Objective:To prepare decellularized extracellular matrix (dECM)-gelatin methacryloyl (GelMA) composite hydrogels and to study their effects on hepatocyte proliferation.Methods:Hepatic dECM was prepared by elution, and GelMA hydrogel and 10%, 30% and 50% dECM-GelMA composite hydrogels were prepared by pepsin solubilization. The morphology of normal liver and dECM liver was observed by eyes and scanning electron microscopy using hematoxylin-eosin, Sirius red and periodate-Schiff staining, respectively. The internal structure of the dECM-GelMA composite hydrogels was observed by scanning electron microscopy, and the pore diameter was measured. Liver HL-7702 cells were co-cultured with GelMA hydrogel and 10%, 30% and 50% dECM-GelMA composite hydrogels, and the cell proliferation viability was determined by cell counting kit-8. The expression of proliferating cell nuclear antigen (PCNA), Wnt family protein 5a (Wnt5a), β-catenin, extracellular-regulated protein kinase 1/2 (ERK1/2) and phosphorylated ERK1/2 (p-ERK1/2) were detected by Western blotting. Comparisons were made using independent sample t-test or one-factor analysis of variance. Results:After decellularization, the hepatocyte morphology showed rounded depressions, and the extracellular matrix structure was intact. The GelMA hydrogel and 10%, 30% and 50% dECM-GelMA composite hydrogels showed inernally porous structures. The pore diameter increased from (3.06±1.35) μm in the GelMA hydrogel to (16.01±4.02) μm in the 50% dECM-GelMA composite hydrogel. On the 3rd, 5th and 7th day, the relative cell proliferation was higher in the 50% dECM-GelMA composite hydrogel group than that in the GelMA hydrogel group (1.89±0.04 vs 1.53±0.01, 9.36±0.04 vs 3.89±0.09, 7.15±0.27 vs 4.89±0.15, all P<0.05). The relative expression levels of PCNA, Wnt5a, β-catenin, and p-ERK1/2/ERK1/2 proteins in the 50% dECM-GelMA composite hydrogel group were higher than those in the GelMA hydrogel group (2.14±0.04 vs 1.00±0.03, 2.36±0.09 vs 1.00±0.08, 1.45±0.03 vs 1.00±0.04, 1.43±0.04 vs 1.00±0.01, all P<0.05). Conclusions:A dECM-GelMA composite hydrogel can be prepared, which may promote hepatocyte proliferation by upregulating the phosphorylation of ERK1/2 and activating Wnt/β-catenin signaling pathway.

To investigate the role of the DPP4-nestin axis in liver fibrosis induced by alveolar cyst infection,a murine model was established using C57BL/6 mice via hepatic portal vein injection.Liver histopathological changes were assessed using HE staining,while immunohistochemistry and immunofluorescence were employed to evaluate the expression levels of nestin and DPP4 in infected mouse livers.In vitro,J S1 cell line was stimulated with recombinant DPP4 protein to es-tablish a cellular model,and qPCR,Western blot,and shRNA lentivirus interference techniques were utilized to examine the involvement of the DPP4-nestin axis in hepatic stellate cell activation.The findings demonstrated that compared to the Sham group,liver tissue structure disruption and collagen deposition were evident along with significantly increased expressions of nestin and DPP4(P＜0.050 0),which colocalized with nesin and α-SMA.Furthermore,stimulation with recombi-nant DPP4 protein significantly enhanced JS1 cell activation(P＜0.050 0)as well as upregulated nestin expression(P＜0.050 0)when compared to control group cells.Notably,shRNA lentivirus-mediated inhibition of nestin expression effectively suppressed the activating effects exerted by re-combinant DPP4 protein on JS1 cells(P＜0.050 0).Collectively,these results highlight the crucial regulatory role played by the DPP4-nestin axis in hepatic stellate cell activation triggered by alveo-lar infection;thus,targeting this axis may represent a novel therapeutic strategy for treating alveo-lar infection-induced liver fibrosis.

To investigate the role of the DPP4-nestin axis in liver fibrosis induced by alveolar cyst infection,a murine model was established using C57BL/6 mice via hepatic portal vein injection.Liver histopathological changes were assessed using HE staining,while immunohistochemistry and immunofluorescence were employed to evaluate the expression levels of nestin and DPP4 in infected mouse livers.In vitro,J S1 cell line was stimulated with recombinant DPP4 protein to es-tablish a cellular model,and qPCR,Western blot,and shRNA lentivirus interference techniques were utilized to examine the involvement of the DPP4-nestin axis in hepatic stellate cell activation.The findings demonstrated that compared to the Sham group,liver tissue structure disruption and collagen deposition were evident along with significantly increased expressions of nestin and DPP4(P＜0.050 0),which colocalized with nesin and α-SMA.Furthermore,stimulation with recombi-nant DPP4 protein significantly enhanced JS1 cell activation(P＜0.050 0)as well as upregulated nestin expression(P＜0.050 0)when compared to control group cells.Notably,shRNA lentivirus-mediated inhibition of nestin expression effectively suppressed the activating effects exerted by re-combinant DPP4 protein on JS1 cells(P＜0.050 0).Collectively,these results highlight the crucial regulatory role played by the DPP4-nestin axis in hepatic stellate cell activation triggered by alveo-lar infection;thus,targeting this axis may represent a novel therapeutic strategy for treating alveo-lar infection-induced liver fibrosis.

Objective To elucidate whether increased palmitic acid(PA)content after obesity can inhibit KLF4 expression through up-regulation of DNMT1/3b,which in turn regulates the biological behavior of endometrial cancer(EC)cells.Methods General data were collected from non-EC control individuals(n=120)and EC patients(n=30)to analyze the differences in obesi-ty-related phenotypes between the two groups;Endometrial tissues from non-EC control individuals(n=20)and cancer tissues from EC patients(n=20)were collected,and the expression of KLF4 and DNMT1/3b was detected by immunohistochemistry in the endometrial tissues;invitro culture of EC cells was carried out;mRNA and protein expression levels of DNMTs/KLF4/MMP2 and other factors in the cells were detected by qRT-PCR and Western blotting.Transwell and scratch assay were used to detect the proliferation,migration and invasion ability of EC cells;An EC hormonal tumor model was constructed in obese mice,and the tumor-forming ability of EC cells or EC cells overexpressing KLF4 was observed in mice.Results The weight,body mass index,plasma LDL and free fatty acid content of EC patients were significantly higher than that of non-cancer subjects(P＜0.05);The percentage of overweight/obese individuals was higher in EC patients than that of non-cancer subjects(73.33％vs.50.00％,P＜0.05);The expression level of KLF4 in tumor tissues of EC patients was significantly lower,and the expres-sion level of DNMT1/3b was significantly higher.The expression level of KLF4 was significantly negatively correlated with the level of FFAs in serum.After PA treatment,the expression level of KLF4 in EC cells was significantly reduced,the expression levels of DNMT1/3b and MMP2 were significantly increased,and the cell migration and invasion ability was significantly en-hanced,with no significant effect on the cell proliferation ability.Up-regulation of KLF4 significantly reduced the expression lev-el of MMP2 and weakened the migration and invasion ability of EC cells;Down-regulation of KLF4 significantly increased the expression level of MMP2 and enhanced the migration and invasion ability of EC cells;PA treatment with concomitant up-regu-lation of KLF4 significantly reversed the promotional effect of PA treatment on the MMP2 expression level as well as migration and invasion ability of EC cells.The expression levels of KLF4 in EC cells were both significantly increased after down-regula-tion of DNMT1 and DNMT3b;PA treatment with simultaneous down-regulation of DNMT1 and DNMT3b,significantly re-versed these results.The tumor-forming ability of EC cells was significantly enhanced in high-fat diet-induced obese mice,whereas tumor-forming ability of EC cells was significantly reduced in high-fat diet-induced obese mice after overexpression of KLF4.Conclusion Increased plasma PA content after obesity promotes endometrial cancer cell migration,invasion,and in vivo tumor-forming ability by inhibiting KLF4 expression through down-regulation of DNMT1/DNMT3b.

Liver fibrosis is a significant health burden,marked by the consistent deposition of collagen.Unfortunately,the currently available treatment approaches for this condition are far from optimal.Lysyl oxidase-like protein 2(LOXL2)secreted by hepatic stellate cells(HSCs)is a crucial player in the cross-linking of matrix collagen and is a significant target for treating liver fibrosis.Mesenchymal stem cell-derived small extracellular vesicles(MSC-sEVs)have been proposed as a potential treatment option for chronic liver disorders.Previous studies have found that MSC-sEV can be used for microRNA delivery into target cells or tissues.It is currently unclear whether microRNA-4465(miR-4465)can target LOXL2 and inhibit HSC activation.Additionally,it is uncertain whether MSC-sEV can be utilized as a gene therapy vector to carry miR-4465 and effectively inhibit the progression of liver fibrosis.This study explored the effect of miR-4465-modified MSC-sEV(MSC-sEVmiR-4465)on LOXL2 expression and liver fibrosis development.The results showed that miR-4465 can bind specifically to the promoter of the LOXL2 gene in HSC.Moreover,MSC-sEVmiR-4465 inhibited HSC activation and collagen expression by downregulating LOXL2 expression in vitro.MSC-sEVmiR-4465 injection could reduce HSC activation and collagen deposition in the CCl4-induced mouse model.MSC-sEVmiR-4465 mediating via LOXL2 also hindered the migration and invasion of HepG2 cells.In conclusion,we found that MSC-sEV can deliver miR-4465 into HSC to alleviate liver fibrosis via altering LOXL2,which might provide a promising therapeutic strategy for liver diseases.

Objective To elucidate whether increased palmitic acid(PA)content after obesity can inhibit KLF4 expression through up-regulation of DNMT1/3b,which in turn regulates the biological behavior of endometrial cancer(EC)cells.Methods General data were collected from non-EC control individuals(n=120)and EC patients(n=30)to analyze the differences in obesi-ty-related phenotypes between the two groups;Endometrial tissues from non-EC control individuals(n=20)and cancer tissues from EC patients(n=20)were collected,and the expression of KLF4 and DNMT1/3b was detected by immunohistochemistry in the endometrial tissues;invitro culture of EC cells was carried out;mRNA and protein expression levels of DNMTs/KLF4/MMP2 and other factors in the cells were detected by qRT-PCR and Western blotting.Transwell and scratch assay were used to detect the proliferation,migration and invasion ability of EC cells;An EC hormonal tumor model was constructed in obese mice,and the tumor-forming ability of EC cells or EC cells overexpressing KLF4 was observed in mice.Results The weight,body mass index,plasma LDL and free fatty acid content of EC patients were significantly higher than that of non-cancer subjects(P＜0.05);The percentage of overweight/obese individuals was higher in EC patients than that of non-cancer subjects(73.33％vs.50.00％,P＜0.05);The expression level of KLF4 in tumor tissues of EC patients was significantly lower,and the expres-sion level of DNMT1/3b was significantly higher.The expression level of KLF4 was significantly negatively correlated with the level of FFAs in serum.After PA treatment,the expression level of KLF4 in EC cells was significantly reduced,the expression levels of DNMT1/3b and MMP2 were significantly increased,and the cell migration and invasion ability was significantly en-hanced,with no significant effect on the cell proliferation ability.Up-regulation of KLF4 significantly reduced the expression lev-el of MMP2 and weakened the migration and invasion ability of EC cells;Down-regulation of KLF4 significantly increased the expression level of MMP2 and enhanced the migration and invasion ability of EC cells;PA treatment with concomitant up-regu-lation of KLF4 significantly reversed the promotional effect of PA treatment on the MMP2 expression level as well as migration and invasion ability of EC cells.The expression levels of KLF4 in EC cells were both significantly increased after down-regula-tion of DNMT1 and DNMT3b;PA treatment with simultaneous down-regulation of DNMT1 and DNMT3b,significantly re-versed these results.The tumor-forming ability of EC cells was significantly enhanced in high-fat diet-induced obese mice,whereas tumor-forming ability of EC cells was significantly reduced in high-fat diet-induced obese mice after overexpression of KLF4.Conclusion Increased plasma PA content after obesity promotes endometrial cancer cell migration,invasion,and in vivo tumor-forming ability by inhibiting KLF4 expression through down-regulation of DNMT1/DNMT3b.

Objective: To compare the clinical results between arthroscopic anchor suture bridge and Ethibond suture bone tunnel for anterior cruciate ligament (ACL) tibial avulsion fractures. Methods: A retrospective case control study was conducted to analyze the clinical data of 18 patients with ACL tibial avulsion fracture admitted to Wenzhou Central Hospital from June 2012 to June 2017. There were 14 males and four females, aged 12-57 years, with an average age of 31.4 years. According to the Meyers-McKeever classification, there were six patients with type II and 12 patients with type III. Seven patients underwent anchor suture bridge fixation (anchor group), and 11 patients underwent Ethibond suture bone tunnel fixation (Ethibond suture group). The operation time, range of motion (ROM) of knee joint, Lysholm knee score and International Knee Documentation Committee (IKDC) knee score of the two groups were compared before operation and 3, 6 and 12 months after operation. Results: All patients were followed up for 12-36 months, with an average of 20.18 months. The operation time of anchor group [(87.14±8.59)minutes]was longer than that of Ethibond suture group [(71.1±11.5)minutes](P<0.05). The ROM of anchor group and Ethibond suture group was (127.1±8.6)° and (124.1±7.4)° respectively at 3 months after surgery, showing no statistical difference between the two groups (P>0.05). But the functional knee scores (Lysholm score and the IKDC score) revealed better clinical results in anchor group [(91.9±3.0)points, (85.6±4.1) points respectively] than Ethibond suture group [(88.6±3.3)points, (79.9±4.9)points respectively] at 3 months after surgery (P<0.05). There were significant differences between before and after operation within two groups regarding the knee function scores including ROM, Lysholm knee score and IKDC score (P<0.05). Three months after operation, there were significant differences between the two groups in terms of the Lysholm score and IKDC score (P<0.05), but there were no statistical differences between two groups at 6 months and 12 months after surgery (P>0.05). Conclusions Both arthroscopic anchor and Ethibond suture can achieve plane fixation of fracture, but there is no significant difference in the long-term effect of improving knee joint mobility and restoring knee joint function between the two approaches. However, the anchor group can achieve early recovery of knee joint function.

Objective To investigate the value of neurite orientation dispersion and density imaging (NODDI) in quantitative evaluation of the microstructural changes in basal ganglia and thalamus in Wilson's disease (WD) patients,and to assess the diagnostic efficacy of NODDI.Methods Totally 27 WD patients (WD group) and 26 age-and sex-matched controls (control group) were enrolled.All subjects underwent MR scanning with NODDI.Parameters of NODDI,including intracellular volume fraction (Vic),orientation dispersion index (ODI) and isotropic volume fraction (Viso) of bilateral caudate nucleus,globus pallidus,putamen and thalamus were compared between the 2 groups.Correlation analysis was performed between each parameter of NODDI and clinical Young scores.Random Forest model was used to assess the relative importance of each parameter and to evaluate the diagnostic efficacy.Results The Vic and ODI of bilateral caudate nucleus,globus pallidus and putamen in WD patients were significantly lower than those in normal controls (all P＜0.05),while Viso was significantly higher than that in normal controls (all P＜0.05).The Vic of bilateral thalamus was lower,while Viso was higher in WD patients than those in normal controls (all P＜0.05),and ODI had no significant difference between the 2 groups (P=0.055).In WD patients,Vic and ODI of bilateral caudate nucleus,globus pallidus and putamen were negatively correlated with clinical Young scores.Viso of globus pallidus and putamen were positively correlated with clinical scores.The prediction accuracy of NODDI was 96.23％,and the area under ROC curve was 0.96.Conclusion NODDI can effectively evaluate changes in microstructures and metabolism during copper deposition in WD patients,and it may be useful in detecting changes of brain deep nuclei and assessing the progression of WD.

AIM: To investigate the effects of safflor injection(SI) on expression of cyclooxygenase-2 mRNA during lung ischemia/reperfusion injury(PIRI) in rabbits.METHODS: Rabbit lung model of ischemia/reperfusion injury was constructed in vivo.The rabbits were randomly divided into three groups: sham-operation group(group S),ischemia-reperfusion group(group I/R) and ischemia/reperfusion plus safflor injection group(group SI).The lung tissue sampled at the end of the experiment was assayed for wet/dry weight ratio(W/D),injured alveoli rate(IAR) and observed ultrastructure changes under electron microscope.The expressions of COX-1 and COX-2 were measured by immunohistochemistry(IHC).The expression of COX-1 mRNA and COX-2 mRNA were observed by in situ hybridization(ISH).RESULTS: The value of W/D and IAR was much higher in I/R group,but decreased in SI group.Electron microscope showed obvious ultrastructure injury brought by PIRI in I/R group,which was greatly attenuated in SI group.The IHC and ISH demonstrated that COX-2 and COX-2 expressions in pulmonary tissue of I/R group were significantly higher than those in S group(P