ObjectiveThis paper aims to investigate the mechanism of Jinyang Dingtong plaster in improving the peripheral pain sensitization and synovial fibrosis in rats with knee osteoarthritis （KOA） by blocking the ion channels of transient receptor potentials （TRPs）. MethodsThe active components in the transdermal absorption solution of Jinyang Dingtong plaster were identified by using ultra-high performance liquid chromatography-electrospray ionization-quadrupole ion trap tandem mass spectrometry （UPLC-MS/MS） technology. A KOA rat model was established through intra-articular injection of monoiodoacetic acid. The rats were randomly divided into blank control group， KOA group， compound Nanxing Zhitong plaster Group， and Jinyang Dingtong plaster group， with eight rats per group. Among them， the rats in the compound Nanxing Zhitong plaster group and the Jinyang Dingtong plaster group were intervened with external application treatment. After the intervention period， the cold and mechanical stimulus pain thresholds of rats in each group were detected， and the transverse diameter of the knee joint was measured. The levels of inflammatory factors in the serum such as interleukin-1β （IL-1β）， tumor necrosis factor-α （TNF-α）， nerve growth factor （NGF）， and calcitonin gene-related peptide （CGRP） were determined by enzyme-linked immunosorbent assay （ELISA）. Protein expression levels of transient receptor potential ankyrin 1 （TRPA1）， transient receptor potential melastatin 8 （TRPM8）， transient receptor potential vanilloid 1 （TRPV1）， transient receptor potential vanilloid 4 （TRPV4）， transforming growth factor-β （TGF-β）， and vascular endothelial growth factor （VEGF） in synovial tissue were detected by Western blot. Histopathological changes in synovial tissue were observed by using hematoxylin and eosin （HE）， Masson， and Sirius red staining， while the expression of type Ⅰ collagen and alpha-smooth muscle actin （α-SMA） was detected by multiplex immunofluorescence. ResultsA total of 35 active components in the transdermal absorption solution of Jinyang Dingtong plaster were identified by UPLC-MS/MS， including phenolic acids， flavonoids， quinones， alkaloids， terpenes， lignans， and coumarins. Among them， the constituents such as berberine， paeoniflorin， ferulic acid， and caffeic acid exhibit clear anti-inflammatory， analgesic， and anti-fibrotic pharmacological effects. Compared to the blank control group， rats in the KOA group showed a significant decrease in cold and mechanical stimuli pain thresholds （P<0.01）. After 14 and 28 days of Jinyang Dingtong plaster intervention， the pain threshold in this group was significantly increased compared to that in KOA group （P<0.01）， showing no significant difference from that in compound Nanxing Analgesic plaster group. Additionally， Jinyang Dingtong plaster reduced the levels of IL-1β， TNF-α， NGF， and CGRP in the serum of KOA rats （P<0.01）， lowered the expression of TRPA1， TRPM8， TRPV1， TRPV4， TGF-β， and VEGF proteins in synovial tissue （P<0.01）， improved synovial pathological damage in KOA rats， and significantly decreased fluorescence intensity of type Ⅰ collagen and α-SMA （P<0.01）. ConclusionJinyang Dingtong plaster can improve the peripheral pain sensitization and synovial fibrosis in KOA rats by downregulating the expression of ion channels of TRPs and related inflammatory and fibrotic factors.

ObjectiveThis paper aims to investigate the mechanism of Jinyang Dingtong plaster in improving the peripheral pain sensitization and synovial fibrosis in rats with knee osteoarthritis （KOA） by blocking the ion channels of transient receptor potentials （TRPs）. MethodsThe active components in the transdermal absorption solution of Jinyang Dingtong plaster were identified by using ultra-high performance liquid chromatography-electrospray ionization-quadrupole ion trap tandem mass spectrometry （UPLC-MS/MS） technology. A KOA rat model was established through intra-articular injection of monoiodoacetic acid. The rats were randomly divided into blank control group， KOA group， compound Nanxing Zhitong plaster Group， and Jinyang Dingtong plaster group， with eight rats per group. Among them， the rats in the compound Nanxing Zhitong plaster group and the Jinyang Dingtong plaster group were intervened with external application treatment. After the intervention period， the cold and mechanical stimulus pain thresholds of rats in each group were detected， and the transverse diameter of the knee joint was measured. The levels of inflammatory factors in the serum such as interleukin-1β （IL-1β）， tumor necrosis factor-α （TNF-α）， nerve growth factor （NGF）， and calcitonin gene-related peptide （CGRP） were determined by enzyme-linked immunosorbent assay （ELISA）. Protein expression levels of transient receptor potential ankyrin 1 （TRPA1）， transient receptor potential melastatin 8 （TRPM8）， transient receptor potential vanilloid 1 （TRPV1）， transient receptor potential vanilloid 4 （TRPV4）， transforming growth factor-β （TGF-β）， and vascular endothelial growth factor （VEGF） in synovial tissue were detected by Western blot. Histopathological changes in synovial tissue were observed by using hematoxylin and eosin （HE）， Masson， and Sirius red staining， while the expression of type Ⅰ collagen and alpha-smooth muscle actin （α-SMA） was detected by multiplex immunofluorescence. ResultsA total of 35 active components in the transdermal absorption solution of Jinyang Dingtong plaster were identified by UPLC-MS/MS， including phenolic acids， flavonoids， quinones， alkaloids， terpenes， lignans， and coumarins. Among them， the constituents such as berberine， paeoniflorin， ferulic acid， and caffeic acid exhibit clear anti-inflammatory， analgesic， and anti-fibrotic pharmacological effects. Compared to the blank control group， rats in the KOA group showed a significant decrease in cold and mechanical stimuli pain thresholds （P<0.01）. After 14 and 28 days of Jinyang Dingtong plaster intervention， the pain threshold in this group was significantly increased compared to that in KOA group （P<0.01）， showing no significant difference from that in compound Nanxing Analgesic plaster group. Additionally， Jinyang Dingtong plaster reduced the levels of IL-1β， TNF-α， NGF， and CGRP in the serum of KOA rats （P<0.01）， lowered the expression of TRPA1， TRPM8， TRPV1， TRPV4， TGF-β， and VEGF proteins in synovial tissue （P<0.01）， improved synovial pathological damage in KOA rats， and significantly decreased fluorescence intensity of type Ⅰ collagen and α-SMA （P<0.01）. ConclusionJinyang Dingtong plaster can improve the peripheral pain sensitization and synovial fibrosis in KOA rats by downregulating the expression of ion channels of TRPs and related inflammatory and fibrotic factors.

AIM:To investigate the mechanism of flavonoid extract from Dracocephalum rupestre hance(DRHF)in the treatment of gouty arthritis through network pharmacology,molecular docking and animal experiment.METHODS:Literature re-trieval was used to explore the main active chemi-cal components and targets of DRHF.Gouty arthri-tis disease targets were obtained using Gene Cards and OMIM databases,and drug-disease intersect-ing targets were obtained using Wayne online tools.protein-protein interactions(PPI)and other related network diagrams were constructed using Cytoscape software.GO and KEGG enrichment analyses were performed on the shared intersect-ing targets using Metascape database.A rat model of gouty arthritis was established by Coderre meth-od;the swelling degree of ankle joint,gait behav-iour scores of rats were observed,and hematoxylin-eosin(HE)staining was performed.ELISA and real-time PCR were used to detect the key targets pre-dicted by the network pharmacology,and the ef-fects of DRHF on the molecular mechanism and key targets of gouty arthritis were observed.RESULTS:A total of 7 active compounds and 129 candidate targets for the treatment of GA were obtained,in-cluding IL-6,IL-1β,RELA,TNF,PPARG,etc.and the KEGG enrichment results suggested that DRHF may be involved in PI3K-Akt,TNF,IL-17 and other signal transduction pathways.Animal results:HE staining showed that the thickening of synovial tissue was not obvious in each administered group,and syno-vial cell proliferation and inflammatory cell infiltra-tion were significantly improved;compared with the normal group,the serum levels of TNF,IL-6,and IL-1β in the model group were significantly higher(P＜0.05),and the mRNA of PPARG,IL-6,and RELA in the synovial tissues were significantly high-er;compared with the model group,the levels of TNF,IL-6,and IL-1β were significantly lower(P＜0.05)in the low group of DRHF(0.45 g/kg)and high group of DRHF(0.9 g/kg),TNF,IL-6,IL-1β lev-els were significantly reduced(P＜0.05);PPARG,IL-6,RELA mRNA in synovial tissue were significantly reduced.CONCLUSION:DRHF inhibits IL-17/PI-3K/TNF signaling pathway by down-regulating the ex-pression of IL-6,PPARG and RELA mRNA,decreas-ing the levels of IL-6,IL-1β and TNF,and then treat-ing gouty arthritis.

AIM:To explore the mechanism of BLJZF in the treatment of abnormal lipid metabo-lism based on network pharmacology,molecular docking andin vivo animal experiments.METHODS:TCMSP database,Swiss Target Prediction database,STITCH database and literature search were used to collect and query the chemical composition infor-mation of BLJZF and the corresponding target of drug chemical composition.Disease targets of lipid abnormalities were collected through GeneCards and OMIM databases.Metascape database was used to analyze the gene ontology function and the Kyoto Encyclopedia gene and genome pathway en-richment of common intersection targets.Cyto-scape software was used to construct the correla-tion network diagram of components and targets,so as to select major components and targets for molecular docking study.The hyperlipidemia model was induced by high fat diet,and the control group,model group,positive group and BLJZF group were set up.The serum lipid index contents of triglyceride(TG),total cholesterol(TC),low lipo-protein cholesterol(LDL-C)and high lipoprotein cholesterol(HDL-C)were detected after continuous administration for 4 weeks.The contents of oxida-tive stress index were detected:alanine amino-transferase(ALT)and aspartate aminotransferase(AST).The contents of superoxide dismutase(SOD)and malondialdehyde(MDA)were detected by ELI-SA.Hematoxylin-eosin(HE)staining was used to de-tect the pathological changes of liver tissue.RE-SULTS:A total of 25 components and 315 corre-sponding targets of BLJZF were obtained,1729 tar-gets of lipid abnormalities and 116 common tar-gets of BLJZF,among which the core targets were AKT1,TNF,IL1β,CASP3,etc.GO and KEGG enrich-ment analysis suggested that BLJZF may play a role through the lipid and atherosclerotic pathway,PI3K-Akt,AGE-RAGE in diabetic complications and other signaling pathways.Molecular docking showed that most of the core targets had high binding activity with the active ingredients.Animal experiments showed that compared with model group,TC,TG,LDL-C,ALT,AST and MDA in BLJZF group were sig-nificantly decreased,HDL-C and SOD were signifi-cantly increased,and the degree of liver fat defor-mation was reduced.CONCLUSION:BLJZF has a therapeutic effect on lipid abnormalities.It can treat lipid metabolism abnormalities through multi-component,multi-target and multi-pathway,and provide reference for subsequent drug research on BLJZF.

AIM:To investigate the mechanism of flavonoid extract from Dracocephalum rupestre hance(DRHF)in the treatment of gouty arthritis through network pharmacology,molecular docking and animal experiment.METHODS:Literature re-trieval was used to explore the main active chemi-cal components and targets of DRHF.Gouty arthri-tis disease targets were obtained using Gene Cards and OMIM databases,and drug-disease intersect-ing targets were obtained using Wayne online tools.protein-protein interactions(PPI)and other related network diagrams were constructed using Cytoscape software.GO and KEGG enrichment analyses were performed on the shared intersect-ing targets using Metascape database.A rat model of gouty arthritis was established by Coderre meth-od;the swelling degree of ankle joint,gait behav-iour scores of rats were observed,and hematoxylin-eosin(HE)staining was performed.ELISA and real-time PCR were used to detect the key targets pre-dicted by the network pharmacology,and the ef-fects of DRHF on the molecular mechanism and key targets of gouty arthritis were observed.RESULTS:A total of 7 active compounds and 129 candidate targets for the treatment of GA were obtained,in-cluding IL-6,IL-1β,RELA,TNF,PPARG,etc.and the KEGG enrichment results suggested that DRHF may be involved in PI3K-Akt,TNF,IL-17 and other signal transduction pathways.Animal results:HE staining showed that the thickening of synovial tissue was not obvious in each administered group,and syno-vial cell proliferation and inflammatory cell infiltra-tion were significantly improved;compared with the normal group,the serum levels of TNF,IL-6,and IL-1β in the model group were significantly higher(P＜0.05),and the mRNA of PPARG,IL-6,and RELA in the synovial tissues were significantly high-er;compared with the model group,the levels of TNF,IL-6,and IL-1β were significantly lower(P＜0.05)in the low group of DRHF(0.45 g/kg)and high group of DRHF(0.9 g/kg),TNF,IL-6,IL-1β lev-els were significantly reduced(P＜0.05);PPARG,IL-6,RELA mRNA in synovial tissue were significantly reduced.CONCLUSION:DRHF inhibits IL-17/PI-3K/TNF signaling pathway by down-regulating the ex-pression of IL-6,PPARG and RELA mRNA,decreas-ing the levels of IL-6,IL-1β and TNF,and then treat-ing gouty arthritis.

AIM:To explore the mechanism of BLJZF in the treatment of abnormal lipid metabo-lism based on network pharmacology,molecular docking andin vivo animal experiments.METHODS:TCMSP database,Swiss Target Prediction database,STITCH database and literature search were used to collect and query the chemical composition infor-mation of BLJZF and the corresponding target of drug chemical composition.Disease targets of lipid abnormalities were collected through GeneCards and OMIM databases.Metascape database was used to analyze the gene ontology function and the Kyoto Encyclopedia gene and genome pathway en-richment of common intersection targets.Cyto-scape software was used to construct the correla-tion network diagram of components and targets,so as to select major components and targets for molecular docking study.The hyperlipidemia model was induced by high fat diet,and the control group,model group,positive group and BLJZF group were set up.The serum lipid index contents of triglyceride(TG),total cholesterol(TC),low lipo-protein cholesterol(LDL-C)and high lipoprotein cholesterol(HDL-C)were detected after continuous administration for 4 weeks.The contents of oxida-tive stress index were detected:alanine amino-transferase(ALT)and aspartate aminotransferase(AST).The contents of superoxide dismutase(SOD)and malondialdehyde(MDA)were detected by ELI-SA.Hematoxylin-eosin(HE)staining was used to de-tect the pathological changes of liver tissue.RE-SULTS:A total of 25 components and 315 corre-sponding targets of BLJZF were obtained,1729 tar-gets of lipid abnormalities and 116 common tar-gets of BLJZF,among which the core targets were AKT1,TNF,IL1β,CASP3,etc.GO and KEGG enrich-ment analysis suggested that BLJZF may play a role through the lipid and atherosclerotic pathway,PI3K-Akt,AGE-RAGE in diabetic complications and other signaling pathways.Molecular docking showed that most of the core targets had high binding activity with the active ingredients.Animal experiments showed that compared with model group,TC,TG,LDL-C,ALT,AST and MDA in BLJZF group were sig-nificantly decreased,HDL-C and SOD were signifi-cantly increased,and the degree of liver fat defor-mation was reduced.CONCLUSION:BLJZF has a therapeutic effect on lipid abnormalities.It can treat lipid metabolism abnormalities through multi-component,multi-target and multi-pathway,and provide reference for subsequent drug research on BLJZF.

Macrophages are important cells of the immune system. Tumor-associated macrophages are enriched macrophages near tumor cells or tissues. Their role is mainly to promote the construction of tumor inflammatory microenvironment and inhibit tumor immune response. Cell co-culture system is a symbiotic culture system formed by mimicking the internal environment of the body in vitro. The co-culture condition is relatively consistent with the environment in vivo, enabling better information exchange and material exchange between cells, which is a supplement to the monolayer cell culture and animal experiments. Tumor-associated macrophages and tumor cells co-exist in the tumor microenvironment. Thus, constructing a co-culture system for tumor-associated macrophages and tumor cells would be conducive to studying the antitumor effect of tumor-associated macrophages and developing new immunotherapy drugs. The co-culture system would provide a new direction for treating malignant tumors. This article mainly reviewed the co-culture patterns of macrophages and the antitumor effects of different phenotypes of macrophages, and highlighted the importance of using immunotherapy to treat malignant tumors in the tumor microenvironment.

With the appearance of the disadvantages of traditional tumor treatment, immunotherapy has entered people′s horizons as modern emerging treatment strategies, among which plant polysaccharides have received much more attention due to their antitumor activity and significant immunomodulatory effects. Tumor-associated macrophages (TAMs), as a component of tumor microenvironment, are important factors affecting tumors, and the regulation of TAMs by plant polysaccharides is one of the effective immunotherapy to treat tumor. In this review, we mainly described the regulation of TAMs by plant polysaccharides and the underlying mechanisms, and then gave an outlook on the research interests and the development of plant polysaccharides as immune adjuvants, aiming to provide reference for the study of plant polysaccharides in the immunotherapy for tumors.

Objective To investigate the proliferative inhibition effect and mechanism of total saponins from marsdenia tenacissima on human liver cancer HepG2 cells. Methods Human liver cancer HpeG2 cells cultured conventionally were divided into the marsdenia tenacissima saponins A control group, the experimental group and the blank control group. The experimental group was treated with different mass concentrations of total saponins from marsdenia tenacissima (0.14, 0.29, 0.58, 1.15, 2.13 mg/ml), while the marsdenia tenacissima saponins A control group was treated with marsdenia tenacissima saponins A (1.0 mg/ml), and the blank control group was established stimultaneously. The methyl thiazolyl tetrazolium (MTT) method was used to detect the effect of total saponins from marsdenia tenacissima on the activity of HepG2 cells, and the cell morphology was observed by using inverted microscopy. The cell apoptosis rate was detected by using flow cytometry from Annexin V-FITC/PI staining. The expressions of bcl-2, bax and p53 were analyzed by using Western blot after the drug effect. Results The results of cell activity test showed that total saponins from marsdenia tenacissima could inhibit the proliferation of HepG2 cells at the concentration of 0.29, 0.58, 1.15, 2.13 mg/ml, which was positively correlated with the concentration. The half maximal inhibitory concentration (IC 50) of cell growth inhibition of total saponins from marsdenia tenacissima on HepG2 cells was 0.75 mg/ml. Under inverted microscopy, the adherent cells were significantly reduced, the cells fell off into clusters and the debris was increased after the effect of total saponins from marsdenia tenacissima. Moreover, flow cytometry showed that total saponins from marsdenia tenacissima could increase the late apoptosis rate of HepG2 cells (P< 0.01). Western blot showed that the expression of bcl-2 was 0.62±0.16, 0.31±0.15, 0.84±0.09 and 1.00±0.11 respectively in the experimental group (total saponins from marsdenia tenacissima 0.58 mg/ml and 2.13 mg/ml), the marsdenia tenacissima saponins A control group and the blank control group;the expression of bax protein was 0.75±0.10, 0.83±0.12, 1.00±0.14 and 0.15±0.02, respectively in the above four groups; the expression of p53 protein was 0.63±0.08, 0.78±0.11, 1.00±0.13 and 0.18±0.02 respectively in the above four groups. The protein expression of bcl-2 was decreased and the protein expressions of bax and p53 were increased in the experiment group, and there was a statistical difference between the experiment group and the blank control group (P< 0.05). Conclusions Total saponins from marsdenia tenacissima can upregulate the expression of bax by upregulating the expression of p53 gene, and inhibit the expression of bcl-2, which would cause the cascade reaction to induce the apoptosis of HepG2 cells. Its inhibitory effect can be realized through mitochondrial pathmay to induce apoptosis.

Objective To establish the quality standard for Sanyuan Rupixiao Gel Paste. Methods Sparganii Rhizoma, Gleditsiae Sinensis Fructus, Cyperi Rhizoma and Impatientis Semen were identified by TLC method. The content of tetrahydropalmatine was determined by HPLC. Waters symmetry column was used with the mobile phase of acetonitrile-0.1% phosphatic acid in a gradient manner (pH was adjusted to 6.4 by triethylamine) (55:45) at the detection wavelength of 280 nm. The flow rate was 1.0 mL/min at the column temperature of 30 ℃. Results The spots in TLC were clear without any interference;tetrahydropalmatine showed a good linear relation in the range of 0.092–1.84 μg;the average recovery was 100.15%with RSD of 1.58%(n=6). Conclusion The method is simple and accurate with high reproducibility, which can be used for the quality control of Sanyuan Rupixiao Gel Paste.