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With a global rise in morbidity rates， obesity has become a pressing public health issue. With increased adipocyte number and volume as the main characteristics， obesity is also manifested by metabolic disorders to varying degrees. At the same time， obesity is a risk factor for diabetes， hypertension， stroke， cancer， and cardiovascular diseases， imposing burdens on society and families. Influenced by lifestyle， environment， behavior， and genetics， obesity is caused by the interaction of many factors， and its pathological process is complex， involving inflammation， autophagy， and intestinal dysbiosis. The mitogen-activated protein kinase （MAPK） cascade reaction， a pivotal signaling pathway， plays a crucial role in cellular processes such as proliferation， differentiation， apoptosis， and stress responses. Both Chinese and international studies indicate that the MAPK signaling pathway can effectively regulate obesity through various pathways， including the modulation of adipocyte differentiation and apoptosis， appetite control， and inflammation improvement. Moreover， traditional Chinese medicine （TCM） has demonstrated significant efficacy in preventing and treating obesity， leveraging advantages such as multiple targets， diverse components， and minimal adverse effects. Research indicates that the MAPK signaling pathway is a primary focus of TCM regulation in this context， although a systematic review in this field is currently lacking. Therefore， this paper， by reviewing the latest Chinese and international research， provided a concise overview of the basic structure of the MAPK pathway， with a specific emphasis on recent progress in TCM interventions targeting the MAPK pathway for obesity treatment. The results indicate that regulating adipose tissue formation， differentiation， and thermogenesis， reducing inflammation and oxidative stress levels， and improving insulin sensitivity and metabolic disorders seem to be the main ways for TCM to regulate the MAPK pathway to prevent and treat obesity. However， it is necessary to find more research methods and explore potential mechanisms underlying TCM formulations based on the MAPK pathway for obesity prevention and treatment.
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ObjectiveThe antitumor activity of sesquiterpenoid M36 isolated from Myrrha against human hepatoma HepG2 cells was investigated in this study. MethodHepG2 cells were treated with M36 at different concentrations （0， 2， 4， 6， 8， 10 μmol·L-1）. Firstly， the effects of M36 on the proliferation of human hepatoma HepG2 cells were detected by methyl thiazolyl tetrazolium （MTT）， colony formation assay， and EdU proliferation assay. Hoechst staining， flow cytometry analysis， and Western blot were used to explore the effect of M36 on the apoptosis of human hepatoma HepG2 cells. Acridine orange staining and western blotting were used to examine the effect of M36 on autophagy in HepG2 cells. Finally， Western blot was used to detect protein expression of cancer-related signaling pathways. ResultCompared with the blank group， M36 treatment significantly inhibited the proliferation of human hepatoma HepG2 cells （P<0.01）， and the half inhibitory concentration （IC50） value of M36 for 48 h was 5.03 μmol·L-1， in a dose- and time-dependent manner. M36 was also able to induce apoptosis and autophagy in human hepatoma HepG2 cells. After treatment with 8 μmol·L-1 M36 for 48 hours， the apoptosis rate of HepG2 cells was （42.03±9.65）% （P<0.01）. Compared with the blank group， HepG2 cells treated with 4 and 8 μmol·L-1 M36 for 48 h had a significant increase in cleaved poly ADP-ribose polymerase （cleaved-PARP） protein levels （P<0.01）. Acridine orange staining showed that autophagy was significantly activated in HepG2 cells treated with 4 and 8 μmol·L-1 M36 for 48 h compared with the blank group （P<0.01）， which was further verified by the up-regulation of microtubule-associated protein 1 light chain 3 Ⅱ （LC3 Ⅱ）. Western blot results showed that compared with the blank group， the levels of phosphorylated extracellular regulated protein kinase （p-ERK）， phosphorylated p38 mitogen-activated protein kinase （p-p38 MAPK）， phosphorylated c-Jun N-terminal kinase （p-JNK）， and its downstream nuclear transcription factors c-Jun and p-c-Jun protein were significantly increased in M36 group （P<0.05， P<0.01）. The mechanism may be related to the up-regulation of MAPK signaling pathway. ConclusionThe sesquiterpenoid M36 isolated from Myrrha inhibits the proliferation of human hepatoma HepG2 cells and promotes apoptosis and autophagy， which may be related to the activation of the MAPK signaling pathway.
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ObjectiveTo observe the effects and underlying mechanisms of Fagopyri Dibotryis Rhizoma extract on the proliferation， apoptosis， and autophagy of human colorectal cancer HCT-116 cells. MethodFirstly， cellular activity was detected by the cell proliferation and activity-8 （CCK-8） assay， and the half inhibition rate （IC50） was calculated. Blank group and Fagopyri Dibotryis Rhizoma group （2， 4， 8 mg·L-1） were set. The effect of Fagopyri Dibotryis Rhizoma on the proliferation of HCT-116 cells was observed by cloning experiments， and that of Fagopyri Dibotryis Rhizoma on apoptosis was observed by flow cytometry. The expressions of autophagy-related proteins， p38 mitogen-activated protein kinase （p38 MAPK）， extracellular signal-regulated kinase （ERK）， c-Jun amino-terminal kinase （JNK）， phosphorylated （p）-p38， p-ERK， p-JNK， and other proteins were detected by Western blot. Finally， flow cytometry instrumentation and fluorescence microscopy were used to analyze the changes in reactive oxygen species （ROS）， and a ROS scavenger （NAC） was adopted for verification. ResultCompared with the blank group， the activity of HCT-116 cells was significantly decreased in the Fagopyri Dibotryis Rhizoma group （P<0.05， P<0.01）. The apoptosis rate of HCT-116 cells in the Fagopyri Dibotryis Rhizoma group was significantly increased （P<0.01）. The expression of autophagy-related protein ubiquitin-binding protein （p62） was decreased， but that of autophagy-specific genes （Beclin1） and autophagic microtubule-associated protein 1 light-chain 3B （LC3B） was enhanced （P<0.05， P<0.01）. Compared with the Fagopyri Dibotryis Rhizoma group， the apoptosis rate of HCT-116 cells in the Fagopyri Dibotryis Rhizoma + NAC group was significantly reduced （P<0.01）. The expression of related autophagy protein Beclin1 was significantly reduced （P<0.01）， and that of LC3B protein was reduced （P<0.01）. In addition， the expression of MAPK pathway-related proteins ERK and JNK was decreased in the Fagopyri Dibotryis Rhizoma group （P<0.05， P<0.01）， and that of p-ERK and p-JNK was enhanced （P<0.05， P<0.01）. ConclusionFagopyri Dibotryis Rhizoma can inhibit the proliferation of HCT-116 cells and induce apoptosis and autophagy through the ROS/MAPK signaling pathway.
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Osteoporosis （OP） is a systemic metabolic disease that affects the health of middle-aged and elderly people by crosslinking multiple signaling pathways. With the increasing aging of the population， the incidence of OP is also increasing year by year. Because of a series of problems such as high incidence， difficulty in treatment， and poor prognosis， it has been widely studied and reported by scholars in China and abroad. At present， the drugs used by western medicine are mainly divided into two categories： Bone resorption inhibitors and bone formation promoters. Although the efficacy is reliable， there are still deficiencies such as poor dependence of patients on the drug， uncontrollable side effects， and high costs. However， in recent years， with the continuous deepening and innovation of traditional Chinese medicine （TCM） research， the treatment of OP by TCM has been widely recognized in clinical practice. Many scholars have found that the mechanism of TCM in the treatment of OP includes the widespread involvement of mitogen-activated protein kinase （MAPK） signaling pathway， which mainly promotes the differentiation of bone marrow mesenchymal stem cells （BMSCs） to osteoblasts （OB）， inhibits the differentiation of osteoclasts （OC）， and improves the expression of osteogenesis-related factors alkaline phosphatase （ALP）， Runt-associated transcription factor 2 （Runx2）， type Ⅰ collagen （ColⅠ） to treat OP. Although the current research on the TCM treatment of OP through the MAPK pathway is deepening， there are still certain deficiencies in the study of its molecular mechanism. Therefore， this paper reviewed the relationship between the MAPK signaling pathway and key target protein factors and OP to clarify the important role of the MAPK signaling pathway in OP. At the same time， the targeted regulation of MAPK signaling pathways by TCM to treat OP was systematically summarized in order to provide a scientific basis for the further accurate treatment of OP in TCM.
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ObjectiveTo observe the protective effect of Baoshen prescription against renal fibrosis and explore its underlying mechanism through network pharmacology， molecular docking， and in vivo experiments. MethodAll mice were randomly divided into sham surgery group， model group， low-， medium-， and high-dose Baoshen prescription groups， and a benazepril hydrochloride group. Unilateral ureteral obstruction （UUO） was performed to establish a renal fibrosis model， and the administration of Baoshen prescription at low， medium， and high doses （0.455， 0.91， and 1.82 g·kg-1）， and benazepril hydrochloride （1.68 mg·kg-1） or distilled water began on the same day as model preparation. Mice in the model group and the sham surgery group were given an equal volume of distilled water. The intervention was carried out once daily for 14 days. Mouse serum levels of blood urea nitrogen （BUN） and creatinine （Cr） were measured. Hematoxylin-eosin （HE） staining and Masson staining were used to observe renal pathological changes. Western blot and immunohistochemistry were used to assess the expression of fibronectin （FN）， α-smooth muscle actin （α-SMA）， and E-cadherin， which are related to renal fibrosis. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was used to measure the mRNA expression of transforming growth factor-β1 （TGF-β1）， tumor necrosis factor-α （TNF-α）， NOD-like receptor protein 3 （NLRP3）， and monocyte chemoattractant protein-1 （MCP-1） in renal tissues. The mechanism of Baoshen prescription in improving renal fibrosis was explored through network pharmacology， molecular docking， and Western blot experiments. ResultCompared with the sham surgery group， the model group showed significantly increased levels of BUN and Cr （P<0.01）. The model group exhibited abnormal renal glomerular morphology， loss of tubular brush borders， tubular dilation， and an enlarged area of blue collagen fibers. Mice in the model group showed significantly elevated levels of FN and α-SMA （P<0.01）， significantly decreased expression of E-cadherin （P<0.01）， and significantly increased expression of TGF-β1， TNF-α， NLRP3， and MCP-1 mRNA （P<0.05， P<0.01）. Compared with the model group， the Baoshen prescription groups showed significantly reduced BUN and Cr levels （P<0.01）， alleviated renal pathological damage， improved fibrosis， reduced expression of FN and α-SMA （P<0.01）， increased E-cadherin expression （P<0.01）， and downregulated mRNA expression of TGF-β1， TNF-α， NLRP3， and MCP-1 （P<0.05， P<0.01）. Network pharmacology and molecular docking predicted that Baoshen prescription could potentially improve renal fibrosis through the extracellular signal-regulated kinase （ERK）/p38 mitogen-activated protein kinase （MAPK） signaling pathway. Pharmacological research showed that compared with the sham surgery group， the model group exhibited significantly increased expression of phosphorylated （p）-ERK and p-p38 （P<0.05， P<0.01）. Compared with the model group， medium- and high-dose Baoshen prescription groups showed significantly downregulated expression of p-ERK and p-p38 proteins （P<0.05， P<0.01）. ConclusionBaoshen prescription can effectively improve renal fibrosis induced by UUO in mice， and its mechanism of action may be related to the ERK/p38 MAPK signaling pathway.
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ObjectiveTo investigate the feasibility of ethyl acetate fraction of Ipomoea muricatum （IM-EA） in the prevention and treatment of alcoholic gastric ulcer （GU） and explore its mechanism of action based on network pharmacology and experimental verification. MethodForty SD rats were randomly divided into a control group， a model group， a ranitidine group （2.7 mg·kg-1）， and low- and high-dose IM-EA groups （30，60 mg·kg-1） after adaptive feeding for 7 days. The GU model was replicated by hydrochloric acid in absolute ethanol （150 mmol·L-1） in rats after prophylactic administration for one week. Hematoxylin-eosin （HE） staining and periodic acid-Schiff （PAS） staining were used to preliminarily evaluate the efficacy of IM-EA in the prevention and treatment of GU. Lead compounds of IM-EA were screened out by ADMET， and the SwissTarget platform was used to identify the potential targets for these compounds. GU-related targets were collected through DisGeNET， OMIM， and GeneCards databases， which were mapped to potential IM-EA targets to obtain the potential targets of IM-EA against GU. The STRING database was used to construct the protein-protein interaction （PPI） network to screen the hub targets， and the DAVID platform was used to annotate the biological functions of common targets to explore the underlying mechanism of IM-EA against GU. Autodock Vina software was used for the preliminary verification of the computer simulation. The serum levels of tumor necrosis factor （TNF）-α and interleukin （IL）-6 and the content of prostaglandin E2 （PGE2）， matrix metalloproteinase-9 （MMP-9）， and superoxide dismutase （SOD） in the gastric tissues were determined by enzyme-linked immunosorbent assay （ELISA）. The relative expression levels of core proteins in the mitogen-activated protein kinase （MAPK） signaling pathway， such as Jun oncoprotein， extracellular signal-regulated kinase （ERK）， and p38， in the gastric tissues were detected by Western blot. ResultAs revealed by the results of animal experiments， compared with the control group， the model group showed significantly damaged gastric tissues and reduced secretion of gastric mucus. Compared with the model group， the groups with drug intervention showed reduced ulcer areas in the gastric tissues （P<0.01） and improved gastric histopathological status and gastric mucus secretion， suggesting that IM-EA was effective in the prevention and treatment of GU. Sixteen lead compounds of IM-EA were screened out by ADMET， and 257 potential targets of IM-EA against GU were obtained. The hub nodes in the PPI network included targets of TNF-α， protein kinase B1 （Akt1）， tumor protein 53 （TP53）， epidermal growth factor receptor （EGFR）， and ERK. Biological functional annotation and molecular docking results suggested that the MAPK signaling pathway potentially played a key role in the prevention and treatment of GU by IM-EA， which was synergistic with the vascular endothelial growth factor （VEGF） signaling pathway， phosphoinositide 3-kinase （PI3K）/Akt signaling pathway， and nuclear factor （NF）-κB signaling pathway in anti-inflammation， anti-oxidation， and damage repair. The pharmacological experiment results showed that compared with the control group， the model group showed increased serum IL-6 content （P<0.01）， an increasing trend of TNF-α content， increased MMP-9 content in the gastric tissues （P<0.01）， and decreased SOD content （P<0.05）. Compared with the model group， the IM-EA groups showed decreased TNF-α and IL-6 levels in the serum and PGE2 and MMP-9 levels in the gastric tissues （P<0.01）， and increased SOD content in the gastric tissues （P<0.01）. Compared with the control group， the model group showed up-regulated expression of p-p38， p-Jun， and p-ERK in the gastric tissues （P<0.01） and up-regulated p38 and Jun （P<0.01）. Compared with the model group， the IM-EA groups showed down-regulated p-p38， p-Jun， p-ERK， and p38 in the gastric tissues （P<0.01） and up-regulated relative expression of Jun and ERK （P<0.05）. ConclusionIM-EA has a remarkable effect in the prevention and treatment of alcoholic gastric injury， which may be achieved through the mechanisms of anti-inflammation， anti-oxidation， and wound repair mediated by the MAPK signaling pathway.
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ObjectiveTo explore the anti-inflammatory mechanism of Huangqintang based on the inflammation model in RAW264.7 cells. MethodHuangqintang was prepared and the safe dose to RAW264.7 cells was screened out. The RAW264.7 cells were seeded in 24-well plates and incubated with Huangqintang and lipopolysaccharide （LPS）， successively. The concentrations of nitric oxide （NO）， interleukin （IL）-6， tumor necrosis factor （TNF）-α， and prostaglandin E2 （PGE2） were measured by Griess assay and enzyme-linked immunosorbent assay （ELISA）， respectively. Meanwhile， RAW264.7 cells were inoculated in 6-well plates， and normal group， LPS group， LPS+Huangqintang group， nuclear factor-κB （NF-κB） p65 inhibitor PDTC group， p38 mitogen-activated protein kinase （MAPK） inhibitor SB203580 group， extracellular signal-regulated kinase （ERK） inhibitor PD98059 group， c-Jun N-terminal kinase （JNK） inhibitor SP600125 group， and Janus kinase （JAK） inhibitor AG490 group were set up. After the cells were incubated with corresponding inhibitors and Huangqintang and stimulated by LPS， RNA and protein were extracted. The mRNA and protein expression levels of NF-κB p65， p38 MAPK， ERK， JNK， and JAK were detected by Real-time fluorescence-based quantitative polymerase chain reaction （Real-time PCR） and Western blot， respectively， to explore the anti-inflammatory mechanism of Huangqintang by regulating the NF-κB， MAPK， and JAK/signal transducer and activator of transcription protein （STAT） signaling pathways. ResultAfter stimulation with LPS， the concentrations of NO， IL-6， TNF-α， and PGE2 in the cells of the model group increased significantly（P<0.05，P<0.01）. Compare with the model group， after incubation with Huangqintang， the secretion of NO， IL-6， TNF-α， and PGE2 showed a downward trend （P<0.05，P<0.01）. Compared with the normal group， the model group showed increased mRNA expression of p38 MAPK， ERK， JNK， JAK， and NF-κB p65 and total protein expression in cells after stimulation with LPS （P<0.05，P<0.01）. Compare with the model group，after incubation with Huangqintang， the total protein and mRNA expression of p38 MAPK， ERK， JNK， JAK， and NF-κB p65 in inflammatory cells decreased （P<0.05，P<0.01）. Meanwhile， the expression of NF-κB p65 total protein and mRNA in each inhibitor group showed a downward trend （P<0.05，P<0.01）. ConclusionHuangqintang can inhibit the inflammatory response through the NF-κB， MAPK， and JAK-STAT signaling pathways.
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ObjectiveTo investigate the regulatory effects of Xuanfu Daizhetang on a mouse model of allergic asthma induced by ovalbumin （OVA）. MethodSixty female BALB/c mice （6-8 weeks old， SPF） were randomly divided into groups. Ten mice were assigned to the normal group and given 0.2 mL of saline， while the remaining groups received intraperitoneal injections of Al（OH）3 at 5 g·L-1 and OVA at 1 g·L-1. The mice were divided into normal group （10 mL·kg-1 saline）， OVA model group （10 mL·kg-1 saline）， dexamethasone group （OVA+DEX， 1 mg·kg-1）， OVA+ low-dose Xuanfu Daizhetang group （OVA+XL， 7.065 g·kg-1）， OVA+ medium-dose Xuanfu Daizhetang group （OVA+XM， 14.13 g·kg-1）， and OVA+ high-dose Xuanfu Daizhetang group （OVA+XH， 28.26 g·kg-1）. An OVA-induced asthma model was established in mice. Hematoxylin-eosin （HE） and periodic acid-Schiff （PAS） staining methods were used to observe bronchial tissue pathological changes. Enzyme-linked immunosorbent assay （ELISA） was performed to measure the levels of immunoglobulin E （IgE）， interleukin-4 （IL-4）， IL-5， IL-13， IL-17A， and γ interferon （IFN-γ） in bronchoalveolar lavage fluid. Western blot was used to detect the phosphorylation of extracellular signal-regulated kinase （ERK）， p38 mitogen-activated protein kinase （MAPK）， and c-Jun N-terminal kinase （JNK） proteins in lung tissue. ResultCompared with the normal group， the OVA model group showed increased inflammatory cell infiltration in mouse alveoli， elevated levels of IL-4， IL-5， IL-13， IL-17A， IFN-γ in bronchoalveolar lavage fluid， and IgE in serum （P<0.05， P<0.01）， and promoted phosphorylation of MAPK signaling pathway-related proteins. Compared with the model group， the OVA+XL， OVA+XM， and OVA+XH groups showed reduced inflammatory cell infiltration in mouse alveoli， decreased levels of IL-4， IL-5， IL-13， IL-17A， IFN-γ in bronchoalveolar lavage fluid， and IgE in serum （P<0.05， P<0.01）， and inhibited phosphorylation of MAPK signaling pathway-related proteins. ConclusionThe results of this study suggest that Xuanfu Daizhetang has potential anti-allergic asthma activity， providing a theoretical basis for its future clinical application.
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ObjectiveTo explore the mechanism of Fuzi Lizhongwan alleviating the damage of chemotherapy-induced peripheral neuropathy （CIPN） mice caused by cisplatin based on mitogen-activated protein kinase （MAPK） signaling pathway. MethodA total of 40 female KM mice were randomized into blank group （distilled water， ig）， model group （distilled water， ig）， Fuzi Lizhongwan group （3.5 g·kg-1， ig）， and aspirin group （0.026 g·kg-1， ig）. Cisplatin （3 mg·kg-1， ip， 5 days） was used to induce CIPN in mice. Administration began while modeling and lasted 12 days. The general conditions and behaviors of mice were observed. After the last administration， samples were collected. Pathological changes of the soles were observed based on hematoxylin-eosin （HE） staining. Biochemical assay was employed to determine the levels of serum superoxide dismutase （SOD）， hydrogen peroxide （H2O2）， malondialdehyde （MDA）， and nitric oxide （NO）， enzyme-linked immunosorbent assay （ELISA） the content of interleukin-6 （IL-6）， interleukin-1β （IL-1β）， tumor necrosis factor-α （TNF-α）， and glutathione peroxidase-3 （GPX-3） in kidney tissue， and Western blotting the expression of extracellular signal-regulated kinase1/2 （ERK1/2）， phosphorylated-ERK1/2 （p-ERK1/2）， p38 MAPK， and phosphorylated-p38 MAPK （p-p38 MAPK） in kidney tissue. ResultCompared with the blank group， model group demonstrated obvious pathological damage on the soles， hyperkeratosis of the epidermis with a basketweave pattern， atrophy of stratum spinosum， reduction of cells， and intracellular edema. Compared with the model group， Fuzi Lizhongwan significantly alleviated the pathological damage of the skin tissue of the soles. The model group showed lower body weight， mechanical pain threshold， thermal pain threshold （P<0.01）， and SOD activity （P<0.05）， higher content of H2O2， MDA， and NO （P<0.01）， and higher expression of IL-6， IL-1β， and TNF-α （P<0.01） than the blank group. Fuzi Lizhongwan group demonstrated higher body weight， mechanical pain threshold， thermal pain threshold （P<0.01）， and SOD activity （P<0.05）， lower content of H2O2， MDA， and NO （P<0.05）， and lower expression of IL-6， IL-1β， and TNF-α （P<0.01） than the model group. The expression of ERK1/2， p-ERK1/2， p38 MAPK， and p-p38 MAPK increased significantly （P<0.01） in the model group compared with that in the blank group， while the expression decreased significantly （P<0.01） in the Fuzi Lizhongwan group compared with that in the model group. ConclusionFuzi Lizhongwan can relieve the neurological injury of cisplatin-induced CIPN mice and increase the pain threshold of mice， possibly by regulating the MAPK signaling pathway and inhibiting inflammatory response and oxidative stress.
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ObjectiveTo observe the repair effect of Dahuanglingxian prescription （DHLX） on bile duct epithelial cells of rats. To explore whether its mechanism of action is to adjust the mutual binding of transforming growth factor -β （TGF-β） activated kinase 1（TAK1） and tumor necrosis factor receptor-associated factor 6 （TRAF6）， and regulate the activation of the nuclear transcription factor -κB （NF-κB）/mitogen-activated protein kinase （MAPK） signaling pathway. MethodThe 20 SD rats were randomly divided into normal group and DHLX group， 10 rats in each group， were given saline and DHLX （320 mg·kg-1·d-1） for 8 days， to prepare normal serum and DHLX serum. Biliary epithelial cells were extracted from normal SD rats and divided into 9 groups： Normal group， model group （20 mg·L-1）， LPS+DHLX group （20 mg·L-1+10% DHLX）， LPS+PDTC group （20 mg·L-1+200 μmol·L-1）， LPS+SB203580 group （20 mg·L-1+0.5 μmol·L-1）， LPS+PDTC+SB203580 group （20 mg·L-1+200 μmol·L-1+0.5 μmol·L-1）， LPS+PDTC+DHLX group （20 mg·L-1+200 μmol·L-1+10% DHLX serum）， LPS+SB203580+DHLX group （20 mg·L-1+0.5 μmol·L-1+10% DHLX serum）， LPS+PDTC+SB203580 +DHLX group （20 mg·L-1+200 μmol·L-1+0.5 μmol·L-1+10% DHLX serum）. The microscopic observation of morphological changes in each group of cells after drug intervention. Enzyme-linked immunosorbent assay（ELISA） was used to detect the expression of （IL）-1β and IL-6 in each group of cells. Western blot detected the expression levels of TAK1 and TRAF6 proteins in each group of cells， Co-IP detected the interaction between TAK1 and TRAF6， and further observed the distribution and co-localization of TAK1 and TRAF6 using Laser confocal microscope. ResultAfter the action of LPS， the cell synapses are reduced， the cell body becomes significantly rounded and smaller， but the cell morphology of each group tends to be normal after medication. Compared with normal group， the expression levels of IL-1β and IL-6 in model group were significantly increased （P<0.05）， while the expression level of TAK1 was decreased while the expression level of TRAF6 was increased （P<0.05）. The content of TAK1-TRAF6 protein complex showed a decreasing trend， and the two proteins co-located in the cytoplasm. Compared with model group， the expression levels of IL-1β and IL-6 in LPS+DHLX group were significantly decreased （P<0.05）， the expression level of TAK1 was increased and the expression level of TRAF6 was decreased （P<0.05）， the content of TAK1-TRAF6 protein complex was significantly increased （P<0.01）， and the two proteins were significantly co-located in cytoplasm. Compared with LPS+DHLX group， the expression levels of IL-1β and IL-6 in other groups were significantly decreased （P<0.05，P<0.01）. TAK1-TRAF6 protein complex content in each group was significantly decreased after pathway blocker intervention （P<0.05）， while TAK1-TRAF6 protein complex content in each group was significantly increased after pathway blocker combined with DHLX intervention （P<0.05）. Co-localization of the TAK1-TRAF6 in cytoplasm was not obvious. ConclusionIn the LPS-induced inflammatory response of bile duct cells， the binding of TAK1 and TRAF6 showed a weakening trend， but DHLX could reverse the phenomenon， we think the mechanism of action may be related to promoting the mutual binding of TAK1 and TARF6 to inhibit the activation of the NF-κB/MAPK signaling pathway.
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ObjectiveProteoglycan TPG-1 isolated from Trametes robiniophila（Huaier） has proved to have anti-hepatoma activity， and this paper aims to explore the molecular mechanism. MethodHuman hepatoma SK-HEP-1 cells were treated with TPG-1 （0， 0.05， 0.1， 0.25， 0.5， 1 g·L-1）. Then cell survival was detected by methyl thiazolyl tetrazolium （MTT） and apoptosis by flow cytometry. In addition， expression of genes in SK-HEP-1 cells treated with or without TPG-1 was examined by DNA microarray to preliminarily explore the anti-hepatoma molecular mechanism of TPG-1. ResultTPG-1 inhibited the proliferation of SK-HEP-1 cells as compared with the blank group （P<0.01）. After treatment with 1 g·L-1 TPG-1 for 48 h， the apoptosis rate of SK-HEP-1 cells increased （P<0.01）， and TPG-1 promoted the cleavage of cysteinyl aspartate specific proteinase （Caspase）-3 and Caspase-7， the key mediators of apoptosis （P<0.01）. Additionally， TPG-1 （1 g·L-1） suppressed the migration of SK-HEP-1 cells （P<0.05）. A total of 971 differentially expressed genes （DEGs） were identified in SK-HEP-1 cells after treatment with TPG-1， with 486 up-regulated and 485 down-regulated. These DEGs were mainly involved in the Gene Ontology （GO） terms of interleukin-6 （IL-6） biosynthesis， antigen processing and presentation， superoxide dismutase activity， positive regulation of mitogen-activated protein kinase kinase kinase （MAPKKK） cascade， nature killer （NK） cell chemotaxis， and chemokine biosynthesis， and the Kyoto Encyclopedia of Genes and Genomes （KEGG） pathways of nucleotide-binding oligomerization domain （NOD）-like receptor signaling pathway， apoptosis， Toll-like receptor signaling pathway， retinoic acid-inducible gene-Ⅰ （RIG-Ⅰ）-like receptor signaling pathway， T-cell receptor signaling pathway， and chemokine signaling pathway. Western blot results showed that TPG-1 （1 g·L-1） activated mitogen-activated protein kinase （MAPK） signaling pathway in SK-HEP-1 cells （P<0.01）. ConclusionProteoglycan TPG-1 inhibited the proliferation and migration， and induced apoptosis of human hepatoma SK-HEP-1 cells. Up-regulation of MAPK signaling pathway may be responsible for the growth inhibition of human hepatoma SK-HEP-1 cells by TPG-1.
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ObjectiveTo study the effect of apigenin on the proliferation and apoptosis of human colon cancer CL187 cells and the underlying mechanisms. MethodHuman colorectal cancer CL187 cells were treated with different concentrations of apigenin （0， 30， 45， 60 mg·L-1） according to the results of the preliminary experiment. The proliferation of CL187 cells was detected by methyl thiazolyl tetrazolium （MTT） and colony formation assays， and the apoptosis was observed via Hoechst 33258 staining. Real-time fluorescence quantitative PCR was conducted to determine the mRNA levels of cysteine protease-3 （Caspase-3）， B-cell lymphoma-2 （Bcl-2）， and Bcl-2-associated X protein （Bax） in the CL187 cells treated with apigenin. Western blot was employed to measure the protein levels of Caspase-3， Bcl-2， and Bax associated with apoptosis， protein kinase B （Akt） and phosphorylated Akt （p-Akt） in phosphatidylinositol 3-kinase/protein kinase B （PI3K/Akt） pathway， and extracellular signal-regulated kinases 1/2 （ERK1/2）， p-ERK1/2， c-Jun N-terminal kinase （JNK）， p-JNK， p38 mitogen-activated protein kinase （MAPK）， and p-p38 MAPK protein in MAPK pathway. ResultCompared with the blank group， the apigenin groups had low cell survival rates and high inhibition rates on cell proliferation （P<0.01）. Apigenin decreased the cell clone number and clone formation rate， and increased the inhibition rate on clone formation （P<0.01）. After CL187 cells were treated with different concentrations of apigenin for 48 h， typical apoptosis characteristics such as nuclear pyknosis， chromatin condensation， and enhanced fluorescence reaction were observed. Compared with blank group， 45， 60 mg·L-1 apigenin treatments down-regulated the mRNA level of anti-apoptotic gene Bcl-2 （P<0.01） and all the apigenin treatments up-regulated those of the pro-apoptotic genes Bax and Caspase-3 （P<0.05， P<0.01）. Similarly， apigenin treatments down-regulated the protein level of Bcl-2 （P<0.05， P<0.01） and up-regulated those of Caspase-3 （P<0.05， P<0.01） and Bax （P<0.01， 45， 60 mg·L-1）. The blank group had higher protein level of Akt than the 60 mg·L-1 apigenin group （P<0.01）， higher protein levels of p-Akt， ERK1/2， and p-ERK1/2 than the 45， 60 mg·L-1 apigenin groups （P<0.01）， and higher protein levels of JNK and p-JNK than the apigenin groups （P<0.05， P<0.01）. Compared with blank group， 60 mg·L-1 apigenin up-regulated the protein level of p38 MAPK （P<0.05）， and all the apigenin groups up-regulated that of p-p38 MAPK （P<0.01）. Furthermore， apigenin lowered the p-Akt/Akt ratio （P<0.05， P<0.01） and p-ERK1/2/ERK1/2 ratio （P<0.01）， while it increased the p-JNK/JNK ratio （45， 60 mg·L-1； P<0.05， P<0.01） and p-p38 MAPK/p38 MAPK ratio （P<0.05， P<0.01）. ConclusionApigenin can inhibit the proliferation and promote the apoptosis of CL187 cells by inhibiting the PI3K/Akt signaling pathway and regulating the expression of proteins in the MAPK signaling pathway.
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ObjectiveTo study the effect of apigenin on the proliferation and apoptosis of human colon cancer CL187 cells and the underlying mechanisms. MethodHuman colorectal cancer CL187 cells were treated with different concentrations of apigenin （0， 30， 45， 60 mg·L-1） according to the results of the preliminary experiment. The proliferation of CL187 cells was detected by methyl thiazolyl tetrazolium （MTT） and colony formation assays， and the apoptosis was observed via Hoechst 33258 staining. Real-time fluorescence quantitative PCR was conducted to determine the mRNA levels of cysteine protease-3 （Caspase-3）， B-cell lymphoma-2 （Bcl-2）， and Bcl-2-associated X protein （Bax） in the CL187 cells treated with apigenin. Western blot was employed to measure the protein levels of Caspase-3， Bcl-2， and Bax associated with apoptosis， protein kinase B （Akt） and phosphorylated Akt （p-Akt） in phosphatidylinositol 3-kinase/protein kinase B （PI3K/Akt） pathway， and extracellular signal-regulated kinases 1/2 （ERK1/2）， p-ERK1/2， c-Jun N-terminal kinase （JNK）， p-JNK， p38 mitogen-activated protein kinase （MAPK）， and p-p38 MAPK protein in MAPK pathway. ResultCompared with the blank group， the apigenin groups had low cell survival rates and high inhibition rates on cell proliferation （P<0.01）. Apigenin decreased the cell clone number and clone formation rate， and increased the inhibition rate on clone formation （P<0.01）. After CL187 cells were treated with different concentrations of apigenin for 48 h， typical apoptosis characteristics such as nuclear pyknosis， chromatin condensation， and enhanced fluorescence reaction were observed. Compared with blank group， 45， 60 mg·L-1 apigenin treatments down-regulated the mRNA level of anti-apoptotic gene Bcl-2 （P<0.01） and all the apigenin treatments up-regulated those of the pro-apoptotic genes Bax and Caspase-3 （P<0.05， P<0.01）. Similarly， apigenin treatments down-regulated the protein level of Bcl-2 （P<0.05， P<0.01） and up-regulated those of Caspase-3 （P<0.05， P<0.01） and Bax （P<0.01， 45， 60 mg·L-1）. The blank group had higher protein level of Akt than the 60 mg·L-1 apigenin group （P<0.01）， higher protein levels of p-Akt， ERK1/2， and p-ERK1/2 than the 45， 60 mg·L-1 apigenin groups （P<0.01）， and higher protein levels of JNK and p-JNK than the apigenin groups （P<0.05， P<0.01）. Compared with blank group， 60 mg·L-1 apigenin up-regulated the protein level of p38 MAPK （P<0.05）， and all the apigenin groups up-regulated that of p-p38 MAPK （P<0.01）. Furthermore， apigenin lowered the p-Akt/Akt ratio （P<0.05， P<0.01） and p-ERK1/2/ERK1/2 ratio （P<0.01）， while it increased the p-JNK/JNK ratio （45， 60 mg·L-1； P<0.05， P<0.01） and p-p38 MAPK/p38 MAPK ratio （P<0.05， P<0.01）. ConclusionApigenin can inhibit the proliferation and promote the apoptosis of CL187 cells by inhibiting the PI3K/Akt signaling pathway and regulating the expression of proteins in the MAPK signaling pathway.
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Objective:To investigate the protective effect of quercetin （Qu） on articular cartilage of knee osteoarthritis and its mechanism by inhibiting p38 mitogen activated protein kinase （MAPK） signaling pathway. Method:Through the network pharmacology technology，we scientifically predicted and analyzed the target factors and signal pathways of Qu in the protection of articular cartilage in patients with osteoarthritis. We selected a prediction pathway closely related to osteoarthritis and validated it by cell experiment <italic>in vitro</italic>. The best intervention concentration of the drug was selected by cell counting kit-8 （CCK-8） method. The osteoarthritis and its closely related inflammatory factors interleukin（IL）-1<italic>β</italic> and tumor necrosis factor（TNF）-<italic>α</italic> were detected by enzyme linked immunosorbent assay（ELISA）. The expression of related mRNA and protein in p38 signal pathway after Qu intervention were detected by quantitative real time polymerase chain reaction（Real-time PCR） and Western blot. Result:It was predicted that MAPK signal pathway was closely related to osteoarthritis by network pharmacology，and p38 MAPK pathway，which was most closely related to osteoarthritis，was selected for study. The results showed that 100 μmol·L<sup>-1</sup> Qu had the most obvious effect in decreasing the expression of related inflammatory factors，inhibited the expression of p38，phosphorylated（p）-p38，matrix metalloproteinase-13（MMP-13），A disintegrin-like and metalloproteinase with thrombospondin type-1 motifs-4（ADAMTS-4） in the pathway，and promoted the expression of CollagenⅡ. Conclusion:Qu could decrease the expression of cartilage inflammatory factors in the prevention and treatment of osteoarthritis，and the effect can be well developed by intervening and inhibiting p38 MAPK pathway related factor expression level. All the results show that Qu can decrease osteoarthritis inflammatory factors and protect articular cartilage in patients with osteoarthritis.
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Objective:To study the effects of licochalcone A （LCA） on the proliferation and apoptosis of rheumatoid arthritis fibroblast-like synoviocytes （MH7A） as well as the related inflammatory factors， also to reveal the relevance between mitogen activated protein kinase （MAPK） signaling pathway and LCA regulation of MH7A cell proliferation and apoptosis. Method:MH7A cells were cultured and divided into blank group， LCA groups （10，20，40 μmol·L-1）. The proliferation of MH7A cells was detected by methylthiazolyldiphenyl-tetrazolium bromide（MTT）and immunofluorescence staining. The cell cycle of MH7A cells was determined by flow cytometry after PI staining and apoptosis was detected by flow cytometry after Annexin V/PI staining. The effect of LCA on interleukin-1β（IL-1β） mRNA was detected by Real-time fluorescence quantitative polymerase chain reaction（Real-time PCR）. Western blot was used to detect the effect of LCA on the key proteins of MAPK signaling pathway， meanwhile， PD98059， a specific ERK inhibitor， was used to observe the expression levels of p-ERK and IL-1β. Result:Compared with blank group， LCA could inhibit the proliferation of MH7A cells in a dose-dependent manner， and the number of living cells decreased significantly（P<0.01）， while the number of early apoptotic cells increased significantly（P<0.01）. Compared with the tumor necrosis factor-α （TNF-α，10 μg·L-1）group， LCA could reverse the expression of IL-1β mRNA induced by TNF-α（P<0.01）. and compared with the blank group， LCA also promoted the phosphorylation of ERK， JNK and p38 in a dose-dependent manner（P<0.01）. After ERK inhibitor PD98059 inhibited ERK phosphorylation， the inhibitory effect of LCA 10， 20 μmol·L-1 on IL-1β disappeared. Conclusion:LCA can inhibit the proliferation and induce apoptosis of MH7A cells， which may be related to the phosphorylation of MAPK pathway related proteins， and then inhibit the expression of inflammatory factors.
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Objective: To investigate the protective effect of Perillae Folium with aqueous extract (PFAE) on some key factors of Adriamycin (ADR)-induced oxidative injury in human renal tubular epithelial cells(HK-2), including the survival rate, oxidative injury indexes and cell apoptosis,in order to define the underlying mechanism. Method: A model of ADR-induced HK-2 cells oxidative injury was established in vitro, then cell viability was detected by cell counting kit-8 (CCK-8) after intervention with positive reference N-acetylcysteine (NAC) or PFAE (5,15,45 g·L-1) at different concentrations. According to the morphological changes under microscopy, the optimum concentration of PFAE was screened out for the follow-up experiments. Then, the experiments were divided into six groups:blank group, ADR (0.05 g·L-1) group, PFAE (15 g·L-1) group, ADR+PFAE (0.05+15) g·L-1 group, NAC (0.81 g·L-1) group, and ADR+NAC (0.05+81) g·L-1 group. After that, malondialdehyde (MDA), superoxide dismutase (SOD), total antioxidant capacity(TAC) were measured in the cell homogenate after 24 h administration. The level of reactive oxygen species (ROS) was detected by 2',7'-dichloroflurescin diacetate (DCFH-DA) fluorescence probe. Flow cytometry and TdT-mediated dUTP Nick-End Labeling (TUNEL) were used to monitor the cell apoptosis. Western blot was used to observed the expressions of mitochondrial apoptosis-associated proteins, like B lymphocyte tumor-2 gene (Bcl-2), Bcl-2 related X protein (Bax), cysteine aspartate protease-9 (Caspase-9), cysteine aspartate protease-3 (Caspase-3) and poly ADP-ribose polymerase (PARP), as well as their shear bodies. In addition, the phosphorylation protein expressions of p38 mitogen-activated protein kinase (p38 MAPK), extracellular signal-regulated kinase (ERK), c-Jun amino-terminal kinase (JNK) in mitogen-activated protein kinase (MAPK) signaling transduction pathway were detected by Western blot. Result: Compared with blank group, ADR group showed a decreased cell viability (PPPPPPPP-1. The ATC and SOD levels were increased in ADR+PFAE group and ADR+NAC group (PPConclusion: PFAE could alleviate the oxidative injury of HK-2 cells induced by ADR, and have an antioxidant effect, which inhibited cell apoptosis through mitochondrial apoptotic pathway and ERK/p38 MAPK signaling pathway.
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Objective: To explore the effect of Da Chengqitang on the lung index,lung index inhibition rate,lung histological morphology,classification changes of inflammatory cells and mitogen-activated protein kinase (MAPK) signal pathway in mice with allergic asthma.Method: Forty female C57BL/6 mice were randomly divided into normal control group,model group,dexamethasone group (0.005 g·kg-1) and Da Chengqitang group (19 g·kg-1).Murine allergic asthma model was established by sensitization and nebulization of ovalbumin (OVA).In brief,asthmatic mice were first sensitized by OVA and Al (OH)3 mixture ip on day 0 and day 14,and then nebulized by OVA from day 21 to 27.At the same time,each mouse in the dexamethasone and Da Chengqitang groups were intragastrically administered with 0.2 mL corresponding medicine one hour before the nebulization challenge,while the normal control group was given with the same amount of normal saline.On day 28,pulmonary morphology was detected by htoxylin eosin (HE) staining and inflammatory cells from the brachial alveolar lavage fluid were counted by Diff staining.The expression levels of key proteins in MAPK signaling pathway were detected by Western blot.Result: As compared with the normal control group,the lung indexes were significantly increased in model group (PP0.01),with a predominant percentage of eosinophils,moreover,the expression levels of phosphorylated p38 MAPK and extracellular signal-regulated kinase 1/2(ERK1/2) were increased obviously in asthmatic mice.After treatment by Da Chengqitang,lung indexes and pulmonary inflammation were significantly decreased,with an inhibitory rate of 68.4% for lung indexes,and inflammatory pathology of lung tissues was obviously improved and inflammatory cell exudation was alleviated,with the obviously lower levels of phosphorylated p38 MAPK and ERK1/2 protein.Conclusion: Da Chengqitang based on "Pulmonary Intestinal Treatment" can effectively improve lung inflammation in mice with allergic asthma,which may be related to the expression of phosphorylated p38 MAPK and ERK1/2 protein.