Inhibitory Effect of Sesquiterpenoid M36 from Myrrha on Growth of Human Hepatoma Cells / 中国实验方剂学杂志

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.

Antitumor Effect of Fagopyri Dibotryis Rhizoma Extract on Colorectal Cancer Cells Through ROS/MAPK Signaling Pathway / 中国实验方剂学杂志

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.

Comparison on Ameliorating Effect of Arisaematis Rhizoma and Arisaematis Rhizoma Preparatum on Allergic Asthma in Rats / 中国实验方剂学杂志

ObjectiveTo investigate the therapeutic effects and difference in the effects of Arisaematis Rhizoma （AR） before and after processing （i.e.， Arisaematis Rhizoma Preparatum， ARP） with Zingiberis Rhizoma Recens-Alumen on allergic asthma in rats and to provide a basis for the theory of processing improving the efficacy. MethodA rat model of allergic asthma was established in 70 SD rats by intraperitoneal injection of ovalbumin （OVA）-aluminum hydroxide. The rats were administrated with the aqueous extracts of AR （1.2， 0.3 g∙kg-1） and ARP （1.2， 0.3 g∙kg-1） aqueous extracts by gavage， and montelukast sodium （0.001 g∙kg-1） was used as the positive drug. The T helper cell type 1/type 2 （Th1/Th2） ratio in the serum and bronchoalveolar lavage fluid （BALF） and percentages of inflammatory cells in BALF were determined. Polymerase chain reaction （PCR） was employed to determine the mRNA level of mucin 5AC （MUC5AC） in the lung tissue. The pathological changes in the lung tissue were observed by hematoxylin-eosin （HE） staining and PAS staining. Immunohistochemical assay was employed to measure the expression of c-Jun amino-terminal kinase （JNK）， extracellular signal regulated protein kinase （ERK）， and p38 mitogen-activated protein kinase （p38 MAPK） in rat lung tissue. Western blot was employed to determine the protein levels of ERK， p-ERK， JNK， p-JNK， p38， p-p38 in the lung tissue. The effects of AR and ARP were compared based on overall desirability. ResultCompared with the blank group， the levels of interleukin-12 （IL-12） and γ interferon （IFN-γ） in serum and BALF of rats in the model group were significantly lower （P<0.05， P<0.01）， and the levels of interleukin-4 （IL-4）， interleukin-5 （IL-5） and interleukin-13 （IL-13） were significantly higher （P<0.05， P<0.01）. Compared with the model group， the serum and BALF contents of IL-12 and IFN-γ in rats in the montelukast sodium group， high-dose AR group and high-dose ARP group were significantly higher （P<0.05， P<0.01）， and the contents of IL-4， IL-5 and IL-13 were significantly lower （P<0.05， P<0.01）， and the serum contents of IFN-γ in rats in the low-dose AR group and low-dose ARP group were in BALF was significantly higher （P<0.05） and IL-4 and IL-13 were significantly lower （P<0.05， P<0.01）， the percentages of macrophages， lymphocytes， neutrophils， and eosinophils were reduced in BALF， and the expression of JNK/ERK/p38 MAPK signaling pathway and MUC5AC protein was inhibited in lung tissues. Overall assessment of the normalized analysis revealed that the ARP group was slightly more potent than the AR group after administration of the same dose. ConclusionAR and ARP can effectively treat allergic asthma by inhibiting JNK/ERK/p38 MAPK signaling pathway， and the effect is better after concoction， which can provide data support for its "concoction efficiency".

Shenling Baizhusan Alleviates Intestinal Inflammation in Rat Model of Crohn's Disease via p38 MAPK Pathway / 中国实验方剂学杂志

ObjectiveTo observe the effect of Shenling Baizhusan on the intestinal inflammatory reaction in the rat model of Crohn's disease （CD） and study its relationship with p38 mitogen-activated protein kinase （MAPK） signaling pathway， so as to provide an experimental and theoretical basis for the clinical application of this prescription. MethodA total of 72 SD rats （36 males and 36 females） were randomized into a normal group （n=12） and a modeling group （n=60）. The rats in the modeling group were treated with 2，4，6-trinitrobenzene sulfonic acid （TNBS， 3 mL·kg-1） and then randomized into model， mesalazine （0.21 g·kg-1·d-1）， and low-， medium-， and high-dose （5.88， 11.76， 23.59 g·kg-1·d-1， respectively） Shenling Baizhusan groups. The rats in the drug intervention groups were administrated with corresponding agents by gavage for 14 days， and those in the normal and model groups with an equal volume of distilled water. The disease activity index （DAI） score of inflammatory bowel disease （IBD） and the colon mucosal damage index （CMDI） score of rats in each group were assessed after gavage. Hematoxylin-eosin （HE） staining was used to observe the histopathological changes in the colon， and enzyme-linked immunosorbent assay （ELISA） to measure the levels of tumor necrosis factor alpha （TNF-α）， interleukin-1 （IL-1）， and interleukin-6 （IL-6） in the serum. Western blotting was employed to determine the protein levels of p38 MAPK， phosphorylated p38 MAPK （p-p38 MAPK）， p65 nuclear factor （NF）-κB， and phosphorylated-p65 NF-κB （p-NF-κB p65） in the colon tissue. Quantitative real-time polymerase chain reaction was conducted to determine the miRNA levels of p38 MAPK and NF-κB p65 in the colon tissue. ResultThe model group had higher DAI and CMDI scores than the normal group （P<0.01） and showed damaged epithelial cells in the colon mucosa， disarrangement of glands， damaged simple tubular glands， local necrosis， infiltration of a large number of inflammatory cells and lymphocytes in each layer， and presence of ulceration. Compared with the normal group， the model group showed elevated levels of TNF-α， IL-1， and IL-6 in the serum （P<0.01） and up-regulated protein levels of p-p38 MAPK and p-NF-κB p65 and miRNA level of p38 MAPK in the colon tissue （P<0.01）. Compared with the model group， mesalazine and high- and medium-dose Shenling Baizhusan decreased the DAI and CMDI scores （P<0.05， P<0.01）， repaired the mucosal epithelium of the colon tissue， increased the glands and goblet cells， lowered the levels of TNF-α， IL-1， and IL-6 in the serum （P<0.05， P<0.01）， and down-regulated the protein levels of p-p38 MAPK and p-NF-κB p65 and the miRNA level of p38 MAP in the colon mucosa （P<0.01， P<0.05）. ConclusionShenling Baizhusan can reduce intestinal inflammation of CD rats and promote the repair of colon mucosa by down-regulating the protein levels of p-p38 MAPK and pNF-κB p65 and the miRNA level of p38 MAPK to inhibit the p38 MAPK pathway.

Mechanism of Xuanfu Daizhetang in Alleviating Allergic Asthma by Inhibiting Secretion of Inflammatory Factors / 中国实验方剂学杂志

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.

Lycium barbarum Polysaccharide Regulates Activation of RAW264.7 Macrophages Through MGL/TLR Pathway / 中国实验方剂学杂志

ObjectiveTo investigate the mechanism of Lycium barbarum polysaccharides （LBP） in promoting the activation of RAW264.7 macrophages. MethodRAW264.7 macrophages were stimulated with LBP at different concentrations （50， 100， 200 mg·L-1）， and those stimulated with lipopolysaccharide （LPS） at 100 μg·L-1 and galactose （Gal） at 100 mg·L-1 as positive controls. After 24 h of LBP stimulation， the cell counting kit-8 （CCK-8） was used to detect the survival rate of RAW264.7 macrophages treated with LBP （0， 50， 100， 200， 400， 800 mg·L-1）. The levels of interleukin-6 （IL-6） and interleukin-12 （IL-12） in cell culture supernatant were detected by enzyme-linked immunosorbent assay （ELISA）. The protein and mRNA expression of p38 mitogen-activated protein kinase （MAPK）， extracellular signal-regulated kinase （ERK）， c-Jun N-terminal kinase （JNK）， and nuclear factor κB （NF-κB） in Toll-like receptor 4 （TLR4）/Toll-like receptor 2 （TLR2）/macrophage galactose-type lectin （MGL） pathway of RAW264.7 macrophages was detected by Real-time fluorescence-based quantitative polymerase chain reaction （Real-time PCR） and Western blot. ResultCCK-8 results showed that compared with the results in the blank group， the survival rate of RAW264.7 macrophages decreased in the 400， 800 mg·L-1 LBP groups （P<0.05）. ELISA results showed that compared with the blank group， 50 mg·L-1 LBP could promote the secretion of IL-12 in RAW264.7 macrophages （P<0.01）. Compared with the blank group， 100 mg·L-1 LBP and 200 mg·L-1 LBP could promote the secretion of IL-6 in RAW264.7 macrophages （P<0.05， P<0.01）. Western blot results showed that compared with the blank group， the LBP groups （50， 100， 200 mg·L-1） enhanced protein expression levels of MAPK key molecules （p-p38 MAPK， p-ERK， p-NF-κB， and p-JNK） in TLR4， TLR2， and MGL pathways （P<0.05， P<0.01）. Compared with the model group， the 200 mg·L-1 LBP group could promote the expression level of p-NF-κB protein in RAW264.7 macrophages （P<0.01）. Real-time PCR results showed that compared with the blank group， the LBP groups （50， 100， and 200 mg·L-1） enhanced the mRNA expression levels of MAPK key molecules （p38 MAPK， ERK， NF-κB， and JNK） in TLR4 and TLR2 pathways （P<0.05， P<0.01）. Compared with the model group， the 50 and 200 mg·L-1 LBP groups could promote the mRNA expression levels of JNK and ERK2 in RAW264.7 macrophages （P<0.05， P<0.01）. ConclusionLBP can regulate the activation of RAW264.7 macrophages and participate in the immune response through the TLR2/TLR4/MGL pathway.

Anti-inflammatory Effect and Mechanism of Duhuo Jishengtang on Rheumatoid Arthritis Rats Based on TLR2/p38 MAPK/NF-κB Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo explore the anti-inflammatory effect of Duhuo Jishengtang （DHJST） on collagen-induced arthritis （CIA） model rats and its effect on the Toll-like receptor 2 （TLR2）/p38 mitogen-activated protein kinase （MAPK）/nuclear factor-κB （NF-κB） signaling pathway. MethodForty-eight male SD rats were randomly divided into the following six groups （n=8）： normal group， model group， methotrexate （MTX） group， low-dose DHJST （DHJST-L） group， medium-dose DHJST （DHJST-M） group， and high-dose DHJST （DHJST-H） group. The CIA model was established by injecting bovine type Ⅱ collagen into the rat tail root with the collagen antibody induction method. After model induction， rats were treated with drugs by gavage. The rats in the MTX group received MTX at 2.0 mg·kg-1， three times a week， and those in the DHJST groups received DHJST at 3.8， 7.6， 15.2 g·kg-1·d-1 for 28 days. The rats in the normal group and the model group were given the same dose of normal saline. The weight of the rats was recorded， and the paw swelling degree was observed. The arthritis index and immune organ index were measured， and the changes in the microcirculation indexes of the rats were detected with a microcirculation detector. Hematoxylin-eosin （HE） staining was used to detect the pathological morphologic changes in rat synovial tissues and the apoptosis rate of synovial cells was detected by flow cytometry to determine the therapeutic effect of DHJST on rheumatoid arthritis. Enzyme-linked immunosorbent assay （ELISA） was used to detect the changes in serum levels of tumor necrosis factor-α （TNF-α）， interleukin （IL）-1β， IL-17A， and interferon-γ （IFN-γ）. The protein expression of TLR2， NF-κB p65， phosphorylated NF-κB p65 （p-NF-κB p65）， p38 MAPK， and p-p38 MAPK was detected by Western blot. ResultCompared with the normal group， the model group showed reduced body weight （P<0.01）， increased paw swelling degree， arthritis index， and immune organ index （P<0.01）， increased comprehensive microvascular score and vascular resistance （P<0.01）， significant hyperplasia of synovial tissues and massive infiltration of inflammatory cells as revealed by pathological sections， and up-regulated expression levels of TNF-α， IL-1β， IL-17A， and IFN-γ in serum， and TLR2， p-NF-κB p65/NF-κB p65 and p-p38 MAPK/p38 MAPK in synovial tissues （P<0.01）. Compared with the model group， the DHJST groups showed increased body weight of rats （P<0.01）， decreased paw swelling degree， arthritis index， and immune organ index （P<0.05， P<0.01）， reduced comprehensive microvascular score and vascular resistance （P<0.05， P<0.01）， improved synovial histopathological injury， increased apoptosis rate of synovial cells （P<0.01）， and down-regulated levels of TNF-α， IL-1β， IL-17A， and IFN-γ in serum （P<0.05， P<0.01） and TLR2， p-NF-κB p65/NF-κB p65 and p-p38 MAPK/p38 MAPK in synovial tissues （P<0.05， P<0.01）. ConclusionDHJST may alleviate the inflammatory reaction in CIA rats by regulating the TLR2/p38 MAPK/NF-κB signaling pathway， thus exerting its anti-rheumatoid arthritis effect.

Expression of TUBB4B in mouse primary spermatocyte GC-2 cells and its regulatory effect on NF-κB and MAPK signaling pathway / 南方医科大学学报

OBJECTIVE@#To explore the interaction between Tubulin beta 4B class IVb (TUBB4B) and Agtpbp1/cytosolic carboxypeptidase- like1 (CCP1) in mouse primary spermatocytes (GC-2 cells) and the role of TUBB4B in regulating the development of GC-2 cells.@*METHODS@#Lentiviral vectors were used to infect GC-2 cells to construct TUBB4B knockdown and negative control (NC-KD) cells. The stable cell lines with TUBB4B overexpression (Tubb4b-OE) and the negative control (NC-OE) cells were screened using purinomycin. RT-qPCR and Western blotting were used to verify successful cell modeling and explore the relationship between TUBB4B and CCP1 expressions in GC-2 cells. The effects of TUBB4B silencing and overexpression on the proliferation and cell cycle of GC-2 cells were evaluated using CCK8 assay and flow cytometry. The signaling pathway proteins showing significant changes in response to TUBB4B silencing or overexpression were identified using Western blotting and immunofluorescence assay and then labeled for verification at the cellular level.@*RESULTS@#Both TUBB4B silencing and overexpression in GC-2 cells caused consistent changes in the mRNA and protein expressions of CCP1 (P < 0.05). Similarly, TUBB4B expression also showed consistent changes at the mRNA and protein after CCP1 knockdown and restoration (P < 0.05). TUBB4B knockdown and overexpression had no significant effect on proliferation rate or cell cycle of GC-2 cells, but caused significant changes in the key proteins of the nuclear factor kappa-B (NF-κB) signaling pathway (p65 and p-p65) and the mitogen-activated protein kinase (MAPK) signaling pathway (ErK1/2 and p-Erk1/2) (P < 0.05); CCP1 knockdown induced significant changes in PolyE expression in GC-2 cells (P < 0.05).@*CONCLUSIONS@#TUBB4B and CCP1 interact via a mutual positive regulation mechanism in GC-2 cells. CCP-1 can deglutamize TUBB4B, and the latter is involved in the regulation of NF-κB and MAPK signaling pathways in primary spermatocytes.

Human menstrual blood-derived stem cells alleviate autoimmune hepatitis via JNK/MAPK signaling pathway in vivo and in vitro / 医学前沿

Autoimmune hepatitis (AIH) is a severe globally distributed liver disease that could occur at any age. Human menstrual blood-derived stem cells (MenSCs) have shown therapeutic effect in acute lung injury and liver failure. However, their role in the curative effect of AIH remains unclear. Here, a classic AIH mouse model was constructed through intravenous injection with concanavalin A (Con A). MenSCs were intravenously injected while Con A injection in the treatment groups. The results showed that the mortality by Con A injection was significantly decreased by MenSCs treatment and liver function tests and histological analysis were also ameliorated. The results of phosphoproteomic analysis and RNA-seq revealed that MenSCs improved AIH, mainly by apoptosis and c-Jun N-terminal kinase/mitogen-activated protein signaling pathways. Apoptosis analysis demonstrated that the protein expression of cleaved caspase 3 was increased by Con A injection and reduced by MenSCs transplantation, consistent with the TUNEL staining results. An AML12 co-culture system and JNK inhibitor (SP600125) were used to verify the JNK/MAPK and apoptosis signaling pathways. These findings suggested that MenSCs could be a promising strategy for AIH.

Efficacy Evaluation of Ethyl Acetate Fraction of Ipomoea muricatum in Prevention and Treatment of Alcoholic Gastric Ulcer / 中国实验方剂学杂志

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.

Anti-inflammatory Effect of Huangqintang on LPS-induced RAW264.7 Inflammatory Cells / 中国实验方剂学杂志

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.

Relationship Between MAPK Pathway and Osteoporosis and Traditional Chinese Medicine Intervention： A Review / 中国实验方剂学杂志

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.

Effect of Shikonin on Synovitis in Mice with Collagen-induced Arthritis Through MAPK1/MAPK Pathway / 中国实验方剂学杂志

ObjectiveTo observe the effect of shikonin （SKN） on synovitis in DBA/1 mice with collagen-induced arthritis （CIA）. MethodThirty-six DBA/1 mice were randomly divided into a normal group， a CIA group， low-， medium-， and high-dose SKN groups （1， 2， and 4 mg·kg-1）， and a methotrexate （MTX， 0.5 mg·kg-1） group， with 6 mice in each group. Mice in the CIA group， the SKN groups， and the MTX group were immunized with an equal volume of bovine type Ⅱ collagen and complete Freund's adjuvant on day 1. On day 21， those mice received a second immunization with an equal volume of bovine type Ⅱ collagen and incomplete Freund's adjuvant to establish the CIA model. On the day of the second immunization， mice were treated with drugs by gavage. Mice in the MTX group received oral administration three times a week， while others received once per day， for 28 days. On day 22， the symptoms of arthritis， such as redness and swelling of joints， in CIA mice were observed， and arthritis scores were recorded. On day 49 after sample collation， histopathological examination of synovial inflammation in CIA mice was performed using hematoxylin-eosin （HE） staining. Immunofluorescence （IF） double labeling was used to detect the expression of vimentin and mitogen-activated protein kinase 1 （MAPK1） in the synovium of CIA mice. Network pharmacology predicted that the target of SKN in rheumatoid arthritis （RA） was MAPK1， which was verified by molecular docking. Western blot was used to detect the expression of extracellular signal-regulated kinase （ERK）， c-Jun N-terminal kinase （JNK）， p38， phosphorylated （p）-ERK， p-JNK， and p-p38 proteins in the synovial membrane of mice. ResultCompared with the normal group， the CIA group showed significantly higher arthritis scores， morbidity， and synovial inflammation， severely disrupted joint structure， evident articular cartilage and bone destruction， severe bone erosion （P<0.01）， increased expression of vimentin and MAPK1 in the synovium of mice， and increased protein expression of p-ERK/ERK， p-JNK/JNK， and p-p38/p38 in the synovium of mice （P<0.01）. Compared with the CIA group， the SKN groups and the MTX group showed relatively normal joint structure， with milder bone erosion and bone destruction， and smoother articular surfaces. Molecular docking results confirmed that the target of SKN was MAPK1. In the SKN groups and the MTX group， the expression of vimentin and MAPK1 in the synovial membrane was significantly reduced （P<0.01）， and the protein expression of p-ERK/ERK， p-JNK/JNK， and p-p38/p38 in the synovium of mice was significantly reduced （P<0.01）. ConclusionSKN can target MAPK1 to inhibit the protein expression of p-ERK， p-JNK， and p-p38 in CIA mice， thereby treating RA.

Active Ingredients of Chinese Medicine Against Esophageal Squamous Cell Carcinoma by Regulating MAPK Signaling Pathway： A Review / 中国实验方剂学杂志

Esophageal squamous cell carcinoma （ESCC）， the predominant histological strain of esophageal cancer in China， is complex in pathogenesis and may be associated with mutations in several genes and dysregulation of the mitogen-activated protein kinase （MAPK） pathway， the phosphatidylinositol 3-kinase （PI3K） pathway， etc. MAPK signaling pathway can be activated by growth factors， proto-oncogenes， and oxidative stress， thus participating in biological functions such as cell proliferation， differentiation， migration， and apoptosis. More evidence shows that the MAPK signaling pathway plays an important role in the occurrence and development of ESCC and is expected to become an effective target for the treatment of ESCC. The classical MAPK family consists of extracellular signal-regulated kinases 1/2 （ERK1/2）， c-Jun N-terminal kinases 1/2/3 （JNK1/2/3）， p38 α/β/γ/δ， and extracellular signal-regulated kinase 5 （ERK5）. Each pathway consists of three cascade sequentially phosphorylated and activated protein kinase systems. The activation of the ERK1/2 pathway is related to the proliferation， migration， drug resistance， and apoptosis inhibition of ESCC. JNK， p38， and ERK5 pathways seem to show bidirectional regulation， and there is signal integration between MAPK internal pathways. Chemotherapeutic drugs for esophageal cancer often have side effects and are prone to drug resistance， so it has become a new idea to find effective and low-toxic drug alternatives. Studies have found that flavonoids， terpenoids， alkaloids， saponins， phenols， and other active ingredients in Chinese medicine can play an anti-ESCC effect by targeting the MAPK pathway， which is mainly reflected in inhibiting proliferation， migration， and invasion， inducing cycle arrest， promoting apoptosis， reversing drug resistance， etc. Therefore， this paper reviewed the regulatory role of the MAPK signaling pathway in ESCC and the research progress in active ingredients of Chinese medicine in regulating MAPK pathway against ESCC to provide references for the mechanism research and new drug development of Chinese medicine in the prevention and treatment of esophageal cancer.

Effect and Mechanism of Baoshen Prescription in Alleviating Renal Fibrosis in UUO Mice by Regulating ERK/p38 MAPK Signaling Pathway / 中国实验方剂学杂志

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.

Da Chaihutang Inhibits Hepatocellular Carcinoma by Regulating p38 MAPK/IL-6/STAT3 Signaling Pathway / 中国实验方剂学杂志

ObjectiveThis study aims to investigate the efficacy and underlying mechanism of Da Chaihutang （DCHT） in treating hepatocellular carcinoma （HCC） in vitro and in vivo. MethodWe employed methyl thiazolyl tetrazolium （MTT） assay and crystal violet staining to observe the proliferation of Hepa1-6 liver cancer cells treated with DCHT at different doses （0， 125， 250， 500， 1 000 mg·L-1） for different time periods （1， 2， 4， 8 days）. The orthotopic liver cancer model was established by injection of 1×106 Hepa1-6 cells into mouse， and then the model mice were randomly assigned into six groups： blank， model， DCHT （0.21， 0.625， 1.875 g·kg-1， ig， qd）， and positive control （5-fluorouracil， 25 mg·kg-1， ip， qod）. After 14 days of administration， the mice were sacrificed， and the liver samples were collected and fixed in 4% paraformaldehyde for hematoxylin-eosin （HE） staining. The Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform （TCMSP）， Cytoscape 3.7.2， STRING， and DAVID were used for the searching of the key targets of DCHT in treating HCC， the construction of protein-protein interaction （PPI） network， and the Kyoto Encyclopedia of Genes and Genomes （KEGG） pathway enrichment. Quantitative real-time PCR was performed to determine the mRNA level of interleukin-6 （IL-6） in Hepa1-6 cells and liver tissue. Western blotting was employed to measure the protein levels of the proteins involved in the mitogen-activated protein kinase （MAPK） and signal transducers and activators of transcription 3 （STAT3） signaling pathways. ResultDCHT （500， 1 000 mg·L-1） treatment for 4 and 8 days inhibited the proliferation of Hepa1-6 cells in a dose- and time-dependent manner （P<0.05）. The in vivo assay showed that DCHT （high dose， 1.875 g·kg-1） treatment for 14 days led to high differentiation and unobvious heterogeneity of HCC cells and small necrotic area compared with the model group. Network pharmacology analysis predicted that the potential targets of DCHT in the treatment of HCC were mainly the inflammation cytokines such as IL-6， interleukin-1β （IL-1β）， and tumor necrosis factor-alpha （TNF-α） in HCC microenvironment. The potential signaling pathways involved in the treatment were mainly associated with HCC growth and differentiation， including MAPK and STAT3 signaling pathways. Compared with the blank group， DCHT （1 000 mg·L-1） treatment for 1， 2， 4， and 8 days down-regulated the mRNA level of IL-6 in Hepa1-6 cells （P<0.05）. Similar results were observed in the livers of mice treated with DCHT （0.625， 1.875 g·kg-1）. The in vitro assay demonstrated that DCHT （1 000 mg·L-1） treatment for 4 and 8 days and DCHT （500， 1 000 mg·L-1） treatment inhibited the phosphorylation of extracellular signal-regulated kinases 1/2 （ERK1/2）， c-Jun NH2-terminal kinase/stress-activated protein kinase （JNK）， p38 MAPK， and STAT3 in a dose- and time-dependent manner （P<0.05）. The in vivo assay showed that DCHT （0.625 and 1.875 g·kg-1） treatment only inhibited the phosphorylation of p38 MAPK and STAT3 （P<0.05）. ConclusionThe present study indicates that DCHT can inhibit liver cancer cell proliferation by regulating p38 MAPK/IL-6/STAT3 signaling pathway.

Baicalin Alleviates LPS-Induced Acute Lung Injury in Rats Through p38 MAPK/NLRP3 Pathway / 中国实验方剂学杂志

ObjectiveTo investigate the effects and mechanism of baicalin （BA） on lipopolysaccharide （LPS）-induced acute lung injury in rats. MethodEighty healthy male SD rats were randomly divided into the control group， model group， low-dose BA （BA-L） group， medium-dose BA （BA-M） group， high-dose BA （BA-H） group， dexamethasone （DEX） group， SB203580 group， and BA + SB203580 group， with 10 rats in each group. The rats in the BA-L， BA-M， and BA-H groups were injected intraperitoneally with different doses （10， 50， 100 mg·kg-1） of BA solution， the ones in the DEX group with 5 mg·kg-1 DEX solution， the ones in the SB203580 group with 0.5 mg·kg-1 SB203580 solution， the ones in the BA + SB203580 group with 100 mg·kg-1 BA solution and 0.5 mg·kg-1 SB203580， and those in both the control group and model group with the same volume of normal saline， once per day， for seven successive days. One hour after the last administration， rats in all groups except for the control group were given 5 mg·kg-1 LPS via intratracheal instillation for inducing the acute lung injury， whereas those in the control group received the same volume of normal saline solution. Twelve hours later， the lung tissues were sampled and stained with htoxylin-eosin （HE） for observing the pathological changes， followed by the counting of the total number of cells and neutrophils in bronchoalveolar lavage fluid （BALF）. The wet/dry weight ratio of the lung tissue and the contents of serum superoxide dismutase （SOD） and malondialdehyde （MDA） were measured. The activity of reactive oxygen species （ROS） in the lung tissue was detected by immunofluorescence and the levels of interleukin-1β （IL-1β）， interleukin-18 （IL-18）， interleukin-6 （IL-6）， and tumor necrosis factor-α （TNF-α） in BALF by enzyme-linked immunosorbent assay （ELISA）. Immunohistochemistry （IHC） was conducted to determine the relative expression of p-p38 mitogen-activated protein kinase （MAPK） and Western blotting was carried out to detect the protein expression levels of p-p38 MAPK， thioredoxin interacting protein （TXNIP）， NOD-like receptor protein 3 （NLRP3）， and cysteinyl aspartate specific protease-1 （Caspase-1） in the lung tissue. ResultCompared with the control group， the model group displayed inflammatory pathological changes in lung tissue， elevated wet/dry weight ratio， total number of cells and neutrophils in BALF， and ROS and MDA levels （P<0.01）， decreased SOD activity （P<0.01）， and up-regulated IL-1， IL-18， IL-6， TNF-α， p-p38 MAPK， NLRP3， and Caspase-1 expression （P<0.01）. Compared with the model group， BA at different doses， SB203580， and BA + SB203580 all effectively alleviated the pathological changes in lung tissue induced by LPS， reduce the lung wet/dry weight ratio， the total number of cells and neutrophils in BALF， and ROS and MDA levels （P<0.05，P<0.01）， enhanced the activity of SOD （P<0.05，P<0.01）， and down-regulated the expression of IL-1β， IL-18， IL-6，TNF-α， p-p38 MAPK， NLRP3， and Caspase-1 in lung tissue （P<0.05，P<0.01）. ConclusionBA has a protective effect against LPS-induced acute lung injury， which may be related to its inhibition of p38MAPK/NLRP3 signaling pathway and the improvement of inflammatory response.

Mechanism of Fuzi Lizhongwan Improving Injury in Cisplatin-induced CIPN Mice by Regulating MAPK Signaling Pathway / 中国实验方剂学杂志

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.

Dahuang Lingxian Prescription Regulates Effect of TAK1 and TRAF6 Interaction and Co-localization on Inflammatory Response of Bile Duct Cells / 中国实验方剂学杂志

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.

Effect of Fufang Shelong Capsules on Rats with Membranous Nephropathy Based on p38 MAPK Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo investigate the nephroprotective and anti-inflammatory effects of Fufang Shelong capsules （FFSL） in rats with membranous nephropathy （MN）， and the role of the p38 mitogen-activated protein kinase （MAPK） signaling pathway. MethodMale SD rats of SPF grade were divided into a normal group and an experimental group. The MN model was induced by tail vein injection of cationized bovine serum albumin in the experimental group. After screening， the eligible model rats were included and divided into a positive control group （tripterygium glycosides tablets） and low-， medium-， and high-dose FFSL groups （0.375， 0.75， 1.5g·kg-1）. The rats were treated correspondingly for eight weeks， and urine protein was detected during drug intervention. Renal function and inflammation-related indicators were detected after drug intervention. The changes in 24-hour urine total protein （24 h UP）， interleukin-6 （IL-6）， interleukin-8 （IL-8）， tumor necrosis factor-α （TNF-α）， creatinine （Cr）， blood urea nitrogen （BUN）， total protein （TP）， albumin （Alb）， and total cholesterol （TC） were detected. Flow cytometry was used to detect CD4+/CD8+ changes. Kidney tissues were collected to observe pathological changes under a light microscope and an electron microscope. The protein expression of p38 MAPK and phosphorylated p38 MAPK （p-p38 MAPK） in kidney tissues was detected by Western blot. ResultCompared with the normal group， the model group showed increased 24 h UP （P<0.01）， elevated serum Cr， BUN， TC， IL-6， IL-8， and TNF-α （P<0.05，P<0.01）， decreased serum Alb and TP levels （P<0.05，P<0.01）， increased CD4+/CD8+ in the peripheral blood （P<0.01）， and up-regulated protein expression of p38 MAPK and p-p38 MAPK in kidney tissues （P<0.05）. Additionally， in the model group， immune complex deposition and foot process fusion， accompanied by infiltration of inflammatory cells， were observed on the epithelial side of the basement membrane in the pathological kidney tissues. Compared with the model group， the groups with drug intervention showed declining 24 h UP levels at six weeks （P<0.05，P<0.01）， decreased serum Cr， BUN， TC， IL-6， IL-8， and TNF-α （P<0.05，P<0.01）， increased serum Alb and TP levels （P<0.05，P<0.01）， reduced CD4+/CD8+ in the peripheral blood （P<0.01）， improved renal pathological damage， and down-regulated p38 MAPK and p-p38 MAPK in kidney tissues （P<0.05，P<0.01）. ConclusionFFSL can decrease the expression of inflammatory factors， reduce proteinuria， delay kidney damage， and protect kidney function by inhibiting the expression of the p38 MAPK signaling pathway.