechanism of TCM regulation of TLR4/NF-κB pathway in the treatment of PD from the aspects of TCM monomer， compound， and other TCM therapies， so as to provide some references for the search for new targets of drug therapy and gene therapy and the in-depth study of TCM prevention and treatment of PD.
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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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ObjectiveTo investigate the effects of Tongluo Juanbi granules on chondrocyte apoptosis and Toll-like receptor 4 （TLR4）/myeloid differentiation factor 88 （MyD88）/nuclear factor-κB （NF-κB） signaling pathway of rabbits with knee osteoarthritis （KOA） and study the mechanism of Tongluo Juanbi granules in the prevention and treatment of KOA. MethodThirty New Zealand rabbits were randomly assigned to the following five groups （n=6）： sham group， model group， low-dose and high-dose groups of Tongluo Juanbi granules （4.1 and 8.2 g·kg-1·d-1）， and celecoxib group （10.9 mg·kg-1·d-1）. The KOA model was established by destabilization of the medial meniscus （DMM） for six weeks. Six weeks after the modeling， the drug was given once a day for eight weeks. The pathological changes of cartilago articularis were observed by hematoxylin-eosin （HE） staining and Safranin O-Fast Green staining. Terminal deoxynucleotidyl transferase dUTP nick end labeling （TUNEL） staining was performed to detect chondrocyte apoptosis. Enzyme-linked immunosorbent assay （ELISA） was used to detect the contents of interleukin-1β （IL-1β） and tumor necrosis factor-α （TNF-α） in synovial fluid. The mRNA and protein expression levels of genes related to the TLR4/MyD88/NF-κB signaling pathway were detected by real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） and Western blot， respectively. ResultCompared with the sham group， the cartilago articularis of the model group significantly degenerated. Mankin's score was increased （P<0.01）， and the contents of IL-1β and TNF-α in synovial fluid were increased （P<0.01）. The number of apoptosis of chondrocytes was increased （P<0.01）. The mRNA and protein expressions of TLR4， MyD88， and NF-κB p65 in cartilage tissue were up-regulated （P<0.01）， while the mRNA and protein expressions of Bcl-2 were down-regulated （P<0.01）. Compared with the model group， chondrocyte degeneration in both low-dose and high-dose groups of Tongluo Juanbi granules was improved， and Mankin's score was decreased （P<0.01）. The contents of IL-1β and TNF-α were decreased （P<0.01）， and the number of apoptosis of chondrocytes was decreased （P<0.01）. The mRNA and protein expressions of TLR4， MyD88， and NF-κB p65 in cartilage tissue were down-regulated （P<0.01）， while the mRNA and protein expressions of Bcl-2 were up-regulated （P<0.01）. In addition， in the above observation indicators， the high-dose group of Tongluo Juanbi granules was significantly superior to the low-dose group of Tongluo Juanbi granules. ConclusionTongluo Juanbi granules could inhibit chondrocyte apoptosis in rabbits with KOA and improve cartilage degeneration， which may be related to inhibiting inflammatory responses mediated by TLR4/MyD88/NF-κB signaling pathway.
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ObjectiveTo study the effects of Epimedii Folium polysaccharides on mice with exercise-induced fatigue and explore its possible mechanism of action. MethodICR male mice screened by swimming training were randomly divided into a control group， model group， vitamin C group， and low， medium， and high dose groups of Epimedii Folium polysaccharides， with eight mice in each group. The exercise-induced fatigue model was established by weight-bearing swimming training in each group except for the control group. After two weeks of weight-bearing swimming， the Epimedii Folium polysaccharide groups were given 100， 200， 400 mg∙kg-1 of Epimedii Folium polysaccharides by gavage， and the vitamin C group was given 200 mg∙kg-1 of vitamin C by gavage. The control group and the model group were given equal amounts of saline for 14 d. At the end of the experimental period， the body mass of the mice in each group and the time of last swimming due to exhaustion were recorded. Serum urea nitrogen （BUN）， lactic acid （LA）， lactate dehydrogenase （LDH）， malondialdehyde （MDA）， superoxide dismutase （SOD）， glutathione peroxidation （GSH-Px）， myoglycogen （MG） in skeletal muscle， hepatic glycogen （HG） in the liver were detected by kits. Hematoxylin-eosin （HE） staining was used to observe the pathological changes in muscle tissue. Western blot was used to detect the protein expression of p38 mitogen-activated protein kinase （p38 MAPK）， phosphorylation （p）-p38 MAPK， extracellular signal-regulated kinase1/2 （ERK1/2）， nuclear factor-κB （NF-κB）， p-NF-κB， interleukin-1β （IL-1β）， and interleukin-6 （IL-6） in muscle tissue. The immunofluorescence （IF） method was used to detect the expression of tumor necrosis factor-α （TNF-α） in skeletal muscle tissue of mice in each group. ResultCompared with the control group， the body mass of mice in the model group decreased， and the time of last swimming due to exhaustion decreased （P<0.01）. In addition， there were significantly higher serum levels of the fatigue metabolites LA， LDH， BUN， and lipid peroxidation product MDA （P<0.01） and decreased levels of MG， HG， SOD， and GSH-Px （P<0.01）. The protein expressions of p-p38 MAPK， ERK1/2， p-NF-κB， IL-1β， IL-6， and TNF-α in skeletal muscle tissue were significantly higher than those of the control group （P<0.01）. Compared with the model group， the body mass and time of last swimming due to exhaustion of the mice in the low， medium， and high dose groups of Epimedii Folium polysaccharides and the vitamin C group were increased （P<0.05， P<0.01）， and the contents of LA， LDH， BUN， and MDA were significantly decreased （P<0.05， P<0.01）. The levels of MG， HG， SOD， and GSH-Px increased （P<0.05， P<0.01）， and the protein expression levels of p-p38 MAPK， ERK， p-NF-κB， IL-1β， IL-6， and TNF-α in skeletal muscle tissue decreased （P<0.05， P<0.01）. ConclusionEpimedii Folium polysaccharides can play a role in alleviating exercise-induced fatigue by inhibiting the p38 MARK/NF-κB signaling pathway， thereby reducing the accumulation of metabolites， improving the activity of antioxidant enzymes， increasing the glycogen content of the body， and reducing inflammation in skeletal muscle.
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ObjectiveTo study the anti-inflammatory effects of Blumea balsamifera （L.） DC oil （BBO） based on nuclear factor kappa-B （NF-κB） nonclassical and arachidonic acid （AA） pathway. MethodsEffects of BBO on the production of slow reacting substance of anaphylaxis （SRS-A） were detected by the ileal smooth muscle method. The contents of prostaglandin E2 （PGE2） and leukotriene B4 （LTB4） in lipopolysaccharides （LPS） -induced macrophages were detected by ELISA kit. The expression of COX-2， 5-LOX， FLAP and RelB were detected by qRT-PCR. Western blot was performed to detect the effects of BBO on the level of NF-κB nonclassical pathway proteins TNF receptor associated factor 3 （TRAF3）， TNF receptor associated factor 2 （TRAF2）， NF-κB-inducing kinase （NIK）， p100 and RelB. ResultsThe contractile tension of guinea pig ileum was reduced （P<0.001）， and the SRS-A production inhibition rate reached 65.34% at 1mg·mL-1 BBO concentration. Compared with LPS group， BBO reduced the concentrations of PGE2 （P<0.05） and LTB4 （P<0.05）， and decreased the expressions of COX-2 （P<0.05）， 5-LOX （P<0.05） and FLAP （P<0.05） in AA pathway at concentrations of 40-80 μg·mL-1. Moreover， 40-80 μg·mL-1 BBO decreased the concentrations of TRAF3 （P<0.05）， TRAF2 （P<0.05）， and NIK （P<0.05）， and further inhibited the phosphorylation of p100 （P<0.05）， as well as the level of the transcription factor RelB in genes （P<0.05） and proteins （P<0.05） in nonclassical NF-κB pathway， whereas BBO did not cause such changes. ConclusionBBO may potentially exert its anti-inflammatory effects by suppressing the regulatory proteins TRAF3 and TRAF2 and the transcription factor RelB in NF-κB nonclassical pathway. The inhibitory action extending to the induction kinase function of NIK， further hindering the phosphorylation of p100 and its binding with the transcription factor RelB. Consequently， downstream elements in the AA pathway， including the pivotal rate-limiting enzymes COX-2， 5-LOX and FLAP， were altered. This modulation influences the levels of inflammatory mediators such as PGE2 and LTB4.
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Aim To investigate the effect of histamine H, receptor (HjR) on the immune responses in astrocytes induced by lipopolysaccharide (LPS) and the regulatory mechanism of its signaling pathway. Methods LPS was used to establish an in vitro astrocyte inflammation model. Rat primary astrocytes were divided into the control group, LPS group, LPS + Hj R agonist group (2-pyridylethlamine, Pyri), and HjR agonist group. Astrocytes were treated with Pyri 100 p,mol • L~ for 1 h, then stimulated with LPS at 100 p,g • L~ for 24 h. Cell viability was measured using the CCK-8 assay. The expression of GFAP and HjR was detected by immunofluorescence. Glial morphological changes were observed under a microscope. The levels of proinflammatory mediators (TNF-a and IL-6) were detected by ELISA. The protein expressions of p-Akt, Akt, p-NF-KB p65, and NF-KB p65 were detected by Western blot. Results Compared with the control group, more activated astrocytes with fewer cell processes and branches were observed in the LPS group. Besides, LPS enhanced the GFAP expression level, reduced the H,R expression level and stimulated the production of TNF-a and IL-6 from astrocytes. Pre treatment with Pyri for 1 h ameliorated the glial morphological changes stimulated by LPS, inhibited LPS-induced upregulation of GFAP level and the inflammatory factors secretion. In addition, LPS stimulated astrocytes showed a higher phosphorylation of Akt and NF-KB p65, which was also ameliorated by Pyri. Conclusions H, R agonist can inhibit LPS-induced astrocyte activation and inflammatory factor secretion, and the Akt/NF-KB signaling pathway may be an important pathway for the involvement of H,R in immune regulation.
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Aim To investigate the effect of colchicine on lipopolysaccharide (LPS) induced endothelial to mesenchymal transition (EndMT) in human umbilical vein vascular endothelial cells (HUVECs) and its related mechanisms. Methods The EndMT model was established by treating HUVECs with LPS. Cell proliferation rate was detected by CCK-8 assay, cytotoxicity was detected by LDH assay, and the optimal drug concentration was screened. The cells were divided into the normal control group, the normal control + colchicine (10 nmol • L) group, the LPS (10 mg • L) model group, and the LPS + colchicine (10 nmol • L) group. The morphologic changes of the cells were observed under an inverted microscope, the cell migration ability was detected by Transwell assay, and the ability of tube formation was analyzed by tube formation assay. The expression of endothelial markers (CD31/ VE-cadherin) and mesenchymal cell markers (a-SMA/FSP-1) were detected by Western blot. NF-KB inhibitor was used to detect the changes in related signaling pathways. Results CCK-8 and LDH experiments showed that 10 nmol • L colchicine was the optimal concentration. LPS could induce morphological changes in HUVECs, and colchicine could reverse morphological changes in HUVECs to a certain extent. Transwell experiment showed that the migration ability of HUVECs in the LPS treatment group was significantly enhanced (P < 0. 05), and colchicine could significantly reverse this phenomenon (P < 0. 05) . Tube formation experiment showed that LPS decreased the endothelial tube formation ability of HUVECs (P < 0. 05), while colchicine treatment markedly improved LPS-induced tube formation defects (P < 0. 05) . Western blot assay showed that after colchicine co-cultured with LPS, the expression levels of CD31 and VE-cadherin significantly increased compared with the model group (P < 0. 05), while the expression levels of a-SMA and FSP-1 significantly decreased compared with the model group (P < 0. 05) . During the induction of EndMT by LPS, colchicine could inhibit the activation of the NF-KB/Snail signaling pathway. Conclusions Colchicine can effectively inhibit EndMT induced by LPS, and the mechanism may be related to the regulation of the NF-KB/Snail signaling pathway.
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Aim To anticipate the mechanism of zuka- mu granules (ZKMG) in the treatment of bronchial asthma, and to confirm the projected outcomes through in vivo tests via using network pharmacology and molecular docking technology. Methods The database was examined for ZKMG targets, active substances, and prospective targets for bronchial asthma. The protein protein interaction network diagram (PPI) and the medication component target network were created using ZKMG and the intersection targets of bronchial asthma. The Kyoto Encyclopedia of Genes and Genomics (KEGG) and gene ontology (GO) were used for enrichment analysis, and network pharmacology findings were used for molecular docking, ovalbumin (OVA) intraperitoneal injection was used to create a bronchial asthma model, and in vivo tests were used to confirm how ZKMG affected bronchial asthma. Results There were 176 key targets for ZKMG's treatment of bronchial asthma, most of which involved biological processes like signal transduction, negative regulation of apoptotic processes, and angiogenesis. ZKMG contained 194 potentially active components, including quercetin, kaempferol, luteolin, and other important components. Via signaling pathways such TNF, vascular endothelial growth factor A (VEGFA), cancer pathway, and MAPK, they had therapeutic effects on bronchial asthma. Conclusion Key components had strong binding activity with appropriate targets, according to molecular docking data. In vivo tests showed that ZKMG could reduce p-p38, p-ERKl/2, and p-I
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OBJECTIVE To explore the protective effect and mechanism of Longshengzhi capsules on cerebral ischemia- reperfusion injury in rats. METHODS The model of middle cerebral artery occlusion （MCAO） was established by using the improved thread occlusion method. The experiment was divided into six groups： sham surgery group （only separating blood vessels without inserting thread plugs， given the same volume of normal saline）， model group （modeling， given the same volume of normal saline）， nimodipine group （positive control， modeling， dose of 20 mg/kg）， and low-dose， medium-dose， and high-dose groups of Longshengzhi capsules （modeling， doses of 0.72， 1.44 and 2.88 g/kg， respectively）， with 10 mice in each group. Each group was given corresponding medication solution/normal saline by gavage， once a day， for 7 consecutive days. One hour after the last administration， the Zea Longa scoring method was used to score the neurological deficits in each group of rats， and the ABC enzyme-linked immunosorbent assay was used to detect the serum levels of tumor necrosis factor-α （TNF-α） and interleukin-6 （IL-6） in rats； TTC staining was used to observe the volume of cerebral infarction in rats and calculate the cerebral infarction volume ratio. Hematoxylin eosin staining was used to observe the pathological changes in the brain tissue of rats. Immunohistochemical staining was used to detect the positive expression of NLRP3 protein in the brain tissue of rats. Real-time fluorescence quantitative PCR was used to detect mRNA relative expressions of Toll-like receptor 4 （TLR4） and nuclear factor-κB （NF-κB） in the brain tissue of rats. Western blot assay was adopted to detect the relative expressions of TLR4， NLRP3 and phosphorylated NF-κB （p-NF-κB） protein in the brain tissue of rats and its intracellular NF-κB protein. RESULTS Compared with the sham surgery group， the neural dysfunction score， serum levels of TNF-α and IL-6， cerebral infarction volume ratio， relative expression levels of NF-κB and TLR4 mRNA， as well as protein relative expressions of TLR4， NLRP3 and p-NF-κB in the brain tissue， and relative protein expression of intracellular NF-κB were increased significantly in the model group （P＜0.01）； the enlarged gap and significant edema were observed in cortical nerve cells of brain tissue in rats， with a large amount of inflammatory cell infiltration； the positive expression of NLRP3 protein in brain tissue of rats obviously increased. Compared with the model group， the levels of the above indicators in the medium-dose and high-dose groups of Longshengzhi capsules， as well as the Nimodipine group， were reversed to varying degrees， and most differences were statistically significant （P＜0.05 or P＜0.01）； the pathological morphology observation showed a significant improvement， and the positive expression of NLRP3 protein in the brain tissue of rats was obviously reduced. CONCLUSIONS Longshengzhi capsules may inhibit TLR4/NF-κB/NLRP3 signaling pathway and neuroinflammatory response， thereby achieving a protective effect against cerebral ischemia-reperfusion injury in rats.
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OBJECTIVE To explore the neuroprotective effect of sodium aescinate on rats with Parkinson’s disease by regulating the silent information regulator 1 （SIRT1）/nuclear factor-κB （NF-κB） signaling pathway. METHODS The Parkinson’s disease rat model was constructed by using 6-hydroxydopamine injection method. Forty-eight rats successfully modeled were randomly divided into model group， sodium aescinate low-dose group （1.8 mg/kg）， sodium aescinate high-dose group （3.6 mg/kg）， sodium aescinate+EX527 （sodium aescinate 3.6 mg/kg+SIRT1 inhibitor EX527 5 mg/kg） group， with 12 rats in each group. Another 12 healthy rats were selected as the sham operation group. Each group was injected with the corresponding drug solution intraperitoneally， once a day， for 21 consecutive days. Twenty-four hours after the end of the last administration， the motor and cognitive functions of rats were detected， and the morphology of neurons in the substantia nigra and CA1 region of hippocampal tissue were observed. The content of dopamine （DA） in the nigrostriatal and the expression levels of tyrosine hydroxylase （TH） and α-synuclein （α-Syn） in the substantia nigra were detected. The serum levels of pro-inflammatory factor [interleukin-6 （IL-6）， IL-18]， anti-inflammatory factor （IL-10）， and the expression levels of SIRT1， phosphorylated NF-κB p65 （p-NF-κB p65） and NF- κB p65 protein in nigrostriatal were detected. RESULTS Compared with sham operation group， the neurons in the substantia nigra and CA1 region of hippocampal tissue were seriously damaged in model group； the number of rotations， escape latency， the expression levels of α-Syn in substantia nigra， the levels of serum pro-inflammatory factors， the relative expression ratio of p-NF- κB p65 and NF-κB p65 protein in nigrostriatal were increased or prolonged significantly （P＜0.05）； the target quadrant residence time， the content of DA in nigrostriatal， the expression level of TH in substantia nigra， the serum level of anti-inflammatory factor， and the expression level of SIRT1 protein in substantia nigra striatum were significantly decreased or shortened （P＜0.05）. Compared with model group， the damage degrees of neuron in sodium aescinate groups were alleviated， and the quantitative indicators were significantly improved， which were more significant in the high-dose group （P＜0.05）； EX527 could reverse the improvement effect of high-dose sodium aescinate （P＜0.05）. CONCLUSIONS Sodium aescinate can inhibit the activation of NF-κB signal by up-regulating the protein expression of SIRT1， thereby reducing the neuroinflammation of rats with Parkinson’s disease， improving the motor and cognitive dysfunctions， and finally playing a neuroprotective role.
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Ulcerative colitis （UC） is a chronic inflammatory bowel disease primarily affecting the colon and rectum， with the typical symptoms such as abdominal pain， bloody diarrhea， and tenesmus. The pathogenesis of UC remains to be fully elucidated. The disease is prone to recurrence， seriously affecting the patients' quality of life. Conventional therapies for UC have limitations， including unsatisfactory clinical efficacy， lengthy courses， and adverse reactions. Therefore， there is an urgent need to explore new therapeutic agents. Peroxisome proliferator-activated receptor gamma （PPARγ）， a ligand-dependent nuclear receptor protein that plays a crucial role in maintaining intestinal homeostasis， is closely associated with the onset and development of UC. Traditional Chinese medicine （TCM） has advantages such as multi-targeting and mild side effects in the treatment of UC. Recent studies have shown that TCM can exert the therapeutic effects on UC by modulating PPARγ. The TCM methods for regulating PPARγ include clearing heat， drying dampness， moving Qi， activating blood， resolving stasis， invigorating the spleen， warming the kidney， and treating with both tonification and elimination. On one hand， TCM directly activates PPARγ or mediates signaling pathways such as nuclear factor-κB （NF-κB）， mitogen-activated protein kinase （MAPK）， Toll-like receptor 4 （TLR4）， and regulates helper T cell 17 （Th17）/regulatory T cell （Treg） balance to promote macrophage polarization from the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype， thereby inhibiting intestinal inflammation. On the other hand， TCM regulates the intestinal metabolism to activate PPARγ， lower the nitrate level， and maintain local hypoxia. In this way， it can restore the balance between specialized anaerobes and facultative anaerobes， thereby improving the gut microbiota and treating UC. This article summarizes the role of PPARγ in UC and reviews the research progress of TCM in treating UC by intervening in PPARγ in the last five years， aiming to give insights into the treatment and new drug development for UC.
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OBJECTIVE To study the inhibitory effect and mechanism of total flavonoids from Melicope pteleifolia （TF-MPL） on transplanted tumor of colorectal cancer in nude mice. METHODS The transplanted tumor model of colorectal cancer was induced by injecting 0.2 mL colorectal cancer cell LoVo subcutaneously via the right armpit of nude mice. After successful modeling， nude mice were randomly divided into model group， 5-fluorouracil group （positive control， 10 mg/kg）， TF-MPL high- dose and low-dose groups （25， 12.5 mg/kg）； a normal group （normal saline containing 0.3% carboxymethyl cellulose sodium） without modeling was additionally set up， with 6 mice in each group. Each group was intraperitoneally injected with the corresponding drug solution/solvent for 21 consecutive days. The inhibitory rate of the transplanted tumor， liver and spleen index， and the levels of tumor necrosis factor-α （TNF-α） and interleukin-6 （IL-6） in serum were detected after the last medication； the morphological changes of tumor tissue were observed； immunohistochemical staining was used to detect protein expressions of Toll- like receptor 4 （TLR4） and nuclear factor-κB subunit p65 （NF-κB p65） in tumor tissue of nude mice. Western blot assay was used to detect protein expressions of TLR4， myeloid differentiation factor 88 （MyD88）， TNF receptor-associated factor 6 （TRAF6）， interleukin-1 receptor-associated kinase 1 （IRAK-1）， NF-κB p65 and caspase-3 in tumor tissue of nude mice. RESULTS Compared with the model group， TF-MPL high-dose group showed a significant decrease in tumor weight （inhibitory rate of 36.91%）， liver and spleen index， serum levels of TNF-α and IL-6 and protein expressions of TLR4， MyD88， TRAF6，IRAK-1 and NF- κB p65 （P＜0.05 or P＜0.01）； the expression of caspase-3 protein was increased significantly （P＜0.05）， and more tumor cell shrinkage and deformation， nuclear pyknosis and fragmentation were observed. CONCLUSIONS TF-MPL can significantly inhibit the growth of transplanted tumor of colorectal cancer in nude mice， the mechanism of which may be associated with reducing inflammatory response， inhibiting TLR4/MyD88/NF-κB signaling pathway， and promoting apoptosis in colorectal cancer cells.
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ObjectiveTo investigate the therapeutic effect of Qingjie Huagong decoction （QJHGD） on a mouse model of severe acute pancreatitis （SAP） and the mechanism of action of QJHGD against inflammatory response. MethodsA total of 36 male C57BL/6J mice were randomly divided into blank group， model group， Western medicine group （ulinastatin）， and low-， middle-， and high-dose QJHGD groups， with 6 mice in each group. All mice except those in the blank group were given 5% sodium taurocholate by retrograde pancreaticobiliary injection to establish a model of SAP. After modeling， the mice in the low-， middle-， and high-dose groups were given QJHGD （1， 2， and 4 g/kg， respectively） by gavage， and those in the Western medicine group were given intraperitoneal injection of ulinastatin （5×104 U/kg）， for 7 days in total. HE staining was used to observe the histopathological changes of the pancreas； ELISA was used to measure the levels of α-amylase， lipase， interleukin-1β （IL-1β）， interleukin-6 （IL-6）， interleukin-8 （IL-8）， interleukin-18 （IL-18）， and tumor necrosis factor-α （TNF-α） in mice； RT-qPCR was used to measure the mRNA expression levels of NOD-like receptor protein3 （NLRP3）， Toll-like receptor 4 （TLR4）， and nuclear factor-kappa B （NF-κB） in pancreatic tissue； immunohistochemistry was used to measure the positive expression rates of NLRP3， TLR4， and NF-κB in pancreatic tissue； Western blot was used to measure the protein expression levels of NLRP3， TLR4， NF-κB， IL-1β， and IL-6. An analysis of variance was used for comparison of continuous data between multiple groups， and the least significant difference t-test was used for further comparison between two groups. ResultsCompared with the blank group， the model group had diffuse destruction of pancreatic tissue structure， focal dilatation of pancreatic lobular septum， pancreatic acinar atrophy， and massive inflammatory cell infiltration， as well as significant increases in the content of α-amylase， lipase， IL-1β， IL-6， IL-8， IL-18， and TNF-α （all P<0.05）， the mRNA expression levels and positive expression rates of NLRP3， TLR4， and NF-κB （all P<0.05）， and the protein expression levels of NLRP3， TLR4， NF-κB， IL-1β， and IL-6 （all P<0.05）. Compared with the model group， the low-， middle-， and high-dose QJHGD groups and the Western medicine group had slightly tighter and more intact structure of pancreatic tissue， ordered arrangement of pancreatic acinar cells， a small amount of inflammatory cell infiltration， and hemorrhagic foci of pancreatic lobules， as well as significant reductions in the content of α-amylase， lipase， IL-1β， IL-6， IL-8， IL-18， and TNF-α （all P<0.05）， the mRNA expression levels and positive expression rates of NLRP3， TLR4， and NF-κB （all P<0.05）， and the protein expression levels of NLRP3， TLR4， NF-κB， IL-1β， and IL-6 （all P<0.05）. ConclusionQJHGD may exert a protective effect on the pancreatic tissue of SAP mice by inhibiting the activation of NLRP3/TLR4/NF-κB signaling pathway-related proteins， reducing the release of inflammatory mediators， and preventing the enhancement of inflammatory cascade response.
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OBJECTIVE To investigate the effects of Setaria italica extract on improving insomnia model mice and to explore its potential mechanisms. METHODS The mice were randomly assigned into blank group， model group， positive control group （diazepam， 2.6 mg/kg）， and S. italica extract low-dose， medium-dose and high-dose groups （1.2， 2.4， 4.8 g/kg）， with 10 mice in each group. Except for the blank group， all other groups received intraperitoneal injection of para-chlorophenylalanine （PCPA） to establish the insomnia model. After modeling， the blank group and model group were given a constant volume of normal saline intragastrically， and administration groups were given relevant medicine intragastrically， with a volume of 0.01 mL/g， once a day， for 7 consecutive days. After the administration， the open-field test was conducted to observe the praxiological changes of mice， and to determine the levels of 5-hydroxytryptamine （5-HT） and 5-hydroxyindoleacetic acid （5-HTAA） in the hippocampal tissue， as well as the contents of 5-HT， brain-derived neurotrophic factor （BDNF）， interleukin-2 （IL-2）， IL-6， B-cell lymphoma-2 （Bcl- 2）， and Bcl-2-associated X protein （Bax） in the serum. The expression of phosphoinositide 3-kinase/protein kinase B/nuclear factor- κB （PI3K/Akt/NF-κB） signaling pathway related protein was determined in the hippocampus of mice. RESULTS Compared with the model group， the total exercise time of mice in S. italica extract high-dose group was significantly prolonged， but the total rest time was significantly shortened （P＜0.01）； the number of standing times and modification times were significantly reduced （P＜ 0.01）. The contents of 5-HT， BDNF， and Bcl-2 in serum， and Bcl-2/Bax were significantly increased， while the contents of IL-2， IL-6， and Bax were significantly reduced （P＜0.05 or P＜ 0.01）. The content of 5-HTAA in the hippocampal tissue and 202104010910029）；the phosphorylation levels of PI3K and Akt proteins were increased significantly， while the phosphorylation level of NF-κB p65 protein was decreased significantly （P＜0.05）.CONCLUSIONS High-dose of S. italica extract demonstrates significant therapeutic effects on insomnia in mice， and the mechanism of which may be associated with the regulation of PI3K/Akt/NF-κB signaling pathway.
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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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@#Objective To develop and verify a rapid detection method for the biological activity of adalimumab based on U937-NF-κB-Luc cell line. Methods Using U937-NF-κB-Luc cell line as the detection cells,a method for detecting the biological activity of adalimumab was developed based on luciferase luminescence principle. The method was optimized for the concentration of tumor necrosis factor-α(TNF-α)(160 ng/mL as initial concentration,2 times serial dilution,10dilutions),the initial concentration of antibody(2 000 ng/mL,2 times serial dilution,20 dilutions),the dilution multiple of antibody(1. 5,2,3,4 times),the inoculation amount(8 × 103,2 × 104,4 × 104,6 × 104cells/well)and the incubation time(0. 5,1,2,3 h),and verified for the specificity,accuracy,precision and linear range. The relative potency of five batches of adalimumab was detected by using the optimized method and TNF-α neutralization activity method based on L929cells respectively. Results The dose-response curve of adalimumab international standard showed a typical S-type,and the data complied with the four-parameter equation y =(A-D)/[1 +(x/C)B]+ D,R2> 0. 99. The optimum concentration of TNF-α was 5 ng/mL,the initial concentration of antibody was 800 ng/mL,the dilution ratio for adalimumab was 1∶2,the inoculation amount was 2 × 104cells/well,and the induction time was 2 h. Three therapeutic monoclonal antibodies of TNF-α target,such as adalimumab,obtained good dose-response curves,while therapeutic monoclonal antibodies of other non-TNF-α targets did not show this curve. The linear regression equation of the logarithmic value of theoretical potency and the logarithmic value of the corresponding measured potency had a slope of 1. 037,and the relative bias was within the range of ± 12%. The geometric coefficient of variation(GCV)of the relative titer measured value of each sample was less than20%. The theoretical potency ranged from 64% to 156%,showing a good linear relationship with the measured values,and the fitting linear regression equation was y = 1. 037 4 x-0. 023 7,R2= 0. 998 4. There was no significant difference in the relative potency measured results of five batches of adalimumab by the two methods(t = 1. 198,P = 0. 265 1). Conclusion The developed detection method for adalimumab biological activity based on U937-NF-κB-Luc cell line has good specificity,accuracy and precision with short time consumption(3 h),which can be used as a rapid evaluation method for the biological activity of adalimumab.
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@#Objective To study the effect of ankyrin repeat domain 49(ANKRD49)on the migration of human lung adenocarcinoma cell line NCI-H1299 and its mechanism.Methods NCI-H1299 cells were infected with lentivirus vector carrying ANKRD49 gene and shRNA targeting ANKRD49 to construct the cell models stably overexpressing and knocking down ANKRD49. Meanwhile,the control cell models infected with empty lentivirus vector and lentivirus vector with scramble sequences were constructed respectively. The expression levels of ANKRD49 mRNA and protein were detected by real-time fluorescence quantitative PCR and Western blot. The effect of ANKRD49 on cell migration was measured by scratch test. The mRNA and protein levels of matrix metalloproteinase(MMP)-2/9 and tissue inhibitor of metalloproteinase(TIMP)-1/2 were detected by real-time fluorescence quantitative PCR and Western blot. The protein expression levels of p65,p-p65,IκBα and p-IκBα were detected by Western blot.Results The levels of ANKRD49 mRNA and protein in the ANKRD49 overexpression group were significantly higher than those in the control group(t = 70. 02 and 45. 68,respectively,each P < 0. 001). Compared with the control group,the migration ability of cells in the ANKRD49 overexpression group significantly increased at 24 h and 48 h(t = 5. 343 and 3. 282,P = 0. 005 9 and 0. 030 4,respectively);The mRNA transcription levels and protein expression levels of MMP-2 and MMP-9 significantly increased(t = 9. 304 and 6. 193,P =0. 000 7 and 0. 003 5,respectively),while the mRNA and protein expression of TIMP-1 and TIMP-2 decreased significantly(t = 3. 858 and 3. 517,P = 0. 018 2 and 0. 024 5,respectively),and the values of MMP-2/TIMP-1 and MMP-9/TIMP-2 significantly increased(t = 17. 7 and 9. 682,P < 0. 001 and < 0. 01,respectively);The expression of p-p65 and pIκBα significantly increased,the total protein levels of p65 and IκBα showed no obvious change,and the values of p-p65/p65 and p-IκBα/IκBα significantly increased(t = 3. 962 and 5. 370,P = 0. 016 7 and 0. 005 8,respectively). However,knocking down of ANKRD49 presented the opposite results.Conclusion ANKRD49 promotes the migration of NCI-H1299cells by enhan-cing the expression of MMP-2/9,the values of MMP-9/TIMP-1 and MMP-2/TIMP-2 via activating NF-κB/p65 signa-ling pathway.