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.

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.

Improvement effect and mechanism of “Layers adjusting external application”paste on synovial fibrosis in rats with knee osteoarthritis / 中国药房

OBJECTIVE To investigate the improvement effect and potential mechanism of “Layers adjusting external application” paste on synovial fibrosis （SF） in rats with knee osteoarthritis （KOA）. METHODS Male SD rats were randomly divided into sham operation group， KOA group and Layers adjusting external application group， with 8 rats in each group. KOA model was induced by the anterior cruciate ligament disruption method in KOA group and Layers adjusting external application group. Fourteen days after modeling， the Layers adjusting external application group was given “Layers adjusting external application” paste [Sanse powder （8 g for every 100 cm2）， Compound sanhuang ointment （5 g for every 100 cm2）] on the knee joint， 8 h every day， for 28 d in total. After the last administration， the degree of synovitis and fibrosis in rats was observed， and Krenn scoring was performed in each group. The expressions of collagen Ⅰ， high mobility group protein B1 （HMGB1） and phosphorylated nuclear factor-κB p65 （p-NF-κB p65） were detected in the synovial membrane； the contents of interleukin-1β （IL- 1β）， IL-6 and tumor necrosis factor-α （TNF-α） in serum as well as the expressions of fibrosis-related and HMGB1/Toll-like receptor 4 （TLR4）/NF-κB signaling pathway-related proteins and mRNA were detected in synovial tissue. RESULTS Compared with the sham operation group， the synovial lining cells in the KOA group showed significant proliferation and disordered arrangement， the inflammatory cell infiltration and collagen fiber deposition were obvious； the positive expressing cells of collagen Ⅰ， HMGB1 and p-NF-κB p65 were increased significantly； the contents of IL-1β， IL-6 and TNF-α in serum， the expressions of fibrosis-related protein （transforming growth factor-β， collagen Ⅰ， tissue inhibitor of metalloproteinase 1， α-smooth muscle actin） and their mRNA as well as theexpressions of HMGB1， TLR4 protein and their mRNA， the expressions of p-NF-κB p65 protein and NF-κB p65 mRNA were all increased significantly in synovial tissues of rats （P＜0.01）. Compared with the KOA group， the pathological changes in the synovial tissue of rats in Layers adjusting external application group were significantly improved， and the above quantitative indicators were significantly reversed （P＜0.05 or P＜0.01）. CONCLUSIONS “Layers adjusting external application” paste could significantly improve SF in KOA rats， the mechanism of which may be associated with the inhibition of the activation of HMGB1/ TLR4/NF-κB signaling pathway.

Protective effect of Qingjie Huagong decoction on pancreatic tissue of mice with severe acute pancreatitis by regulating the NOD-like receptor protein 3/Toll-like receptor 4/nuclear factor-kappa B signaling pathway / 临床肝胆病杂志

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.

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.

Research progress on mechanism of TLR4 in carcinogenesis of inflammatory bowel disease / 中国药理学通报

Patients with inflammatory bowel disease (IBD) have an increased risk of colorectal cancer (CRC) compared with the general population. Currently the molecular mechanism of CRC occurrence in the context of IBD is not clear. The inflammation-atypical hyperplasia-cancer process has been widely accepted. Toll-like receptor 4(TLR4) is a key receptor for pathogen recognition and immune activation, and plays a crucial role in inflammatory and carcinogenic transformation of IBD. Therefore, this paper reviews the epidemiology of colitis-associated colorectal cancer (CAC) and the main mechanisms of TLR4 in the development of IBD to CAC, which will help to further understand the carcinogenesis of IBD, detect and better describe CAC at an earlier stage, and provide more effective prevention and treatment for CAC.

Effect of cangfudaotan decoction on TLR4/NF-κB p65 signaling pathway in obese PCOS-IR rats / 中国药理学通报

Aim To explore the regulatory effect of Cangfudaotan Decoction on the ovarian Toll receptor 4 (TLR4)/nuclear transcription factor kBp65 (NF-κB p65) signaling pathway in obese PCOS-IR rats. Methods Forty-eight female rats were randomly divided into normal group (n = 8) and model group (n = 40). The obese PCOS-IR rats were established by letrozole (1 mg · kg

Gut microbiota controls the development of chronic pancreatitis: A critical role of short-chain fatty acids-producing Gram-positive bacteria

Chronic pancreatitis (CP) is a progressive and irreversible fibroinflammatory disorder, accompanied by pancreatic exocrine insufficiency and dysregulated gut microbiota. Recently, accumulating evidence has supported a correlation between gut dysbiosis and CP development. However, whether gut microbiota dysbiosis contributes to CP pathogenesis remains unclear. Herein, an experimental CP was induced by repeated high-dose caerulein injections. The broad-spectrum antibiotics (ABX) and ABX targeting Gram-positive (G+) or Gram-negative bacteria (G-) were applied to explore the specific roles of these bacteria. Gut dysbiosis was observed in both mice and in CP patients, which was accompanied by a sharply reduced abundance for short-chain fatty acids (SCFAs)-producers, especially G+ bacteria. Broad-spectrum ABX exacerbated the severity of CP, as evidenced by aggravated pancreatic fibrosis and gut dysbiosis, especially the depletion of SCFAs-producing G+ bacteria. Additionally, depletion of SCFAs-producing G+ bacteria rather than G- bacteria intensified CP progression independent of TLR4, which was attenuated by supplementation with exogenous SCFAs. Finally, SCFAs modulated pancreatic fibrosis through inhibition of macrophage infiltration and M2 phenotype switching. The study supports a critical role for SCFAs-producing G+ bacteria in CP. Therefore, modulation of dietary-derived SCFAs or G+ SCFAs-producing bacteria may be considered a novel interventive approach for the management of CP.

Gastrin attenuates sepsis-induced myocardial dysfunction by down-regulation of TLR4 expression in macrophages

Myocardial dysfunction is the most serious complication of sepsis. Sepsis-induced myocardial dysfunction (SMD) is often associated with gastrointestinal dysfunction, but its pathophysiological significance remains unclear. The present study found that patients with SMD had higher plasma gastrin concentrations than those without SMD. In mice, knockdown of the gastrin receptor, cholecystokinin B receptor (Cckbr), aggravated lipopolysaccharide (LPS)-induced cardiac dysfunction and increased inflammation in the heart, whereas the intravenous administration of gastrin ameliorated SMD and cardiac injury. Macrophage infiltration plays a significant role in SMD because depletion of macrophages by the intravenous injection of clodronate liposomes, 48 h prior to LPS administration, alleviated LPS-induced cardiac injury in Cckbr-deficient mice. The intravenous injection of bone marrow macrophages (BMMs) overexpressing Cckbr reduced LPS-induced myocardial dysfunction. Furthermore, gastrin treatment inhibited toll-like receptor 4 (TLR4) expression through the peroxisome proliferator-activated receptor α (PPAR-α) signaling pathway in BMMs. Thus, our findings provide insights into the mechanism of the protective role of gastrin/CCKBR in SMD, which could be used to develop new treatment modalities for SMD.

Acacetin protects rats from cerebral ischemia-reperfusion injury by regulating TLR4/NLRP3 signaling pathway / 中国中药杂志

This study aims to investigate the mechanism of acacetin in protecting rats from cerebral ischemia-reperfusion injury via the Toll-like receptor 4(TLR4)/NOD-like receptor protein 3(NLRP3) signaling pathway. Wistar rats were randomized into sham, model, low-and high-dose acacetin, and nimodipine groups, with 10 rats in each group. The rat model of middle cerebral artery occlusion(MCAO) was established with the improved suture method in other groups except the sham group. The neurological deficit score and cerebral infarction volume of each group were evaluated 24 h after modeling. Enzyme-linked immunosorbent assay(ELISA) was employed to measure the levels of interleukin-1β(IL-1β), IL-6, tumor necrosis factor-α(TNF-α), malondialdehyde(MDA), supe-roxide dismutase(SOD), and glutathione(GSH). Western blot was employed to determine the expression levels of B-cell lymphonoma-2(Bcl-2), Bcl-2-associated X protein(Bax), and TLR4/NLRP3 signaling pathway-related proteins(TLR4, p-NF-κB/NF-κB, NLRP3, pro-caspase-1, cleaved caspase-1, pro-IL-1β, and cleaved IL-1β) in the rat brain tissue. Hematoxylin-eosin(HE) staining was employed to reveal the histopathological changes in the ischemic area. Compared with the sham group, the modeling of MCAO increased the neurological deficit score and cerebral infarction volume, elevated the IL-1β, IL-6, TNF-α, and MDA levels and lowered the SOD and GSH levels in the brain tissue(P<0.05). Compared with the MCAO model group, low-and high-dose acacetin and nimodipine decreased the neurological deficit score and cerebral infarction volume, lowered the IL-1β, IL-6, TNF-α, and MDA levels and elevated the SOD and GSH levels in the brain tissue(P<0.05). Compared with the sham group, the model group showed up-regulated protein levels of Bax, TLR4, p-NF-κB/NF-κB, NLRP3, pro-caspase-1, cleaved caspase-1, pro-IL-1β, and cleaved IL-1β and down-regulated protein level of Bcl-2 in the brain tissue(P<0.05). Compared with the MCAO model group, the acacetin and nimodipine groups showed down-regulated protein levels of Bax, TLR4, p-NF-κB/NF-κB, NLRP3, pro-caspase-1, cleaved caspase-1, pro-IL-1β, and cleaved IL-1β and up-regulated protein level of Bcl-2 in the brain tissue(P<0.05). In conclusion, acacetin regulates the TLR4/NLRP3 signaling pathway to inhibit neuroinflammatory response and oxidative stress, thus exerting the protective effect on cerebral ischemia-reperfusion injury in rats.

Morphine promotes migration and lung metastasis of mouse melanoma cells

Abstract Background Morphine is an analgesic agent used for cancer pain management. There have been recent concerns that the immunosuppressant properties of morphine can also promote cancer metastasis. Morphine is an agonist for toll like receptor 4 (TLR4) that has a dual role in cancer development. The promotor or inhibitor role of morphine in cancer progression remains controversial. We investigated the effects of morphine on migration and metastasis of melanoma cells through TLR4 activation. Methods Mouse melanoma cells (B16F10) were treated with only morphine (0, 0.1, 1, and 10 μM) or in combination with a TLR4 inhibitor (morphine10 μM +CLI-095 1μM) for either 12 or 24 hours. Migration of cells was analyzed by transwell migration assays. Twenty C57BL/6 male mice were inoculated with B16F10 cells via the left ventricle of the heart and then randomly divided into two groups (n = 10 each) that received either morphine (10 mg.kg−1, sub-q) or PBS injection for 21 days (control group). Animals were euthanized and their lungs removed for evaluation of metastatic nodules. Results Morphine (0.1, 1, and 10 μM) increased cell migration after 12 hours (p < 0.001) and after 24 hours of treatment with morphine (10 μM) (p < 0.001). Treatment with CLI-095 suppressed migration compared to cells treated with morphine alone (p < 0.001). Metastatic nodules in the morphine-treated group (64 nodules) were significantly higher than in the control group (40 nodules) (p < 0.05). Conclusion Morphine increases the migration and metastasis of mouse melanoma cells by activating TLR4.

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.