Effect of ANKRD49 on migration of human lung adenocarcinoma cells and its mechanism / 中国生物制品学杂志

@#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.

Protective Effect of Zingiberis Rhizoma Recens on LPS-induced Acute Lung Injury in Mice Through TLR4/NF-κB Signaling Pathway / 中国实验方剂学杂志

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.

Study on Mechanism of Linggan Wuwei Jiangxintang in Treating Acute Lung Injury / 中国实验方剂学杂志

ObjectiveTo explore the action mechanism of Linggan Wuwei Jiangxintang on the treatment of lipopolysaccharide （LPS）-induced acute lung injury （ALI） in mice. MethodTraditional Chinese Medicine Systems Pharmacology Database and Analysis Platform （TCMSP）， GeneCards， DisGeNET， and Herb databases were combined with clinical data from Gene Expression Omnibus （GEO） to screen the key targets of Linggan Wuwei Jiangxintang in the treatment of ALI. The protein-protein interaction （PPI） network was constructed to screen the core targets， and gene ontology （GO） and Kyoto encyclopedia of genes and genomes （KEGG） pathway enrichment analyses were performed. The mouse ALI model was established by LPS induction to verify the effect and key targets of Linggan Wuwei Jiangxintang on the treatment of ALI. The expression levels of Toll-like receptor 4 （TLR4）， nuclear transcription factor-κB p65 （NF-κB p65）， and phosphorylated NF-κB p65 （NF-κB p-p65） in lung tissue were detected by Western blot. ResultThe analysis showed that the treatment of ALI with Linggan Wuwei Jiangxintang was related to 10 core targets such as interleukin-6 （IL-6）， tumor necrosis factor-α （TNF-α）， and JUN， involving TNF signaling pathway， Toll-like receptor signaling pathway， NF-κB signaling pathway， etc. The animal experimental results show that Linggan Wuwei Jiangxintang can reduce lung injury， improve the pathological state of ALI mice， significantly reduce the expression of TNF-α and IL-6 in serum， increase the activity of total superoxide dismutase （T-SOD） and catalase （CAT） in lung tissue， and reduce the expression levels of JUN， TLR4， NF-κB p65， and NF-κB p-p65 proteins in lung tissue. ConclusionLinggan Wuwei Jiangxintang can inhibit LPS-induced inflammation and oxidative damage in ALI mice， and its mechanism may be related to the inhibition of TLR4/NF-κB signaling pathway and the reduction of inflammatory factors such as TNF-α and IL-6.

Effect of Gegen Qinliantang-medicated Serum on Nonalcoholic Steatohepatitis Based on Nrf2/TXNIP Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo investigate the effect of Gegen Qinliantang （GGQLT）-medicated serum on free fatty acid （FFA）-induced nonalcoholic steatohepatitis （NASH） in vitro model of human hepatoma cells HepG2. MethodNASH model of HepG2 cells was established in vitro， and the cells were intervened with different volume fractions of GGQLT-medicated serum and resveratrol. Intracellular lipid deposition in each group was detected by oil red O staining， the level of reactive oxygen species （ROS） in each group were detected by flow cytometry， the levels of glutathione peroxidase （GSH-Px）， superoxide dismutase （SOD）， triglyceride （TG） and malondialdehyde （MDA） in each group were detected by kits. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was used to measure the mRNA expression levels of nuclear transcription factor （NF）E2-related factor 2 （Nrf2）， heme oxygenase-1 （HO-1）， quinone oxidoreductase 1 （NQO1）， Kelch-like epichlorohydrin-associated protein-1 （Keap1）， NF-κB， thioredoxin interacting protein （TXNIP）， interleukin-1β （IL-1β） in HepG2 cells of each group. The protein expression of Nrf2， TXNIP in cells of each group was detected by Western blot. ResultFFA induced large accumulation of intracellular lipids. Compared with the normal group， the activities of GSH-Px and SOD were significantly decreased （P<0.01） and the contents of TG， ROS and MDA were significantly increased （P<0.05， P<0.01） in the model group. Compared with the model group， all GGQLT groups and resveratrol group could elevate intracellular SOD activity to different degrees （P<0.05， P<0.01） and significantly reduce the levels of intracellular ROS and MDA （P<0.05， P<0.01）， GGQLD high- and medium-dose groups and resveratrol group significantly elevated GSH-Px activity （P<0.01）， GGQLD medium- and low-dose groups and resveratrol group significantly decreased TG content （P<0.05， P<0.01）. Compared with the model group， GGQLT high- and medium-dose groups and resveratrol group could significantly upregulate the mRNA expression levels of Nrf2， HO-1 and NQO1 （P<0.01）， all GGQLT groups and resveratrol group could significantly downregulate the TXNIP protein expression level， as well as significantly downregulate the mRNA expression levels of Keap1， NF-κB （P<0.05， P<0.01）. Nrf2-siRNA transfection of cells revealed that Nrf2 expression was significantly downregulated （P<0.01） in the Nrf2-siRNA group of cells by comparing with NC-siRNA group at the corresponding dose of drugs， and the inhibitory effects of GGQLT and resveratrol on TXNIP， IL-1β were attenuated. ConclusionFFA induces the production of ROS and inflammatory factors in HepG2 cells， and GGQLT can improve the anti-inflammatory and antioxidant capacities of cells， and its mechanism may be related to the regulation of Nrf2/TXNIP signaling pathway， so as to improve NASH.

Effect of Bushen Huatan Prescription on Serum LPS and TLR4/MyD88/NF-κB Signaling Pathway in Rats with Ovariectomy-induced Osteoporosis / 中国实验方剂学杂志

Objective:To explore the effect of Bushen Huatan prescription on serum lipopolysaccharide （LPS） and Toll-like receptor 4 （TLR4）/ myeloid cell differentiation protein 88 （MyD88）/nuclear transcription factor-<italic>κ</italic>B （NF-<italic>κ</italic>B） signaling pathway in rats with ovariectomy-induced osteoporosis. Method:Sixty SPF 6-month-old female rats were randomly divided into sham operation group， model group， estradiol valerate group and Bushen Huatan prescription low， medium and high dose groups.One week after modeling by bilateral ovariectomy， 8 rats in each group were selected to receive intragastric administration.The estradiol valerate group was given 0.184 mg·kg^-1 by gavage， and Bushen Huatan prescription low， middle and high dose groups were given 4.7， 9.4 and 18.8 g·kg^-1 by gavage， sham operation group and model group were given 0.9% saline 4 mL by gavage respectively.After 12 weeks of intervention， the rats were sacrificed for detection.Serum LPS was detected by enzyme linked immunosorbent assay （ELISA）， while protein expressions of TLR4， MyD88 and phosphorylated （p）-NF-<italic>κ</italic>B p65 in bone tissue were detected by Western blot， and the mRNA expressions of TLR4， MyD88， NF-<italic>κ</italic>B p65， IL-1<italic>β</italic>， and IL-6 in bone tissue were detected by quantitative real-time polymerase chain reaction（PCR）. Result:Compared with sham operation group， the serum LPS level as well as protein expression of TLR4， MyD88， p-NF-<italic>κ</italic>B p65 and mRNA expression of TLR4， MyD88， NF-<italic>κ</italic>B p65， IL-1<italic>β</italic>， and IL-6 significantly increased in model group（<italic>P</italic><0.05）.Compared with the model group， serum LPS level， protein expression of TLR4， MyD88， and p-NF-<italic>κ</italic>B p65， mRNA levels of TLR4， MyD88， and NF-<italic>κ</italic>B p65 in bone tissues as well as downstream inflammatory factors IL-1<italic>β</italic>， IL-6 mRNA expression decreased to different degrees in estradiol valerate group and Bushen Huatan prescription high dose group（<italic>P</italic><0.05）. Conclusion:Bushen Huatan prescription can reduce serum LPS content， regulate mRNA and protein expression of TLR4， MyD88， NF-<italic>κ</italic>B p65 and p-NF-<italic>κ</italic>B p65 in TLR4/MyD88/NF-<italic>κ</italic>B pathway， and down-regulate mRNA levels of IL-1<italic>β</italic> and IL-6 in bone tissues to improve bone microstructure and inhibit the development of postmenopausal osteoporosis （PMOP）.

Effect of Kidney-tonifying Method on OPG/RANK/RANKL Signaling Pathway in Postmenopausal Osteoporosis Animal Model： A Meta-analysis / 中国实验方剂学杂志

Objective:As the problem of global aging intensifies，postmenopausal osteoporosis （PMOP） has become a global health problem among females. At present，the commonly used biological agents have been proved not suitable for long-term use due to multiple adverse reactions. Several Meta-analyses have confirmed the good safety and effectiveness of kidney-tonifying method against PMOP，but its therapeutic mechanism remains unclear. The purpose of this Meta-analysis was to evaluate the effect of kidney-tonifying method on osteoclastogenesis inhibitory factor（OPG）/receptor activator of nuclear transcription factor （NF）-<italic>κ</italic>B （RANK）/receptor activator of NF-<italic>κ</italic>B ligand （RANKL） signaling pathway in PMOP animal model，so as to provide an experimental basis for the treatment of PMOP with kidney-tonifying method. Method:The related articles were retrieved from PubMed，Ovid Medline，Embase，China National Knowledge Infrastructure （CNKI），Chongqing Weipu Database for Chinese Technical Periodicals （VIP），and Wanfang Data Knowledge Service Platform with the retrieval time set from their inception to January 2020. The quality of each included article was evaluated using the SYRCLE's risk of bias tool. Then RevMan 5.3 was utilized for Meta-analysis according to the Cochrane systematic review methodology. Result:Thirty-two studies involving 619 rats were included. The quality score of these studies ranged from 3 to 5 points. The results of the Meta-analysis indicated obvious advantages of kidney-tonifying method in increasing bone mineral density （BMD）［standardized mean difference （SMD）=2.01，95% confidence interval（CI）=1.50-2.52，<italic>P</italic><0.000 01］），serum OPG level （SMD=3.33，95% CI=2.59-4.07，<italic>P</italic><0.000 01），and OPG mRNA expression （SMD=11.81，95% CI=7.49-16.13，<italic>P</italic><0.000 01），promoting OPG protein production （SMD=4.95，95% CI=3.09-6.81，<italic>P</italic><0.000 01），reducing serum RANKL（SMD=-4.88，95% CI=-6.01--3.75，<italic>P</italic><0.000 01） and RANK levels （SMD=-7.30，95% CI=-9.53--5.07，<italic>P</italic><0.000 01），and down-regulating RANKL （SMD=-6.22，95%CI=-8.95--3.49，<italic>P</italic><0.000 01） and RANK mRNA （SMD=-3.18，95% CI=-6.19--0.18，<italic>P</italic><0.05） expression and RANKL protein expression in bone tissue （SMD=-3.99，95% CI=-5.47--2.50，<italic>P</italic><0.000 01）. Conclusion:The kidney-tonifying method has been proved to possess potential advantages in regulating the balance of OPG/RANK/RANKL signaling pathway in PMOP animal model. Nevertheless，more large-sample sized，properly designed，and high-quality animal experiments are still needed for further verification.

Effect of Xiao Chaihutang on NF-κB Signaling Pathway in Synovial Tissue of CFA Rats / 中国实验方剂学杂志

Objective:To observe the expression of tumor necrosis factor receptor-associated death domain (TARDD), nuclear transcription factor-κB inhibiting protein α(IκBα)IκB kinase-α (IKKα) and nuclear transcription factor (NF)-κB p65 protein in the NF-κB signaling pathway of synovial tissues of complete Freund's adjuvant (CFA) rats after treatment with Xiao Chaihutang (XCHT). Method:In animal experiments, SPF health adult female Wistar rats were used to prepare the CFA animal model of rats with rheumatoid arthritis with Freund's complete adjuvant and cattle Ⅱ collagen type. According to the random number table, the rats were randomly divided into the normal group, the model group, the low-dose XCHT group, the medium-dose XCHT group, the high-dose XCHT group, and the Tripterygium glucosides group. The drugs were given at 7 d after the model was built. Both normal group and model group were given water for injection,and low-dose XCHT group(5.94 g·kg-1),medium-dose XCHT group(11.88 g·kg-1),high-dose XCHT group(23.76 g·kg-1),Tripterygium glucosides group(0.006 3 g·kg-1) were given corresponding drugs by gavage for three times a day, 2 mL/time. The histopathology of rat ankle joint was observed, and the protein expressions of TARDD,IKKα,IκBα,NF-κB p65 in the NF-κB signaling pathway in synovial tissue of CFA rats were detected by Western blot. Result:With the increase of the dosage of XCHT, the histopathological score of the right posterior ankle joint of the experimental rats was increased. And in the protein expressions of TARDD,IKKα,IκBα,NF-κB p65 in NF-κB signaling pathway in Synovial Tissue of CFA rats, compared with the model group, the statistical results of the low-dose XCHT group showed decreased protein expressions (PPPα, IκB α, NF-κB p65 in the NF-κB signaling pathway were significantly increased (PPα, IκBα, NF-κB p65 key protein expressions in the NF-κB signaling pathway and protein expressions in low-dose XCHT group were obviously lower (PPConclusion:This study shows that as the dose of Xiao Chaihutang increases, it could effectively improve synovitis, and suppress the expressions of key proteins in the inflammatory signaling pathway of NF-κB, thereby preventing inflammation and suppressing bone erosion.

Research progress on the correlation between TLR4-MyD88-NF-κB signalling pathways and the hepatic inflammation-fibrosis-cancer axis / 国际药学研究杂志

As a critical pathway in the reaction of hepatic disease, Toll-like receptor 4(TLR4)-myeloid differentiation factor 88(MyD88)-nuclear transcription factor(NF-κB)signalling pathway, which widely exists in various tissues and cells, is one of the most important signalling pathways that mediate the expression of inflammatory factor between the intracellular and intercellular. Study found that TLR4-MyD88-NF-κB signalling pathway plays an important role in the regulation of liver immune abnormalities, inflammatory response triggered by the liver damage, activation of hepatic stellate cells and liver fibrosis deterioration and the development of cancer of the liver. The signalling pathway may be an important regulatory point in the dynamic changes of the”hepatic inflammationfibrosis-cancer axis”(IFC axis)and liver cancer metastasis. In this paper, we review the recent advances in the pathological mechanism of TLR4-MyD88-NF-κB signalling pathway participating in IFC axis disease, so as to provide reference for related research in the future.

The relationship between transcription factor NF-E2-related factor 2 and liver diseases / 中华实用儿科临床杂志

The liver is the most exuberant organs of human metabolism,susceptible to oxidative stress injury.More and more studies in recent years show that various types of liver diseases such as hepatitis,liver fibrosis,liver cirrhosis,liver cancer and so on are closely related with oxidative stress injury.The transcription factor NF-E2-related factor 2 (Nrf2) is an important transcription factor regulating oxidative stress reaction.It combines with the antioxidant response element antioxidant response elements (ARE),then activates target gene transcription,and increases the resistance of cell to oxidative stress.Kelch-like ECH-associated protein 1-Nrf2-ARE pathway plays an important role in the pathogenesis of liver diseases.