Improvement effect and mechanism of total flavonoids from Rosa multiflora root on vascular injury in rheumatoid arthritis model rats / 中国药房

OBJECTIVE To study the improvement effect of total flavonoids from Rosa multiflora root on vascular injury in rheumatoid arthritis （RA） model rats and its potential mechanism. METHODS Female Wistar rats were randomly divided into normal control group， model group， aspirin group （positive control， 30 mg/kg）， low-dose and high-dose groups of total flavonoids from R. multiflora root （4.15， 8.30 g/kg， by crude drug）， with 10 rats in each group. Except for the normal control group， the RA model was induced in other groups by collagen induction and high-fat diet. After 14 days of modeling， they were given corresponding drug solution/0.5% sodium carboxymethyl cellulose solution intragastrically， once a day， for 36 consecutive days. The total body score， arthritis index （AI） and swollen joint count （SJC） of the rats were evaluated regularly. After the last medication， serum levels of interleukin-6 （IL-6）， intercellular adhesion molecule-1 （ICAM-1） and vascular cell adhesion molecule- 1 （VCAM-1） were determined. The pathological morphological changes in the vascular tissue of thoracic aorta were observed； the protein expression of Toll-like receptor 4 （TLR4） and the protein phosphorylation levels of Janus kinase 2 （JAK2） and signal transduction and activator of transcription 3 （STAT3） in vascular tissue of thoracic aorta were measured. RESULTS Compared with the normal control group， serum levels of IL-6， ICAM-1 and VCAM-1， protein expression of TLR4， and the protein phosphorylation levels of JAK2 and STAT3 in vascular tissue of thoracic aorta were increased significantly in model group （P＜ 0.01）. The atherosclerotic plaque （atheroma）， cholesterol crystal， lymphocyte infiltration and a small number of unbroken foam cell aggregation could be seen in the vascular tissue of thoracic aorta. Compared with the model group， total body score （except for the low-dose group）， AI and SJC were decreased significantly in groups of total flavonoids from R. multiflora root on the 28th day （P＜0.05 or P＜0.01）； total body score，AI and SJC were decreased significantly in low-dose group of total flavonoids from R. multiflora root on the 49th day （P＜0.05 or P＜0.01）； the other quantitative indicators in serum and vascular tissue were significantly reversed in groups of total flavonoids from R. multiflora root （P＜0.05 or P＜0.01）， and pathological damage of vascular tissue was significantly relieved. CONCLUSIONS Total flavonoids from R. multiflora root can significantly improve vascular injury in RA model rats， and its mechanism may be related to reducing the protein expression of TLR4 in vascular tissue and inhibiting the activation of IL-6/JAK2/ STAT3 signaling pathway.

Traditional Chinese Medicine in Treatment of Parkinson's Disease by Regulating TLR4/NF-κB Signaling Pathway： A Review / 中国实验方剂学杂志

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.

Effect of Tongluo Juanbi Granules on Inflammatory Injury and Apoptosis of Osteoarthritis Based on TLR4/MyD88/NF-κB Signaling Pathway / 中国实验方剂学杂志

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.

Protective effect and mechanism of Longshengzhi capsules on cerebral ischemia-reperfusion injury in rats / 中国药房

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.

Traditional Chinese Medicine Intervention in Acute Pancreatitis Based on TLR4 Signaling Pathway： A Review / 中国实验方剂学杂志

Acute pancreatitis （AP） is one of the most clinically common acute digestive disorders characterized by quick onset，rapid progression，severe condition，and high mortality. If the disease is not timely intervened in the early stage，it can develop into severe AP in the later stage，which damages the long-term quality of life and brings serious economic burden to patients and their families. However， the pathogenesis of this disease is complex and has not been fully explained. The generation and development of AP is closely related to many signaling pathways. Among them，Toll-like receptor 4（TLR4），as a transmembrane signal transduction receptor，can mediate immune response and inflammatory response，and play a key role in the occurrence and development of AP. Traditional Chinese medicine（TCM）can regulate the TLR4 signaling pathway with multiple targets，multiple effects，and multiple administration methods to inhibit inflammatory response，and effectively intervene in the progression of AP， which has gradually become a new craze for preventing and treating AP. Many studies have shown that TCM has obvious advantages in the prevention and treatment of AP. It can effectively treat AP by regulating TLR4 signaling pathway，strengthening immune resistance and defense，and inhibiting inflammatory response. Despite of the research progress，there is still a lack of comprehensive review on TCM regulation of TLR4 signaling pathway in the treatment of AP. Therefore，the literature on TCM regulation of TLR4 signaling pathway published in recent years was systematically reviewed and elaborated，aiming to provide new ideas for the treatment of AP and further drug development.

Role of PPARγ in Ulcerative Colitis and Traditional Chinese Medicine Intervention： A Review / 中国实验方剂学杂志

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.

Inhibitory effect and mechanism of total flavonoids from Melicope pteleifolia on transplanted tumor of colorectal cancer in nude mice / 中国药房

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.

Da Chengqitang Protects Intestinal Barrier in Septic Mice via Endogenous Antimicrobial Peptide mCRAMP / 中国实验方剂学杂志

ObjectiveMolecular docking and animal experiments were employed to explore the protective effect and mechanism of Da Chengqitang （DCQD） on intestinal barrier in septic mice. MethodText mining method was used to screen the active ingredients in DCQD. AutoDock Tools and Discovery Studio were used to study the interactions of active components with the core target proteins ［claudin-1， tumor necrosis factor （TNF）-α， interleukin （IL）-6， endogenous antimicrobial peptide mCRAMP， Toll-like receptor 4 （TLR4）， and myeloid differentiation primary response gene 88 （MyD88）］ in sepsis. Fifty C57BL/6 mice were randomized into sham， model， low- and high-dose （4 g∙kg-1 and 8 g∙kg-1） DCQD， and ulinastatin groups （n=10）. Before， during， and after the day of modeling surgery， each group was administrated with corresponding drugs. The mice in other groups except the model group were subjected to modeling by cecal ligation and puncture. Enzyme-linked immunosorbent assay （ELISA） was used measure the serum level of D-lactic acid to assess intestinal mucosa permeability. Hematoxylin-eosin staining was employed to observe the histopathological changes in the ileum and assess the intestinal mucosal damage and inflammatory infiltration. Western blotting was employed to determine the expression levels of tight junction proteins claudin-1 and occludin in the ileal tissue， which were indicative of the bowel barrier function. The TNF-α and IL-6 levels were measured by ELISA to assess the intestinal inflammation. The expression of mCRAMP in the ileal tissue was observed by immunohistochemistry. The mRNA levels of mCRAMP， TLR4， and MyD88 in mouse ileal tissue were determined by Real-time polymerase chain reaction， on the basis of which the mechanism of DCQD in protecting the intestinal barrier of septic mice was explored. ResultMolecular docking results showed that most of the 10 active ingredients of DCQD that were screened out by text mining could bind to sepsis targets by van der Waals force， hydrogen bonding， and other conjugated systems. The results of animal experiments showed that compared with the model group， low- or high-dose DCQD lowered the D-lactic acid level in the serum （P<0.01）， alleviated damage to the ileal tissue and mucosal edema， protected the small intestine villus integrity， reduced inflammatory cell infiltration， promoted the expression of claudin-1 （P<0.01）， lowered the IL-6 level （P<0.01）， up-regulated the mRNA and protein levels of mCRAMP （P<0.01）， and down-regulated the mRNA and protein levels of TLR4 and MyD88 （P<0.01） in the ileal tissue. In addition， high-dose DCQD lowered the TNF-α level and promoted the expression of occludin in the ileum tissue （P<0.01）， and low-dose DCQD up-regulated the protein level of occludin in the ileum tissue （P<0.05）. ConclusionDCQD has a protective effect on intestinal barrier in septic mice. It can reduce intestinal inflammation， repair intestinal mucosal damage， improve the tight junction protein level， and reduce intestinal mucosal permeability by up-regulating the mRNA and protein levels of mCRAMP and the down-regulating the expression of genes in the TLR4/MyD88 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.

Effects of HMGB1/TLR4 on secretion IL-10 and VEGF in human jaw bone-marrow mesenchymal stem cells

Abstract Objective: We aimed to investigate the regulatory effects of HMGB1/TLR4 signaling pathway on the expression of IL-10 and VEGF in human bone marrow mesenchymal stem cells. Methodology: Human JBMSCs were isolated and cultured. Then, HMGB1 was added into the JBMSCs culture medium, and the protein and mRNA expression levels of IL-10 and VEGF were assessed. Moreover, cells were pretreated with a specific TLR4 inhibitor (TAK-242), and the expression changes of IL-10 and VEGF were compared. Results: Compared with the control group, exposure to HMGB1 in human JBMSCs up-regulated TLR4, IL-10, and VEGF secretion at both protein and mRNA levels (P<0. 05). In addition, the increased expression of IL-10 and VEGF could be restrained in TAK-242 group compared with the HMGB1 group (P<0.05). Conclusions: The results indicated that HMGB1 activate TLR4 signaling pathway in Human JBMSCs, which plays a regulatory role in cytokines expression.

Targeting TLR-4 Signaling to Treat COVID-19-induced Acute Kidney Injury

The newly discovered severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) has turned into a potentially fatal pandemic illness. Numerous acute kidney injury (AKI) cases have been reported, although diffuse alveolar destruction and acute respiratory failure are the major symptoms of SARS-CoV-2 infection. The AKI, often known as a sudden loss of kidney function, carries a greater risk of mortality and morbidity. AKI was the second most frequent cause of death after acute respiratory distress syndrome (ARDS) in critically ill patients with coronavirus disease 2019 (COVID-19). While most patients with COVID-19 have moderate symptoms, some have severe symptoms, such as septic shock and ARDS. Also, it has been proven that some patients have severe symptoms, such as the failure of several organs. The kidneys are often affected either directly or indirectly. The major signs of kidney involvement are proteinuria and AKI. It is hypothesized that multiple mechanisms contribute to kidney injury in COVID-19. Direct infection of podocytes and proximal tubular cells in the kidneys may lead to acute tubular necrosis and collapsing glomerulopathy. SARS-CoV2 may also trigger a cascade of immunological responses that lead to AKI, including cytokine storm (CS), macrophage activation syndrome, and Toll-like receptor type 4 activation (TLR-4). Other proposed processes of AKI include interactions between organs, endothelial failure, hypercoagulability, rhabdomyolysis, and sepsis. Furthermore, ischemic damage to the kidney might result from the decreased oxygen supply. This article focuses on kidney injury’s epidemiology, etiology, and pathophysiological processes. Specifically, it focuses on the CS and the role of TLR-4 in this process. To effectively manage and treat acute kidney damage and AKI in COVID-19, it is crucial to understand the underlying molecular pathways and pathophysiology.

Huoxin Pill Reduces Myocardial Ischemia Reperfusion Injury in Rats via TLR4/NFκB/NLRP3 Signaling Pathway / 中国结合医学杂志

OBJECTIVE@#To explore the protective effect of Huoxin Pill (HXP) on acute myocardial ischemia-reperfusion (MIRI) injury in rats.@*METHODS@#Seventy-five adult SD rats were divided into the sham-operated group, model group, positive drug group (diltiazem hydrochloride, DH), high dose group (24 mg/kg, HXP-H) and low dose group (12 mg/kg, HXP-L) of Huoxin Pill (n=15 for every group) according to the complete randomization method. After 1 week of intragastric administration, the left anterior descending coronary artery of the rat's heart was ligated for 45 min and reperfused for 3 h. Serum was separated and the levels of creatine kinase (CK), creatine kinase isoenzyme (CK-MB) and lactate dehydrogenase (LDH), superoxide dismutase (SOD), and malondialdehyde (MDA), hypersensitive C-reactive protein (hs-CRP) and interleukin-1β (IL-1β) were measured. Myocardial ischemia rate, myocardial infarction rate and myocardial no-reflow rate were determined by staining with Evans blue and 2,3,5-triphenyltetrazolium chloride (TTC). Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) and Bioinformatics Analysis Tool for Molecular mechANism of Traditional Chinese Medicine (BATMAN) databases were used to screen for possible active compounds of HXP and their potential therapeutic targets; the results of anti-inflammatory genes associated with MIRI were obtained from GeneCards, Drugbank, Online Mendelian Inheritance in Man (OMIM), and Therapeutic Target Datebase (TTD) databases was performed; Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment were used to analyze the intersected targets; molecular docking was performed using AutoDock Tools. Western blot was used to detect the protein expression of Toll-like receptor 4 (TLR4)/nuclear factor kappa-B (NFκB)/NOD-like receptor protein 3 (NLRP3).@*RESULTS@#Compared with the model group, all doses of HXP significantly reduced the levels of LDH, CK and CK-MB (P<0.05, P<0.01); HXP significantly increased serum activity of SOD (P<0.05, P<0.01); all doses of HXP significantly reduced the levels of hs-CRP and IL-1β (P<0.05, P<0.01) and the myocardial infarction rate and myocardial no-reflow rate (P<0.01). GO enrichment analysis mainly involved positive regulation of gene expression, extracellular space and identical protein binding, KEGG pathway enrichment mainly involved PI3K-Akt signaling pathway and lipid and atherosclerosis. Molecular docking results showed that kaempferol and luteolin had a better affinity with TLR4, NFκB and NLRP3 molecules. The protein expressions of TLR4, NFκB and NLRP3 were reduced in the HXP group (P<0.01).@*CONCLUSIONS@#HXP has a significant protective effect on myocardial ischemia-reperfusion injury in rats, and its effect may be related to the inhibition of redox response and reduction of the inflammatory response by inhibiting the TLR4NFκB/NLRP3 signaling pathway.

Effects of Buyang Huanwu Decoction and Astragali Radix-Angelicae Sinensis Radix combination on inflammatory responses in atherosclerotic mice / 中国中药杂志

The study aims to observe the effects and explore the mechanisms of Buyang Huanwu Decoction and Astragali Radix-Angelicae Sinensis Radix combination in the treatment of the inflammatory response of mice with atherosclerosis(AS) via the Toll-like receptor 4(TLR4)/myeloid differentiation primary response protein 88(MyD88)/nuclear factor-κB(NF-κB) signaling pathway. Male ApoE~(-/-) mice were randomly assigned into a model group, a Buyang Huanwu Decoction group, an Astragali Radix-Angelicae Sinensis Radix combination group, and an atorvastatin group, and male C57BL/6J mice of the same weeks old were used as the control group. Other groups except the control group were given high-fat diets for 12 weeks to establish the AS model, and drugs were administrated by gavage. Aortic intimal hyperplasia thickness, blood lipid level, plasma inflammatory cytokine levels, M1/M2 macrophage markers, and expression levels of proteins in TLR4/MyD88/NF-κB pathway in the vessel wall were measured to evaluate the effects of drugs on AS lesions and inflammatory responses. The results showed that the AS model was successfully established with the ApoE~(-/-) mice fed with high-fat diets. Compared with the control group, the model group showed elevated plasma total cholesterol(TC), triglyceride(TG), and low-density lipoprotein cholesterol(LDL-c) levels(P<0.05), thickened intima(P<0.01), and increased plasma tumor necrosis factor-α(TNF-α) and interleukin-6(IL-6) levels(P<0.01). Moreover, the model group showed increased expression of vascular cell adhesion molecule-1(VCAM-1) and inducible nitric oxide synthase(iNOS)(P<0.01), inhibited expression of endothelial nitric oxide synthase(eNOS) and cluster of differentiation 206(CD206)(P<0.01), and up-regulated mRNA and protein levels of TLR4, MyD88, NF-κB inhibitor alpha(IκBα), and NF-κB in the vessel wall(P<0.05). Compared with the model group, Buyang Huanwu Decoction and Astragali Radix-Angelicae Sinensis Radix combination lowered the plasma TC and LDL-c levels(P<0.01), alleviated the intimal hyperplasia(P<0.01), and reduced the plasma TNF-α and IL-6 levels(P<0.05). Moreover, the two interventions promoted the expression of eNOS and CD206(P<0.05), inhibited the expression of VCAM-1 and iNOS(P<0.01), and down-regulated the mRNA and protein levels of TLR4, MyD88, IκBα, and NF-κB(P<0.05) in the vessel wall. This study indicated that Buyang Huanwu Decoction and Astragali Radix-Angelicae Sinensis Radix combination could delay the progression of AS, inhibit the polarization of vascular wall macrophages toward M1 type, and attenuate vascular inflammatory response by inhibiting the activation of TLR4/MyD88/NF-κB signaling pathway in the vascular wall. Astragali Radix and Angelicae Sinensis Radix were the main pharmacological substances in Buyang Huanwu Decoction for alleviating the AS vascular inflammatory response.

Naoxintong Capsules Treat Cardiac Injury after Cerebral Ischemia via TLR2/TLR4 Signaling Pathway / 中国实验方剂学杂志

ObjectiveAfter the brain and heart injuries were simulated by myocardial injury caused by acute cerebral ischemia， this study explored the mechanism of Naoxintong capsules in treating brain and heart injuries under cerebral ischemia state with Toll-like receptor （TLR） 2/TLR4 as the breakthrough point. MethodC57BL/6 male mice were randomly assigned into the sham operation， model， Naoxintong， and Ginaton groups. The middle cerebral artery occlusion （MCAO） method was used to establish a mouse model of cerebral ischemia. The neuroethological score， cerebral infarction area， cell apoptosis， ionized calcium-binding adaptor molecule 1 （IBA-1）-positive microglia proportion， and serum levels of N-terminal pro-brain natriuretic peptide （NT-proBNP）， creatine kinase-MB （CK-MB）， and lactic dehydrogenase （LDH） were determined to evaluate the pharmacodynamic effects of Naoxintong capsules on heart and brain injuries after cerebral ischemia in mice. Western blotting was employed to determine the expression of TLR2/TLR4 protein in the brain and heart of mice. ResultCompared with the sham operation group， the model group showed increased cerebral infarction area， neuroethological score， apoptosis rate， IBA-1-positive microglia proportion， and serum levels of NT-proBNP， CK-MB， and LDH （P<0.01）. Naoxintong capsules reduced the cerebral infarction area， neuroethological score， apoptosis rate， IBA-1-positive microglia proportion （P<0.01）， and serum NT-proBNP and CK-MB levels （P<0.05） in mice compared with the model group. Western blotting results showed that Naoxintong Capsules down-regulated the expression levels of TLR2 （P<0.05） in the brain and TLR2 （P<0.01） and TLR4 （P<0.05） in the heart. ConclusionCerebral ischemia can cause myocardial damage， reflecting the pathological process of cardiac injury after cerebral ischemia. Naoxintong capsules can mitigate brain and heart injuries after cerebral ischemia and achieve the simultaneous treatment of the brain and the heart， in which TLR2/TLR4 plays a role.

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.

Derris eriocarpa Extract Treats Gout via TLR4/NF-κB/NLRP3 Pathway / 中国实验方剂学杂志

ObjectiveTo explore the anti-gout effect and mechanism of Derris eriocarpa extract by network pharmacological analysis combined with in vivo and in vitro experimental verification. MethodThe chemical components and candidate targets of D. eriocarpa were obtained from the database. The key targets and potential active components of D. eriocarpa in the treatment of gout were screened by the protein-protein interaction analysis， and then the Gene Ontology （GO） functional annotation and Kyoto Encyclopedia of Genes and Genomes （KEGG） pathway enrichment were performed for the key targets. A mouse model of hyperuricemia was established by intraperitoneal injection of hypoxanthine to observe the effect of D. eriocarpa alcohol extract on hyperuricemia. A rat model of gouty inflammation induced by the injection of microcrystalline sodium urate crystals into the foot and plantar was used to observe the effect of D. eriocarpa alcohol extract on gouty inflammation. A xylene-induced acute inflammation model was established to observe the anti-inflammatory effect of D. eriocarpa alcohol extract. The hot plate test and twisting test were performed to observe the pain-relieving effect of D. eriocarpa. The lipopolysaccharide （LPS）-induced RAW264.7 cells were used to study the anti-gout effect and mechanism of D. eriocarpa alcohol extract. ResultA total of 12 key targets and 15 potential active components were obtained from the D. eriocarpa-component-gout target network. The emodin， betulinic acid， and medicarpin endowed D. eriocarpa with anti-hyperuricemia， anti-inflammatory， and pain-relieving effects by acting on Toll-like receptor 4 （TLR4）， NOD-like reception protein 3 （NLRP3）， and nuclear factor （NF）-κB. Compared with the control group， the model groups showed elevated serum uric acid level in mice （P<0.01）， increased swelling degree of rats （P<0.05， P<0.01）， alleviated the auricular swelling of mice （P<0.05）， reduced the twisting times of mice （P<0.05， P<0.01）， and increased the hot plate pain threshold （P<0.05）. Moreover， the model group showed up-regulated mRNA level of TLR4 and protein levels of TLR4， NF-κB， and NLRP3 in cells （P<0.01）， and elevated levels of TLR4 and NF-κB in the cell supernatant （P<0.05， P<0.01）. Compared with the model group， the alcoholic extracts （20， 10， 5 g·kg-1） of D. eriocarpa lowered the serum uric acid level in hyperuricemic mice （P<0.01）， inhibited foot and plantar swelling in rats （P<0.05， P<0.01）， down-regulated the mRNA level of TLR4 and the protein levels of TLR4， NF-κB， NLRP3 in cells， and lowered the levels of TLR4， TNF-α， NF-κB， and IL-6 in cell supernatants （P<0.05， P<0.01）. ConclusionD. eriocarpa alcohol extract may exert the anti-gout， anti-inflammatory， and pain-relieving effects by regulating the TLR4/NF-κB/NLRP3 signaling pathway.

Dendrobium Polysaccharides Mitigate CSE-induced Inflammatory Damage of Human Bronchial Epithelial Cells via TLR4/NF-κB Pathway / 中国实验方剂学杂志

ObjectiveTo observe the effects of Dendrobium polysaccharides on the secretion of inflammatory cytokines and Toll-like receptor 4 （TLR4）/nuclear factor （NF）-κB pathway in 16HBE cells exposed to cigarette smoke extract （CSE）. MethodThe 16HBE cells were classified into the control， CSE， and CSE+ Dendrobium polysaccharides （100， 200， 400 mg·L-1） groups. The cell-counting kit-8 （CCK-8） assay was employed to measure the cell viability， and a microscope was used to observe the cell morphology. The enzyme-linked immunosorbent assay was employed to measure the levels of interleukin （IL）-8， IL-1β， IL-4， IL-13， and transforming growth factor （TGF）-β in cell culture supernatants. Real-time PCR was carried out to determine the mRNA levels of Toll-like receptor 4 （TLR4）， nuclear factor-κB （NF-κB）， and IL-4. Western blot was employed to determine the protein levels of interleukin-4 receptor （IL-4R）， TLR4， myeloid differentiation primary response protein 88 （MyD88）， NF-κB， phosphorylated nuclear factor-κB （p-NF-κB）， and nucleoproteins nuclear factor-κB （NEs-NF-κB）. The immunofluorescence assay was employed to measure the nuclear translocation of NF-κB. ResultCompared with the control group， the CSE group showed elevated levels of IL-8， IL-1β， IL-4， IL-13， and TGF-β in the cell culture supernatants （P<0.05， P<0.01）， up-regulated expression levels of TLR4， MyD88， NF-κB， p-NF-κB， NEs-NF-κB， and IL-4 （P<0.01）， and significant nuclear translocation of NF-κB. Compared with the CSE group， Dendrobium polysaccharides increased the cell survival rate， recovered the cell activity， lowered the levels of IL-8， IL-1β， IL-4， IL-13， and TGF-β， down-regulated the expression of TLR4， MyD88， NF-κB， p-NF-κB， NEs-NF-κB， and IL-4 （P<0.05， P<0.01）， and reduced the nuclear translocation of NF-κB. ConclusionDendrobium polysaccharides showed significant protective effects on the 16HBE cells exposed to CSE by inhibiting the TLR4/NF-κB signaling pathway.

Huanglian Jiedutang Treats Acute Gouty Arthritis via NLRP3 Inflammasome and TLR4/NF-κB Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo observe the effect of Huanglian Jiedutang on the inflammatory injury in the mouse model of acute gouty arthritis （AGA） and to explore the mechanism of Huanglian Jiedutang in treating AGA. MethodForty male C57BL/6J mice were randomized into blank， model， colchicine （0.83 mg·kg-1）， and Huanglian Jiedutang （5 g·kg-1） groups. The mouse model of AGA was established by injecting monosodium urate （MSU） crystals into the ankle joint. The swelling degree of the right ankle joint of each mouse was measured every day for 7 days， and the pathological changes of the ankle joint were detected by hematoxylin-eosin （HE） staining after 7 days. The other 40 C57BL/6J mice were grouped as above. After 18 hours of modeling， the right ankle joint was collected， and real-time polymerase chain reaction was employed to measure the mRNA levels of interleukin （IL）-1β， tumor necrosis factor （TNF）-α， IL-6， NOD-like receptor protein 3 （NLRP3）， apoptosis-associated speck-like protein containing a CARD （ASC）， and Caspase-1. The expression levels of IL-1β， TNF-α， and IL-6 were measured by the enzyme-linked immunosorbent assay. Western blot was employed to determine the protein levels of NLRP3 inflammasome， Toll-like receptor 4 （TLR4）， and nuclear factor-κB （NF-κB）. ResultCompared with the blank group， the model group showed swelling right ankle joint （P<0.01）， obvious foreign body granuloma in the ankle joint with inflammatory cell infiltration. After the treatment with Huanglian Jiedutang， the ankle joint swelling was relieved （P<0.05， P<0.01）， and the size of foreign body granuloma was reduced. Compared with the blank group， the model group showed up-regulated mRNA levels of IL-1β， TNF-α， and IL-6 in the ankle joint tissue （P<0.01）， up-regulated mRNA levels of NLRP3 and Caspase-1 in the NLRP3 inflammasome （P<0.05， P<0.01）， and up-regulated protein levels of NLRP3， Caspase-1， TLR4， and NF-κB （P<0.05， P<0.01）. Huanglian Jiedutang down-regulated the mRNA levels of IL-1β， TNF-α， IL-6， NLRP3， and Caspase-1 （P<0.05， P<0.01） and the protein levels of IL-1β， TNF-α， IL-6， NLRP3， Caspase-1， TLR4， and NF-κB （P<0.05 or P<0.01）. ConclusionInjecting MSU crystal resulted in local inflammatory injury of the joints in the mouse model of AGA. The treatment with Huanglian Jiedutang may alleviate the inflammatory injury by regulating the NLRP3 inflammasome and TLR4/NF-κB signaling pathway.

Traditional Chinese Medicine Intervention in Ulcerative Colitis Based on TLR4 Signaling Pathway： A Review / 中国实验方剂学杂志

Ulcerative colitis （UC） is a disease that affects the mucosal and submucosal layers of the colon and is characterized by inflammation of the intestinal mucosa. The incidence of UC is increasing year by year， and it is complex and refractory， severely impacting the physical and mental health of patients. The pathological mechanism of this disease is complex， with immune responses and uncontrollable inflammatory reactions in the intestine being important physiopathologic mechanisms. Toll-like receptor 4 （TLR4）， as a transmembrane signaling receptor， plays a key role in mediating immune responses and inflammatory reactions in the development of UC. Currently， the treatment of UC mainly relies on salicylic acids， glucocorticoids， and other agents to reduce intestinal inflammation. While these drugs can partially inhibit the progression of the disease， they often come with significant adverse effects and the potential for relapse upon discontinuation. Traditional Chinese medicine （TCM） offers multiple pathways， effects， and targets for regulating the TLR4 pathway， suppressing inflammatory responses， and effectively intervening in the progression of UC. This approach has become a hot topic in the prevention and treatment of UC. Numerous studies have shown that TCM treatment of UC has unique advantages. TCM can enhance immune defenses， suppress inflammatory responses， promote intestinal mucosal healing， and maintain the balance of the intestinal microbiota by regulating the TLR4 signaling pathway， thereby effectively treating UC， with substantial progress achieved. However， there is currently a lack of comprehensive reviews on the role of TCM in regulating the TLR4 signaling pathway for the treatment of UC. Therefore， this article systematically summarized the relationship between the TLR4 signaling pathway and UC， as well as the role of TCM in this context， by reviewing relevant literature from recent years， aiming to provide new insights into the potential treatment and new drug development for UC.

Impact of interaction between NLRP3, TLR4 gene polymorphisms and triglyceride-glucose index on gout / 中华内分泌代谢杂志

Objective:To explore the effect of triglyceride glucose(TyG) index, single nucleotide polymorphism of Toll-like receptor 4(TLR4) and NOD-like receptor thermal protein domain associated protein 3(NLRP3) genes, and its interaction on the risk of gout.Methods:A total of 315 male patients with gout and 499 men for health checkup at the same period were selected. General data were collected through questionnaires, and peripheral venous blood was collected for biochemical test. Three single nucleotide polymorphisms(SNPs) of NLRP3 and TLR4 were detected with multiplex ligase assay reaction, and logistic regression analysis was applied to compare the correlation between NLRP3 and TLR4 alleles and gout risk. The interaction of SNP and TyG index with gout was analyzed by generalized multi-factor dimensionality reduction(GMDR) model and logistic regression.Results:After adjusting for smoking, drinking, and other factors, the risk of gout increased by 61.1% for each standard deviation increase in TyG index. CC genotypes of rs10754558, rs10759932, and rs7525979 were high risk genotypes of gout in Han ethnicity. GMDR results showed significant differences in the interaction models of rs10754558-TyG index, rs7525979-TyG index, and rs10759932-TyG index between control group and gout group( P<0.05), suggesting an interaction between the three genotypes of SNPs selected and TyG index. Stratified analysis of the three selected SNPs and TyG index showed that after adjusting for age, smoking, and other factors, the high TyG index patients carrying C/C or C/G genotype at rs10754558 displayed an increased risk of gout compared with those carrying GG genotype and low TyG index( OR=2.127, P<0.05). Conclusion:The CC genotypes of rs10754558, rs10759932, and rs7525979 are high risk genotypes for gout in Han ethnicity. The interaction between rs10754558 and TyG index may increase the risk of gout development.