Membrane type-1 matrix metalloproteinase (MT1-MMP or MMP14) plays the pivotal role in tumor development and metastasis, so it is a promising drug target in malignancy. To acquire MT1-MMP specific binding peptides, we first analyzed MMPs sequences to find the divergent and specific sequence of MT1-MMP by bioinformatics approach, then set the specific sequence as the sense peptide target and designed antisense peptide library. Finally, by means of molecular docking, molecular dynamics simulation and in vitro cell assays, we screened the antisense peptide library against MT1-MMP and further studied the obtained specific peptides. Here, we identified the divergent and specific sequence of AYIREGHE (Named MT1-loop) located in MT1-MMP loop by multiple sequence alignment and established the antisense peptides library with capacity of 1 536 sequences. After two rounds of virtual screening, we obtained five antisense peptides with Rerankscores in the top for further screening. They all interacted with MT1-MMP, and docked well at the active site composed of MT1-loop sequence. Analysis of the affinities of these five antisense peptides to other MMPs (MMP1-3, MMP7-13, MMP14 HPX, MMP16) revealed that the peptide FVTFPYIR was more specific to MT1-MMP. Molecular dynamics simulation showed that the peptide FVTFPYIR might affect the stability of MT1-MMP and thus have effects on its activities. Meanwhile, the peptide FVTFPYIR could specifically inhibit the growth of MG63 and MDA-MB-231 tumor cells both of which expressed MT1-MMP. The work provides a new insight and way for the development of antitumor lead peptides targeting MT1-MMP.
Amino Acid Sequence ; Humans ; Matrix Metalloproteinase 14 ; chemistry ; Molecular Dynamics Simulation ; Neoplasms ; Peptide Library ; Peptides ; chemistry

Objective:In this study,a series of experiments were conducted to research the mechanism of anticancer and preliminary molecular effects of PAMs on the HEPG-2 cancer cells.Methods:Morphological observation and MTT assay were used to explore the inhibition and killing effect of PAMs acting on HEPG-2.AO/EB staining and Annexin V-FITC/PI staining were employed to observe the apoptosis of HEPG-2 treated with PAMs.The expression level of Foxm1,bcl-2 and others genes in HEPG-2 cells were detected by using qRT-PCR and western blot.Wound healing and transwell experiments determined if PAMs can inhibit the migration of HEPG-2.Results:PAMs can inhibit and kill HEPG-2 cells in time and dose-dependent manners,and the cytotoxic effects were closely related to the cell apoptosis.The mRNA expression of foxm1,bcl-2 and surviving gene were remarkably decreased in HEPG-2 cells after the treatment of PAMs.PAMs decreased the FoXM1 protein expression in HEPG-2 cells,while up-regulating thep53 protein expression.,and it could also inhibit the migration of cancer cells.Conclusions:The possible molecular mechanism for the killing of HEPG-2 cancer cells by PAMs was proposed.By down-regulating the expression of foxm1 and up-regulating the expression of p53,the transcriptional expression of their downstream target genes survivin and bcl-2 was inhibited or reduced,hence enhancing the cancer cell apoptosis.This study provides an important foundation for the development of anti-cancer Chinese folk medicine based on PAMs.

Objective To investigate the impact and mechanism of Liraglutide on autophagy and apoptosis of human podocyte induced by high glucose.Methods Human podocytes were cultured in vitro,and grouped into normal control group(NC group),high glucose group(HG group),25 nmol/L Liraglutide group(HG+Lir 25 group),50 nmol/L Liraglutide group(HG+Lir 50 group),Liraglutide+LY294002 group(HG+Lir+LY294002 group),and Liraglutide+3-MA group(HG+Lir+3-MA group).The podocyte activity was detected by CCK-8.The apoptosis rate and morphology of podocytes were detected by flow cytometry and Hoechst 33342 staining.The expression of autophagic body and autophagic marker LC3 protein in podocyteswas observed by transmission electron microscopy and immunofluorescence staining.Western blot was applied to detect the expression of apoptosis,autophagy and phosphoinositol 3-kinase(PI3K)/protein kinase B(Akt)/mammalian target of rapamycin(mTOR)pathway related proteins in podocytes.Results Compared with NC group,the activity of podocytes and the expression of Bcl-2,Bcl-2/Bax,LC3 Ⅱ/LC3 Ⅰ,p-PI3K/PI3K,p-Akt/Akt proteins in HG group were decreased(P＜0.05),while the apoptosis rate and the expression of Bax,p62,p-mTOR/mTOR proteins were increased in HG group(P＜0.05).There were many podocytes with pyknotic nuclei,the number of autophagic bodies and the number of green fluorescent spots of LC3 protein were decreased in HG group.Compared with HG group,the activity of podocyte increased,and the expression of Bcl-2,Bcl-2/Bax,LC3 Ⅱ/LC3 Ⅰ,p-PI3K/PI3K,p-Akt/Akt protein increased(P＜0.05),while the apoptosis rate and the expression of Bax,p62,p-mTOR/mTOR protein decreased(P＜0.05)in HG+Lir 25 group and HG+Lir 50 group.The number of podocytes with karyopyknosis was reduced,the number of autophagosomes and the number of green fluorescent spots of LC3 protein were increased in HG+Lir 25 group and HG+Lir 50 group,and the above changes indexes were more obvious in the HG+Lir 50 group group.Compared with HG+Lir 50 group,PI3K/Akt/mTOR pathway could be regulated,and reduce the improvement of Liraglutide on podocyte viability,apoptosis and autophagy induced by high glucose in HG+Lir+LY294002 group and HG+Lir+3-MA group.Conclusion Liraglutide may promote the autophagy of human podocyte induced by high glucose and inhibit its apoptosis through PI3K/Akt/mTOR pathway.

In this study, the leukemia K562 cell line was used as a model to elucidate the anticancer effects and preliminary mechanisms of PAMs. MTT assay showed that PAMs could cause cytotoxicities in K562 cells in dose- and time-dependent manners. AO-EB, Annexin-FITC/PI staining showed that the killing effects of PAMs in K562 cells were related to apoptosis, which was further confirmed by the following molecular and enzymatic assay. The mRNA levels of pro-apoptotic genes caspase-3, caspase-9 and bax were remarkably increased while the anti-apoptotic gene bcl-2 was significantly decreased determined by fluorescent quantitative PCR. Western blotting disclosed that PAMs could up-regulate caspase-3 and down-regulate anti-apoptotic survivin protein expression. The latter was also consistent with the results that PAMs could increase the enzymatic activities of both caspase-3 and caspase-9. All these results suggested that PAMs could effectively inhibit the proliferation of K562 cells and the mechanisms may be closely related to apoptosis induction. The work provides evidence basis for PAMs to be potentially developed as anti-cancer leukemia Chinese medicine.

Objective To investigate the relationship between mean platelet volume/lymphocyte ratio(MPVLR)and left atrial thrombosis in elderly patients with non-valvular atrial fibrillation(NVAF).Methods A total of 178 elderly patients with NVAF admitted to the hospital from January 2019 to December 2022 were enrolled in the study.The patients were divided into thrombosis group(28 cases)and non-throm-bosis group(150 cases)according to the left atrial thrombosis judged by using esophageal echocardiography(TEE).The white blood cell count(WBC),red blood cell count(RBC),lymphocyte count,lymphocyte pro-portion,platelet count(PLT)and mean platelet volume(MPV)were detected by automatic blood cell analy-zer,and MPVLR was calculated.The liver and kidney function indicators and blood lipid indicators were detec-ted by automatic biochemical analyzer.Receiver operating characteristic(ROC)curve was used to evaluate the predictive value of MPV,lymphocyte ratio and MPVLR for left atrial thrombosis in NVAF patients.Multiva-riate Logistic regression was used to analyze the influencing factors of left atrial thrombosis in elderly NVAF patients.Results MPV,lymphocyte proportion and MPVLR in the thrombosis group were higher than those in the non-thrombosis group,and the differences were statistically significant(P＜0.05).ROC curve analysis showed that the area under the curve(AUC)of MPV,lymphocyte ratio and MPVLR for predicting left atrial thrombosis in NVAF patients were 0.821(95％CI:0.764-0.882),0.771(95％CI:0.714-0.842)and 0.901(95％CI:0.861-0.949).respectively.The course of disease in the thrombosis group was longer than that in the non-thrombosis group,the proportion of patients with chronic heart failure,the proportion of patients with stroke,CH A2DS2-VASc score,LAEF,LAD,LVEDV,MPVLR,serum uric acid,MPV,lymphocyte proportion and MPVLR were higher than those in the non-thrombosis group,and LVEF was lower than that in the non-thrombosis group,the differences were statistically significant(P＜0.05).Multivariate Logistic regression a-nalysis showed that disease duration ≥1.93 years(OR=3.050,95％CI:1.928-4.824),chronic heart failure(OR=3.333,95％CI:1.808-6.144),MPVLR≥3.10(OR=3.873,95％CI:1.734-8.650)were independ-ent risk factors for left atrial thrombosis in elderly NVAF patients(P＜0.05).Conclusion The increase of MPVLR is associated with left atrial thrombosis in elderly patients with NVAF,and it can be used as a an in-dicator to predict left atrial thrombosis in patients with NVAF.

Objective To investigate the imaging features of dysplastic nodules with a focus of hepatocellular carcinoma ( DN-HCC ) on contrast-enhanced ultrasound ( CEUS ) and to improve the diagnostic accuracy . Methods The clinical data of 60 patients and CEUS imaging of 62 hepatic nodules [DN-HCCs , n =54 ;dysplastic nodules (DN) , n =8] pathologically proved were reviewed retrospectively . According to Contrast Enhanced Ultrasound Liver Imaging Reporting and Data System (CEUS LI-RADS) , the lesions were categorized . Results Significantly different CEUS patterns between DN-HCCs and DNs were observed ( P < 0 .05) . During the arterial phase ,54 DN-HCC lesions showed various enhancement patterns [ hypervascular ,59 .3％ ( 32/54 ) ;nodule-in-nodule ,9 .3％ ( 5/54 ) ;isovascular ,13 .0％ ( 7/54 ) and hypovascular ,18 .5％ (10/54)] . Of the 54 DN-HCC lesions ,44 .4％ (24/54) showed washout during the late phase .Of the 8 DN lesions ,62 .5％ (5/8) showed iso-enhancement during the arterial phase ,25％ (2/8) showed hypo-enhancement ,and 12 .5％ (1/8) showed hyper-enhancement . No DN lesion showed washout during the late phase .According to CEUS LI-RADS (LR) algorithm ,27 .8％ (15/54) DN-HCCs were LR-5 ,46 .3％ (25/54) DN-HCCs were LR-4 ,25 .9％ (14/54) DN-HCCs and 100％ (8/8) DNs were LR-3 . Regarding hyper-enhancement ( including local hyper-enhancement ) during the arterial phase or hypo-enhancement (including local hypo-enhancement) during the late phase as the diagnostic standard of DN-HCC , the diagnostic sensitivity , specificity and accuracy value were 83 .3％ , 87 .5％ and 83 .9％ , respectively . Conclusions The imaging features of hyper-enhancement during the arterial phase or hypo-enhancement during the late phase on CEUS are useful to diagnose DN-HCCs .

Nonalcoholic fatty liver disease (NAFLD) interacts with the intestinal immune system due to enterohepatic circulation and immune cell recruitment and recirculation. Intestinal immune imbalance promotes liver inflammation and fibrosis in the process of NAFLD, and meanwhile, NAFLD can cause disorders in the number and function of immune cells in the liver and intestinal tract. This article mainly elaborates on the impact of NAFLD on intestinal immune cells and briefly summarizes the new treatment methods for NAFLD targeting at intestinal immune cells, in order to provide a new understanding of the pathogenesis and treatment of NAFLD.

Objective:To investigate the regulatory effect of WNT1-inducible signaling pathway protein 2(WISP2)on macrophage polarization in palmitic acid(PA)and lipopolysaccharide(LPS)-induced inflammation.Methods:The macrophage cell line RAW264.7 was treated with different concentrations of WISP2 protein, and cell viability was determined by means of luminescence assay using Cell-Titer Glo to determine the concentration of WISP2.The cells were divided into control group, palmitic acid group, palmitic acid combined with different concentrations of WISP2 group(10 μg/L and 100 μg/L)and lipopolysaccharide group, lipopolysaccharide combined with different concentrations of WISP2 group(10 μg/L and 100 μg/L). mRNA expression of M1 and M2 macrophages phenotype of each group were detected by real-time quantitative polymerase chain reaction.The protein expression of important inflammatory factors, TNF-α and IL-6, were evaluated by ELISA.Results:Compared with the control group, both 10 μg/L and 100 μg/L WISP2 groups had no effect on the activity of RAW264.7 cells, but significantly up-regulated the expression of various inflammatory factors, including Tnfα(1.877±0.039, 2.202±0.034, F=309.7, P<0.001), Il6(1.418±0.056, 1.506±0.059, F=81.39, P<0.001), Mcp1(1.620±0.014, 1.982±0.125, F=71.45, P<0.001), Ccl3(1.892±0.118, 1.942±0.132, F=32.93, P<0.001), and iNos(1.691±0.201, 1.548±0.090, F=13.60, P<0.05). mRNA in macrophages, and significantly down-regulated the expression of anti-inflammatory factors, including Tgfβ(1.376±0.025, 2.152±0.107, F=1.846, P<0.05), CD206(2.123±0.031, 3.139±1.663, F=8.037, P<0.05), Il4(2.098±0.464, 2.494±0.141, F=48.68, P<0.01), and Il10(1.303±0.216, 1.574±0.274, F=5.774, P<0.05)mRNA, causing M1 type macrophage polarization.Compared with the control group, 100 μmol/L palmitic acid could mildly but significantly increase the expression of inflammatory factors such as TNF-α and IL-6 at the transcriptional and protein levels.Compared with palmitic acid stimulation alone, the combination of palmitic acid and WISP2 further promoted the protein expression of macrophage inflammatory factors TNF-α[(589.4±17.0)ng/L, (692.6±83.4)ng/L, F=56.38, P<0.05], IL-6[(15.13±1.14)ng/L, (13.33±1.22)ng/L, F=23.32, P<0.001]and the mRNA expression of chemokines Mcp1(160±9.796, 140±18.91, F=141.1, P<0.0001)and C cl3(17.76±1.92, 14.41±1.27, F=125.2, P<0.0001). Compared with the control group, 100 μg/L lipopolysaccharide strongly stimulated the expression of inflammatory factors such as TNF-α[(3444±423)ng/L, F=71.20, P<0.0001]and IL-6[(497.0±41.2)ng/L, F=63.50, P<0.0001]in macrophages at the protein level.Compared with lipopolysaccharide stimulation alone, the combination of lipopolysaccharide and WISP2 further significantly up-regulated the mRNA expression of chemokines Mcp1(106.8±8.7, 118.7±4.6, F=251.5, P<0.0001)and Ccl3(35.3±12.5, 116.4±4.5, F=160.1, P<0.0001). Conclusions:The adipokine WISP2 can promote M1 macrophage polarization in palmitic acid and lipopolysaccharide-induced inflammation, and it had distinct regulation in macrophage polarization under different inflammatory response conditions.

Objective:To investigate the effects of various concentrations of recombinant human WISP2 protein(WISP2)on lipid metabolism in HepG2 cells.Methods:HepG2 cells were treated with different concentrations(0, 0.4, 1 and 2 μg/L)of recombinant human WISP2 for 48 hours.Cell viability was detected by Cell-Titer, and enzymatic hydrolysis methods were used to measure intracellular triacylglycerol(TG)and total cholesterol(TC)levels.The mRNA expression was detected by quantitative real-time reverse transcription-PCR(RT-qPCR)and protein expression in HepG2 cells was detected by western blot.Results:Compared with the control group, the WISP2 groups treated with various concentration did not significantly reduce the viability of HepG2 cells.TG and TC in HepG2 cells were significantly increased by recombinant human WISP2 treatment(all P<0.05).The concentrations of TG in the 0.4, 1 and 2 μg/L recombinant human WISP2-treated groups were 1.254±0.039, 1.216±0.028 and 1.174±0.014)times the concentration in the untreated group, respectively( F=6.791, P=0.006).The concentration of TC in the untreated group was 1.264±0.057, 1.394±0.101 and 1.392±0.077), respectively, times the concentration in each of the treated groups( F=7.045, P=0.005).Further experiments found that the mRNA expression of sterol regulatory element binding protein 1(SREBP1), 3-hydroxy-3-methylglutaryl coenzyme A reductase(HMGCR), acetyl-CoA carboxylase(ACC), type 2 diacylglycerol acyltransferase(DGAT2)and the protein expression of SREBP1, ACC and fatty acid synthase(FAS)were significantly increased in the recombinant human WISP2-treated groups, compared with the control group(all P<0.05).However, the expression of lipid transporters such as the low-density lipoprotein receptor(LDLR), ApoB and ApoE and adipose triglyceride lipase(ATGL), a key lipolysis protein, was not significantly affected. Conclusions:Human recombinant WISP2 protein increases lipid levels in hepatocytes and the key underlying mechanisms may be through promoting lipid synthesis.

ObjectiveTo explore the effects and possible mechanism of Wenshen Tongdu Formula （温肾通督方， WTF） on spinal cord injury. MethodsThirty-six C57BL/6 female mice were randomly divided into sham operation group， model group and WTF group， with 12 mice in each group. The spinal cord injury model was established in the model group and the WTF group using the modified Allen's method， while in the sham operation group the spinal cord was only exposed. Since the 1st day after surgery， 50 g/（kg·d） of WTF solution was given to the WTF group by gavage， while 20 ml/（kg·d） of normal saline was given to the sham operation and model group by gavage， all for 14 days. Before surgery and on the 1st， 7th， and 14th days after surgery， the motor function of the mice was evaluated using the inclined plane test and hind limb motor function score （by BMS）. On the 3rd day after surgery， the nerve electrophy-siology was detected through electromyography and motor evoked potential； the spleen length was measured， and B cells in the spleen were sorted by magnetic beads； the differential expression of proteins were detected through proteomics technology； and the protein expression of mitochondrial outer membrane transport porin 20 （Tom20） and downstream cleaved caspase-3 in spleen B cells were measured using Western blotting. On the 14th day after surgery， MRI was used to observe the recovery of the spinal cord. ResultsCompared to those in the sham operation group at the same time， the BMS scores and subscores and the inclined plane test angle in the model group were reduced on the 1st， 7th and 14th days after surgery； the peak value of electromyogram and motor evoked potential were reduced， and the spleen length was shortened， while the expression of Tom20 and cleaved caspase-3 increased in splenic B cells increased （P＜0.05）. Compared to those in the model group at the same time， the BMS subscores on the 14th day and the angle of the inclined plane test on the 7th and 14th days after surgery increased in the WTF group； the peak value of electromyography and motor evoked potential， as well as the length of spleen increased， and the expression of Tom20 and cleaved caspase-3 decreased （P＜0.05）. The proteomics results showed that there were 100 differential proteins in the WTF group versus the model group， of which 37 were up-regulated and 63 were down-regulated. GO enrichment analysis showed that differential proteins mainly played their roles in oxygen binding， exogenous apoptosis negative feedback， zinc ion response， and oxygen transport. KEGG enrichment analysis showed that differential proteins were mainly concentrated in metabolic pathways， Huntington's disease， oxidative phosphorylation and other pathways. Subcellular localization showed that differential proteins were associated with mitochondria. Magnetic resonance imaging on the 14th day after surgery showed that the spinal cord structure of the mice in the sham operation group was intact， and the segments were clear， with normal spinal cord signal； the low signal area in the spinal cord injury area increased in the model group， and the spinal cord became significantly thinner； the injured segment had obvious depression in the WTF group， but the structure was more complete than that in the model group. ConclusionWTF may promote spinal cord injury repair by regulating immune function， and its mechanism may be related to inhibiting pyroptosis of spleen B cells.