Objective:To investigate the preventive and therapeutic effects of iridoid glycosides from Boschniakia rossica(IGBR)on precancerous lesions of liver cancer in rats,and to clarify its possible mechanism.Methods:Thirty Wistar rats were selected and the precancerous liver lesion model was established using the modified Solt-Faber method.The rats were randomly divided into sham operation group,model group,and IGBR group,and there were 10 rats in each group.The morphology of liver tissue of the rats in various groups were observed;the liver weights,liver indexes and liver regeneration degrees of the rats in various groups were measured;HE staining was used to observe the pathomorphology of liver tissue of the rats in various groups;immunohistochemistry method was used to detect the expression of glutathione-S-transferase(GST)-Pi protein in liver tissue of the rats in various groups;colorimetric method was used to detect the activities of γ-glutamyl transpeptidase(γ-GT),catalase(CAT),superoxide dismutase(SOD),glutathione peroxidase(GSH-Px)and glutathione S-transferase(GST),and the level of malondialdehyde(MDA)in liver tissue and mitochondria of the rats in various groups;Western blotting method was used to detect the expression levels of α-smooth muscle actin(α-SMA),collagen type Ⅰ alpha 1 chain(ColⅠα1),matrix metalloproteinase(MMP)13,MMP2,tissue inhibitor of metalloproteinase(TIMP)1,TIMP2,transforming growth factor β1(TGF-β1),TGF-β1 receptor(TβR),mothers against decapentaplegic homolog(Smad)2/3,Smad4,and Smad7 proteins in liver tissue of the rats in various groups.Results:Compared with sham operation group,the liver weight,liver index and degree of liver regeneration of the rats in model group were increased(P<0.05);compared with model group,the liver weight and liver index of the rats in IGBR group showed a decreasing trend,but the difference was not statistically significant(P>0.05).The HE staining results showed that the liver lobule structure in sham operation group was intact and clear,with large hepatocytes,abundant eosinophilic cytoplasm,and hepatocytes arranged in single cords radiating from the central vein;there were irregular hepatic sinusoids between plates,with only a small amount of collagen fibers and inflammatory cell infiltration in the portal area,and no degeneration or necrosis of hepatocytes.In model group,the normal arrangement of hepatocytes disappeared,the liver lobule structure was disrupted,small cell hyperplasia(mainly oval cells)was observed in the portal area,with massive collagen deposition and significant fibrous tissue hyperplasia in the fibrous septum;hepatocytes showed extensive degenerative edema,hydropic degeneration or even ballooning degeneration and focal necrosis;basophilic hepatocytes formed proliferative areas with clear cytoplasm,centrally located nuclei and 1-2 prominent nucleoli;glassy hepatocytes with enlarged nuclei and pale transparent cytoplasm were also observed.In IGBR group,the liver lobule structure was basically preserved,with reduced inflammatory lesions,mild edema,and scattered spotty or focal necrosis;nuclear atypia and pathological mitotic figures or binucleation were observed.The immunohistochemistry results showed GST-Pi protein positive foci with brown-yellow cytoplasmic staining in round or oval nodules.The GST-Pi protein positive foci were observed in liver tissue of the rats in model group,indicating successful establishment of precancerous liver lesion model.The scattered GST-Pi protein positive foci were observed in IGBR group,which were significantly reduced compared with model group.Compared with sham operation group,the activity of γ-GT in liver tissue of the rats in model group was increased(P<0.05);compared with model group,the activity of γ-GT in liver tissue of the rats in IGBR group was decreased(P<0.05).Compared with sham operation group,the GST activity and MDA level in liver tissue and liver mitochondria of the rats in model group were increased(P<0.05),while the activities of SOD,CAT,and GSH-Px were decreased(P<0.05);compared with model group,the GST activity and MDA level in liver tissue and liver mitochondria of the rats in IGBR group were decreased(P<0.05),while the activities of SOD,CAT,and GSH-PX were increased(P<0.05).The Western blotting results showed that compared with sham operation group,the expression levels of α-SMA,ColⅠα1,TIMP1,and TIMP2 proteins in liver tissue of the rats in model group were increased(P<0.05),while the expression levels of MMP13 and MMP2 proteins were decreased(P<0.05),and the ratios of TIMP1/MMP13 and TIMP2/MMP2 were increased(P<0.05);compared with model group,the expression levels of α-SMA,ColⅠα1,and TIMP2 proteins in liver tissue of the rats in IGBR group were decreased(P<0.05),while the expression levels of MMP13 and MMP2 proteins were increased(P<0.05),and the ratios of TIMP1/MMP13 and TIMP2/MMP2 were decreased(P<0.05).Compared with sham operation group,the expression levels of TGF-β1 and Smad2/3 proteins in liver tissue of the rats in model group were increased(P<0.05);compared with model group,the expression levels of TGF-β1,Smad2/3 and Smad 4 proteins in liver tissue of the rats in IGBR group were decreased(P<0.05).Conclusion:IGBR can inhibit precancerous liver lesions and liver fibrosis in rats,and its mechanism may be related to enhancing the antioxidant capacity of liver tissue,inhibiting TGF-β/Smad signaling pathway and regulating TIMP/MMP balance.

ObjectiveTo investigate the effect of iridoid glycosides from Boschniakia rossica （IGBR） on epithelial-mesenchymal transition （EMT） of HepG2 hepatoma cells induced by transforming growth factor-beta 1 （TGF-β1）. MethodsHepG2 hepatoma cells were induced by 10 μg/L TGF-β1 to construct an EMT model of hepatoma cells. The cells were divided into control group （treated with serum-free DMEM）， model group （treated with 10 μg/L TGF-β1）， and IGBR group （treated with 10 μg/L TGF-β1 and 500 mg/L IGBR）， and all cells were cultured for 48 hours. Cell adhesion assay， wound healing assay， and Transwell chamber assay were used to observe the migration and invasion abilities of cells. RT-PCR and Western blot were used to measure the mRNA and protein expression levels of E-cadherin， N-cadherin， and vimentin in cells， and Western blot was used to measure the protein expression levels of Slug， Twist1， ZEB1， p-STAT3， and STAT3. A one-way analysis of variance was used for comparison of continuous data between multiple groups， and the least significant difference t-test was used for further comparison between two groups； the independent-samples t test was used for comparison between two groups. ResultsAfter TGF-β1 induction， HepG2 cells in the model group showed long spindle-shape changes， while those in the control group showed polygonal epithelia-like changes. Compared with the model group， the IGBR group had a significant reduction in cell adhesion rate and significant inhibition of cell migration and invasion abilities （all P<0.05）， as well as significant increases in the mRNA and protein expression levels of E-cadherin （P<0.05）， significant reductions in the mRNA and protein expression levels of N-cadherin and vimentin （all P<0.05）， and significant reductions in the protein expression levels of Slug， Twist1， ZEB1， and p-STAT3 （all P<0.05）. ConclusionIGBR can inhibit TGF-β1-induced EMT process in HepG2 cells， thereby attenuating cell adhesion， migration， and invasion abilities， and it can also upregulate E-cadherin， downregulate N-cadherin and vimentin， and upregulate the protein expression of Slug， Twist1， ZEB1， and STAT3， possibly by inhibiting the STAT3 pathway to downregulate the EMT transcription factors such as Slug， Twist1， and ZEB1.

Objective:To discuss the effect of Boschnikia rossica polysaccharides rapa polysaccharides(BRPS)on lipopolysaccharide(LPS)-induced inflammatory responses in the THP-1 macrophages,and to clarify its mechanism.Methods:The THP-1 monocytes were differentiated into the macrophages,and the inflammation model was established using LPS to induce the THP-1 macrophages.CCK-8 method was used to detect the survial rates of the THP-1 macrophages after treated with different concentrations(0,100,200,500,1 000,and 2 000 μg·L-1)of LPS and different concentrations(0,12.5,25.0,50.0,100.0,and 200.0 mg·L-1)of BRPS to select the concentrations for the subsequent experiments.The THP-1 macrophages were divided into blank group,model group,low dose of BRPS group(25.0 mg·L-1 BRPS),medium dose of BRPS group(50.0 mg·L-1 BRPS),and high dose of BRPS group(100.0 mg·L-1 BRPS).P38 inhibitor SB203580,ERK inhibitor U0126,c-Jun N-terminal kinase(JNK)inhibitor SP600125,and nuclear factor of kappa B(NF-κB)inhibitor BAY11-7082 were used to verify the effects on THP-1 cells.The THP-1 cells were divided into control group,LPS group,inhibitor group,100.0 mg·L-1 BRPS group,and inhibitor+100.0 mg·L-1 BRPS group.ELISA method was used to detect the levels of tumor necrosis factor α(TNF-α),interleukin(IL)-6,and IL-1β in culture fluid of the THP-1 macrophages in various groups;DCFH-DA fluorescence probe method was used to detect the reactive oxygen species(ROS)levels in the THP-1 macrophages in various groups;Hoechst33342/PI fluorescence staining method was used to detect the membrane damage in the THP-1 macrophages in various groups;JC-1 fluorescence staining was used to observe mitochondrial membrane potential in the THP-1 macrophages in various groups;Western blotting method was used to detect the expression levels of cyclooxygenase-2(COX-2),high mobility group protein B1(HMGB1),NOD-like receptor thermal protein domain assciated protein 3(NLRP3),cysteinyl aspartate specific protease(Caspase)-1,gasdermin D(GSDMD)-N,IL-1β,mitogen-activated protein kinase(MAPK),and nuclear factor-kappa B(NF-κB)related proteins in the THP-1 macrophages in various groups.Results:The CCK-8 method results showed that when the LPS concentration was 100-2 000 μg·L-1,the survival rates of the THP-1 macrophages were over 90%.Compared with 0 μg·L-1 LPS group,the IL-6 levels in culture fluid of the THP-1 macrophages in 100,200,500,1 000,and 2 000 μg·L-1 LPS group were increased(P<0.05),indicating a significant enhancement of the inflammatory response in the macrophages,so 100 μg·L-1 LPS was used to construct the inflammation model.After treated with 12.5,25.0,50.0,100.0,and 200.0 mg·L-1 BRPS,the survival rates of the THP-1 macrophage were 91.2%,93.8%,91.4%,90.6%,and 91.8%,respectively,so 25.0,50.0,and 100.0 mg·L-1 BRPS were selected as the drug concentrations for low,medium,and high doses of BRPS groups in the subsequent experiments.The ELISA results showed that compared with blank group,the levels of IL-6,TNF-α,and IL-1β in culture fluid of the THP-1 macrophages in model group were increased(P<0.05);compared with model group,the levels of IL-6,TNF-α,and IL-1β in low,medium,and high doses of BRPS groups were decreased(P<0.05).The DCFH-DA fluorescence probe method results showed that compared with blank group,the ROS level in the THP-1 macrophages in model group was increased(P<0.05);compared with model group,the ROS levels in low,medium,and high doses of BRPS groups were decreased(P<0.05).The Hoechst33342/PI fluorescence staining results showed that compared with blank group,the degree of membrane damage in the THP-1 macrophages in model group was increased;compared with model group,the degrees of membrane damage in low,medium,and high doses of BRPS groups were decreased.The JC-1 fluorescence staining results showed that compared with blank group,the mitochondrial membrane potential in the THP-1 macrophages in model group was decreased significantly;compared with model group,the mitochondrial membrane potential in low,medium,and high doses of BRPS groups were increased gradually.The Western blotting results showed that compared with blank group,the expression levels of COX-2,HMGB1,NLRP3,Caspase 1,GSDMD-N,and IL-1β proteins and the ratios of p-P38/P38,p-ERK/ERK,p-JNK/JNK,and p-NF-κB/NF-κB in the THP-1 macrophages in model group were increased(P<0.05);compared with model group,the expression levels of HMGB1,NLRP3,Caspase-1,GSDMD-N,and IL-1β proteins and the ratios of p-P38/P38,p-ERK/ERK,p-JNK/JNK,and p-NF-κB/NF-κB in the THP-1 macrophages in medium and high doses of BRPS groups were decreased(P<0.05),the expression levels of NLRP3,Caspase-1,and IL-1β proteins in the cells in low dose of BRPS group were decreased(P<0.05),the expression level of COX-2 protein in the cells in high dose of BRPS group was decreased(P<0.05).Compared with control group,the ratios of p-P38/P38,p-ERK/ERK,p-JNK/JNK,and p-NF-κB/NF-κB,and the expression level of IL-1β protein in the THP-1 macrophages in LPS group were increased(P<0.05);compared with LPS group,the ratios of p-P38/P38,p-ERK/ERK,p-JNK/JNK,and p-NF-κB/NF-κB,and the expression level of IL-1β protein in the THP-1 macrophages in inhibitor group,100 mg·L-1 BRPS group,and inhibitor+100 mg·L-1 BRPS group were decreased(P<0.05);compared with inhibitor group,the ratios of p-P38/P38,p-ERK/ERK,p-JNK/JNK,and p-NF-κB/NF-κB in the THP-1 macrophages in inhibitor+100 mg·L-1 BRPS group were decreased(P<0.05).Conclusion:BRPS inhibits the inflammatory response of the THP-1 macrophages,which may be related to the MAPK and NF-κB signaling pathways regulated by BRPS.

Objective To investigate the inhibitory effect of gallic acid(GA)on lipopolysaccharide(LPS)-induced inflammatory responses in human THP1 macrophages.Methods THP1 monocytes were differentiated into macrophages with phorbol myristate acetate.A macrophage inflammation model was established with LPS induction,and GA was added at different concentrations.The safe concentrations of LPS and GA for THP1 cells were screened using the CCK-8 method,and a normal group,model group(100 μg/L LPS),and GA group(100 μg/L LPS+different concentrations of GA)were set up.ELISA was used to detect the levels of interleukin(IL)-6,tumor necrosis factor-α(TNF-α),and IL-1β in the cell culture media of each group.The microplate method was used to detect lactate dehydrogenase(LDH)activity and NO content in the cell cultures,and fluorescence staining was used to detect the reactive oxygen species(ROS)levels,cell damage,and changes in mitochondrial transmembrane potential in each group.Western blot was performed to detect the levels of cyclooxygenase-2(COX-2),HMGB1,c-Jun amino-terminal kinase(JNK),extracellular-regulated protein kinase(ERK),P38 mitogen-activated protein kinase(P38),nuclear transcription factor-KB(NF-κB),NOD-like receptor protein 3(NLRP3),Caspase-1,IL-1β,and gasdermin D(GSDMD).Results Compared with the normal group,the model cell group's secretion of IL-6,TNF-α,IL-1β,and NO was increased(P＜0.05);the secretion of COX-2,HMGB1,p-ERK,p-JNK,and p-P38 and expression of p-NF-KB,NLRP3,Caspase-1,IL-1β were elevated(P＜0.05);expression of GSDMD protein activation fragment was reduced(P＜0.05);ROS generation and cellular damage were significantly increased;mitochondrial transmembrane potential was significantly lower;and LDH activity was elevated(P＜0.05).In comparison with the model group,the GA group cells'secretion of IL-6,TNF-α,IL-1β,and NO was decreased(P＜0.05);expression of COX-2,HMGB1,p-ERK,p-JNK,p-P38,p-NF-κB,NLRP3,Caspase-1,and IL-1β was decreased(P＜0.05);GSDMD protein activation fragment expression level was increased(P＜0.05);ROS generation and cellular damage were decreased;mitochondrial transmembrane potential gradually increased;and LDH activity was decreased(P＜0.05).Conclusions GA had an inhibitory effect on the LPS-induced inflammatory response in THP1 macrophages,and its anti-inflammatory mechanism may involve the MAPK and NF-κB signaling pathways.

Objective: To study the hypoglycemic effects of soybean isoflavones (SI) and soyasaponins (SS) in diabetes, and their inhibitory activities on alpha-glucosidase (EC 3.2.1.20) and alpha-amylase (EC 3.2.1.1). Methods: GK/Jcl type 2 diabetic rats were given diet containing 20 g/kg of soybean hypocotyl extracts (SHE) for 20 weeks, then the blood glucose and oral glucose tolerance test were observed. SI and SS were isolated by ODS column chromatography and high-performance liquid chromatography (HPLC) from SHE. The inhibitory activities of each component of SI and SS against alpha-glucosidase and alpha-amylase were tested by colorimetric method. Results: SHE decreased blood glucose significantly in type 2 diabetic rats and improved glucose tolerance in both normal and diabetic rats. In alpha-glucosidase inhibitory assay, Group B, Group E and DDMP (2,3-dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-one) saponins showed potent inhibitory activities with IC50 values of 10-40 靘ol/L. Isoflavone aglycones also showed potent inhibitory activities against alpha-glucosidase with IC50 values of 20-150 mol/L, while isoflavone glycosides showed a little lower potencies. Inhibition of SI and SS on alpha-amylase was low, and their inhibitory activities were about 10%~20% at the concentration of 1g/L. Conclusion: SI and SS may decrease blood glucose and improve oral glucose tolerance in diabetic rats, probably via the inhibitory effects on alpha-glucosidase and alpha-amylase.