ObjectiveTo investigate whether menaquinone-4 （MK-4） can exert a protective effect against carbon tetrachloride （CCl₄）-induced acute liver injury （ALI） in mice by alleviating ferroptosis. MethodsAfter adaptive feeding， adult male ICR mice， aged 8 weeks， were divided into Control group， MK-4 group， CCl₄ model group （6-hour， 12-hour， and 24-hour）， and MK-4+CCl₄ group （6-hour， 12-hour， and 24-hour）， with 6 mice in each group. The mice in the Control group were given intraperitoneal injection of an equal dose of corn oil； the mice in the MK-4 group were given intraperitoneal injection of 40 mg/kg MK-4 solution， followed by an equal dose of corn oil after 1 hour； the mice in the MK-4+CCl₄ group （6-hour， 12-hour， and 24-hour） were given intraperitoneal injection of 40 mg/kg MK-4 solution， and after 1 hour， the mice in this group and the CCl₄ model group （6-hour， 12-hour， and 24-hour） were given intraperitoneal injection of 0.3 mL/kg CCl₄ solution， with samples collected at 6， 12， and 24 hours. HE staining was used to observe the pathological changes of mouse liver； Prussian blue staining was used to observe iron accumulation in liver tissue； a biochemical analyzer was used to measure the serum levels of aspartate aminotransferase （AST） and alanine aminotransferase （ALT）； related kits were used to measure the levels of tissue iron content and the oxidative stress indices malondialdehyde （MDA） and glutathione （GSH） in liver homogenate； RT-PCR was used to measure the expression levels of ferroptosis marker genes （acyl-CoA synthetase long-chain family member 4 ［ACSL4］， prostaglandin-endoperoxide synthase 2 ［PTGS2］， and glutathione peroxidase 4 ［GPX4］） and iron metabolism-related genes （hemojuvelin ［HJV］， transferrin receptor 1 ［TFR1］， and ferroportin ［FPN］）， and Western blot was used to measure the protein expression level of GPX4. 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. ResultsIn the aging study， compared with the Control group， the CCl₄ model group （6-hour， 12-hour， and 24-hour） had significant increases in liver weight coefficient and the serum levels of ALT and AST （all P<0.05）， and HE staining also showed that liver injury gradually aggravated over time. Meanwhile， compared with the CCl₄ model group （6-hour， 12-hour， and 24-hour）， the MK-4+CCl₄ （12-hour） group had significant reductions in liver weight coefficient and the serum levels of ALT and AST （all P<0.05）， with a reduction in the necrotic area of liver tissue， and therefore， 12-hour mouse tissue samples were used for detection in the following study. Compared with the Control group， the CCl₄ group had a significant increase in MDA and a significant reduction in GSH （both P<0.05）， and compared with the CCl₄ group， the MK-4+CCl₄ group had a significant reduction in MDA and a significant increase in GSH （both P<0.05）. Compared with the Control group， the CCl₄ group had significant increases in the key ferroptosis indices ASCL4 and PTGS2 and a significant reduction in GPX4 （all P<0.05）； compared with the CCl₄ group， the MK-4+CCl₄ group had significant reductions in the mRNA expression levels of ASCL4 and PTGS2 and a significant increase in the mRNA expression level of GPX4 （all P<0.05）. Western blotting showed that compared with the Control group， the CCl₄ group had a significant reduction in the protein expression level of GPX4 （P<0.05）， and compared with the CCl₄ group， the MK-4+CCl₄ group had a significant increase in the protein expression level of GPX4 （P<0.05）. Prussian blue staining showed that compared with the Control group， the CCl₄ group had a significant increase in iron accumulation； after MK-4 intervention， compared with the CCl₄ group， the MK-4+CCl₄ group had a significant reduction in iron accumulation. As for the measurement of iron metabolism genes in mouse liver， compared with the Control group， the CCl₄ group had a significant increase in iron content， significant reductions in the mRNA expression levels of FPN and HJV， and a significant increase in the mRNA expression level of TFR1 （all P<0.05）； after protection with MK-4， there was a significant reduction in iron content， significant increases in the mRNA expression levels of FPN and HJV， and a significant reduction in the mRNA expression level of TFR1 （all P<0.05）. ConclusionMK-4 intervention in advance can alleviate CCl₄-induced ALI in mice， possibly by inhibiting ferroptosis and improving the expression of iron metabolism-related genes in mouse liver.

ObjectiveTo explore the protective effects of Dahuang Tangluo pills on early diabetic kidkdey disease （DKD） in db/db mice. MethodEight db/m mice were selected as the control group. Forty male db/db mice were selected and blood samples were collected via tail vein to measure fasting blood glucose （FBG）. Mice with FBG ≥ 16.7 mmol·L^-1， increased urine output， and persistent albuminuria were considered successful in model establishment. After successful modeling， they were randomly divided into a model group， a dapagliflozin group （1.5 mg·kg^-1·d^-1）， and high， medium， and low dose groups of Dahuang Tangluo pills （3.6， 1.8， 0.9 g·kg^-1·d^-1， respectively）， with eight mice in each group. All medication groups were administered orally， while the control and model groups were given an equal amount of distilled water by gavage daily. After continuous administration for 10 weeks， the survival status of the mice was observed， and their body weight， FBG， and kidney function-related indicators were measured. Inflammatory indicators in renal tissues were determined by enzyme-linked immunosorbent assay （ELISA）. Hematoxylin-eosin （HE） staining， Masson staining， and electron microscopy were used to observe the pathological changes in renal tissues in each group. Immunofluorescence was employed to examine the expression of advanced glycation end products （AGEs） and receptors for advanced glycation end products （RAGE） proteins. Real-time quantitative polymerase chain reaction （Real-time PCR） and Western blot were utilized to detect the gene and protein expression levels of AGEs， RAGE， inhibitor of nuclear factor-κB （NF-κB） kinase （IKK）， and NF-κB in the renal tissues of mice in each group. ResultCompared with control group， the model group showed a significant increase in body weight， FBG， serum creatinine （SCr）， urinary microalbumin/urine creatinine ratio （ACR）， total cholesterol （TC）， and triglycerides （TG） （P<0.05）. The levels of intercellular adhesion molecule-1 （ICAM-1）， interleukin-6 （IL-6）， and tumor necrosis factor-alpha （TNF-α） in renal tissues were significantly elevated （P<0.05）. Renal histopathological staining and electron microscopy revealed loose arrangement， gaps， structural disarray， mesangial proliferation， and significant fibrosis in renal tissues. Real-time PCR results showed a significant increase in the expression of RAGE， IKK， and NF-κB genes in renal tissues （P<0.05）. Immunofluorescence results demonstrated a significant increase in the expression of AGEs and RAGE proteins in renal tissues （P<0.05）. Western blot results showed a significant increase in the expression of AGEs， RAGE， IKK， and NF-κB proteins in renal tissues （P<0.05）. After drug intervention， compared with model group， the dapagliflozin group and the high-dose Dahuang Tangluo pills group showed significant reductions in body weight， FBG， SCr， and ACR （P<0.05）， and a significant decrease in TC in mouse serum （P<0.05）， while the high-dose Dahuang Tangluo pills group showed a significant decrease in TG in mouse serum （P<0.05）. All treatment groups showed a significant reduction in ICAM-1， IL-6， and TNF-α in renal tissues （P<0.05）. Renal histopathological staining and electron microscopy showed improved kidney injury， decreased collagen fiber deposition， and reduced mesangial proliferation in all treatment groups. Real-time PCR results showed a significant decrease in the expression of RAGE， IKK， and NF-κB genes in the dapagliflozin group and the high- and medium-dose Dahuang Tangluo pills groups （P<0.05）. Immunofluorescence results demonstrated a significant decrease in the expression of AGEs and RAGE proteins in the dapagliflozin group and the high- and medium-dose Dahuang Tangluo pills groups （P<0.05）. Western blot results showed a significant decrease in the expression of AGEs， RAGE， IKK， and NF-κB proteins in the dapagliflozin group and the high- and medium-dose Dahuang Tangluo pills groups （P<0.05）. ConclusionDahuang Tangluo pills can improve the pathological structure of the kidneys and reduce renal inflammation in DKD mice， possibly through inhibiting the AGEs/RAGE/IKK/NF-κB pathway.

Objective To investigate the correlation between the expression of long non-coding ribonucleic acid growth arrest specific 5(lncRNA GAS5),phospholysine phosphohistidine inorganic pyrophosphate phos-phatase(LHPP)and epithelial-mesenchymal transition(EMT)in cancer tissues of patients with non-small cell lung cancer(NSCLC)and its clinical significance.Methods Cancer tissues and adjacent tissues of 90 patients with NSCLC who underwent surgical resection in the First Hospital Affiliated to Hebei North College from June 2018 to January 2020 were collected.The expressions of lncRNA GAS5,LHPP and EMT-associated pro-teins[E-calmodulin(E-Cad),N-calmodulin(N-Cad),and vimentin(VIM)]were detected by real-time fluores-cence quantitative polymerase chain reaction.The relationship between lncRNA GAS5 and LHPP mRNA and clinicopathological features in cancer tissues of NSCLC patients was analyzed,and the correlation between ln-cRNA GAS5 and LHPP mRNA and EMT-associated proteins expression in cancer tissues of NSCLC patients was analyzed by Pearson correlation.Kaplan-Meier method was used to plot the survival curves of NSCLC pa-tients with different lncRNA GAS5 and LHPP mRNA expressions,and multivariate Cox regression was used to analyze the prognostic factors of NSCLC patients.Results The expressions of lncRNA GAS5,LHPP mR-NA and E-Cad mRNA in cancer tissues of NSCLC patients were lower than those in adjacent tissues,while the expressions of N-Cad mRNA and VIM mRNA were higher than those in adjacent tissues,with statistical sig-nificance(P＜0.05).Pearson correlation analysis showed that lncRNA GAS5 in cancer tissues of NSCLC pa-tients was positively correlated with E-Cad mRNA expression(r=0.724,P＜0.001),and negatively correla-ted with N-Cad mRNA and VIM mRNA expression(r=-0.699,-0.689).P＜0.001);lncRNA GAS5 was positively correlated with LHPP mRNA expression(r=0.651,P＜0.001).The mRNA expressions of ln-cRNA GAS5 and LHPP in cancer tissues of NSCLC patients with different degrees of differentiation,tumor TNM stage and lymph node metastasis were significantly different(P＜0.05).Kaplan-Meier survival curve a-nalysis showed that the 3-year overall survival rate in the lncRNA GAS5 high expression group[68.18％(30/44)]was higher than that in the lncRNA GAS5 low expression group[36.96％(17/46)].The 3-year overall survival rate in the high LHPP mRNA expression group[67.39％(31/46)]was higher than that in the lowLHPP mRNA expression group[36.36％(16/44)],and the difference was statistically significant(x2=10.274,10.322,P＜0.05).Low differentiation,TNM stage Ⅲ and lymph node metastasis were independent risk factors for death in NSCLC patients,and lncRNA GAS5≥1.32 and LHPP mRNA≥1.12 were independ-ent protective factors(P＜0.05).Conclusion The low expression of lncRNA GAS5 and LHPP mRNA in cancer tissues of patients with NSCLC is related to EMT-associated proteins expression,differentiation de-gree,tumor TNM stage,lymph node metastasis and prognosis,and may become a new target for the diagnosis and treatment of NSCLC.

Objective To investigate the mechanism of bis(2-ethylhexyl)phthalate(DEHP)in inducing cholestasis and liver injury in mice.Methods In the in vivo experiment,adult female ICR mice were randomly divided into control group(corn oil)and DEHP group(200 mg/kg/d),and a model of cholestasis was established by intragastric administration for 4 weeks.After blood and liver tissue samples were collected from all mice,a biochemical analyzer was used to measure the level of total bile acid(TBA)in serum and the liver,and a microplate reader was used to measure alkaline phosphatase(ALP)and gamma-glutamyl transpeptidase(GGT);HE staining was used to observe the pathological changes of the liver;RT-PCR was used to measure the mRNA expression levels of the inflammatory factors interleukin-1β(IL-1β),interleukin-6(IL-6),and tumor necrosis factor-α(TNF-α)in the liver;liquid chromatography/triple quadrupole mass spectrometry was used to measure the bile acid profile in the liver of mice.In the in vitro experiment,AML-12 mouse hepatocytes were cultured and treated with DEHP(250 μmol/L),DCA(125 μmol/L),and CDCA(125 μmol/L)for 24 hours,and RT-PCR was used to measure the mRNA expression levels of the inflammatory cytokines IL-1β,IL-6,and TNF-α.The independent-samples t test was used for comparison of continuous data between two groups;a one-way analysis of variance was used for comparison between multiple groups,and the LSD-t test was used for further comparison between two groups.Results The in vivo experiment showed that compared with the control group,the DEHP group had significant increases in the serum levels of TBA,ALP,and GGT and the level of TBA in the liver(the t values are respectively-4.396,-5.109,-8.504,-3.792 and-7.974,all P<0.05,).Compared with the control group,the DEHP group had significant increases in cholic acid,chenodeoxycholic acid,taurocholic acid,deoxycholic acid,and ursodeoxycholic acid(the t values are respectively-2.802,-3.177,-2.633,-2.874 and-2.311,all P<0.05).HE staining of the liver showed that the mice in the DEHP group had enlargement of the portal area,bile duct deformation,inflammatory cell infiltration around the bile duct,and significant increases in the mRNA expression levels of the inflammatory factors IL-1β,IL-6,and TNF-α in the liver(the t values are respectively-2.539,-2.823 and-4.636,all P<0.05).The in vitro experiment showed that the actual difference in hepatocyte viability after 0-1 000 μmol/L DEHP treatment does not exceed 15%,but there were significant increases in the mRNA expression levels of the inflammatory cytokines IL-1β,IL-6,and TNF-α after treatment with DEHP at different concentrations of 125 μmol/L,250 μmol/L,and 500 μmol/L(all P<0.05).Compared with DEHP stimulation alone,the combined stimulation of CDCA and DEHP upregulates the cytokine in hepatocyte IL-1β mRNA levels(P<0.01);the combined stimulation of DCA and DEHP can significantly increase the cytokine in hepatocyte IL-1β and IL-6 mRNA levels(all P<0.01).Conclusion DEHP exposure can cause cholestasis and induce liver inflammation in mice,possibly by promoting the production of toxic bile acids and the secretion of inflammatory factors.

Objective To explore the value of peripheral blood circulating DNA in predicting the efficacy and prognosis of immunotherapy for advanced non-small cell lung cancer.Method A retrospective study was conducted on 78 NSCLC patients who were admitted to the Respiratory and Critical Care Medicine Department of the First Affiliated Hospital of Hebei North University and were treated with tirelizumab for advanced driver gene negativity from January 2021 to December 2021.After 2 cycles of immunotherapy,the efficacy was evaluated according to the Solid Tumor Efficacy Evaluation Criteria(RECIST 1.1),including complete remission,partial remission,disease stability,and disease progression.CR and PR patients were defined as the experimental group(n=48)Other patients were defined as the control group(n=30),and the ctDNA levels in peripheral blood were measured before and after treatment in both groups.ROC curves were used to analyze the predictive value of periph-eral blood ctDNA levels for achieving objective remission after immunotherapy.All patients were followed up and their progression free survival were calcutated.Using univariate and multivariate regression analysis identified the factors affecting the prognosis of patients after immunotherapy.Using Spearman correlation coefficient analyzed the correlation between ctDNA levels and PFS.Kalplan Meier survival curve were used for survival analysis.Result The peripheral blood ctDNA levels before and after treatment in the experimental group were(4.47±1.21)ng/μL and(2.65±1.14)ng/μL,respectively(t=7.559,P<0.001),while those in the control group were(4.54±1.15)ng/mL and(4.29±1.57)ng/μL,respectively(t=0.699,P=0.487).There was no statistically significant difference in peripheral blood ctDNA levels between the two groups before treatment(t=-0.25,P=0.801).The peripheral blood ctDNA levels in the experimental group decreased compared to the control group after treatment(t=-5.35,P<0.001).The ROC curve analysis showed that the area under the curve for predicting objective remission after immunotherapy based on peripheral blood ctDNA levels was 0.819,with a sensitivity of 81.3%and specificity of 80%.Peripheral blood ctDNA levels were negatively correlated with progression free survival(r=-0.784,P=0.000).Single factor COX regression was used to analyze the clinical and pathological characteristics and ctDNA levels of enrolled patients,and the results showed that the maximum tumor diameter was greater than 5 cm(HR=0.501,95%CI:6.731～35.567)Tumor stage IV(HR=0.392,95%CI:0.227～0.677),treatment approach(HR=15.473,95%CI:6.731～35.567),and ctDNA levels(HR=4.657,95%CI:3.182～6.555)are all influencing factors for PFS in advanced NSCLC patients after immunotherapy.Multiple factor analysis was conducted on the appeal indicators with statistical differences,and the results showed that treatment approach(HR=2.981,95%CI:1.019～8.722)and peripheral blood ctDNA levels(HR=3.918,95%CI:2.619～5.861)It is an independent influencing factor of PFS in advanced NSCLC patients.The Kalplan Meier survival curve was used for analysis,and the results showed that the median PFS of the treatment effective group was 8.4 months,while the median PFS of the control group was 5.4 months.(χ2=49.277,P=0.000).Conclusion Immunotherapy combined with chemotherapy can enhance the ability to kill tumor cells,and peripheral blood ctDNA levels can evaluate the efficacy and prognosis of immunotherapy,which can be used to guide immunotherapy in advanced NSCLC patients.

Objective:To investigate the effect and mechanism of miR-217 targeted regulation of extracellular signal-regulated kinase 2(ERK2)expression on activity and immune escape of non-small cell lung cancer cells(NSCLC).Methods:qRT-PCR was used to detect expression levels of miR-217 and ERK2 mRNA in NSCLC tissues,adjacent tissues,and HLF-1,A549 and HCC827 cell lines.Analyzed prognosis and survival status of NSCLC patients with different miR-217 expression level.Bioinformatics and dual luciferase gene reporting experiments were used to analyze the targeting relationship between miR-217 and ERK2.Cultivated NSCLC A549 cells and divided them into NC group,miR-217 inhibitor group,miR-217 mimic group,miR-217 mimic+ERK2 NC group and miR-217 mimic+ERK2 group.Except for the NC group without any treatment,all other groups were transfected with corresponding plasmids to analyze the proliferation activity and immune escape status of A549 cells in each group,and clarified the mechanism of action.Results:Compared with adjacent tissues,expression of miR-217 in NSCLC tissue was decreased,while expression of ERK2 mRNA was increased(P<0.05).Compared with human normal lung fibroblast HLF-1 cell lines,expression of miR-217 in NSCLC cell lines A549 and HCC827 were decreased,while expression of ERK2 mRNA was increased(P<0.05).Analysis of the relationship be-tween miR-217 and prognosis of NSCLC patients based on Kaplan-Meier Plotter database showed that low expression of miR-217 was associated with poor prognosis of patients(HR=0.90,P=0.033).Dual fluorescein reporter genes showed matching sequences between the 3'UTR regions of miR-217 and ERK2.miR-217 mimic fragment could inhibit ERK2-WT signal,but had no effect on ERK2-MUT.Compared with NC group,cell proliferation activity,PD-L1 and PD-L2 mRNA expression levels of miR-217 inhibitor group were in-creased,while CD8+T cell activity was decreased,and cell proliferation activity,PD-L1 and PD-L2 mRNA expression levels of miR-217 mimic group were decreased,while CD8+T cell activity was increased(P<0.05).Compared with miR-217 mimic group,cell pro-liferation activity,CD8+T cell activity,PD-L1 and PD-L2 mRNA expression levels of miR-217 mimic+ERK2 NC group had no signifi-cant changes(P>0.05),cell proliferation activity,PD-L1 and PD-L2 mRNA expression levels of miR-217 mimic+ERK2 group were increased,while CD8+T cell activity was decreased(P<0.05).Conclusion:Overexpression of miR-217 can reduce the activity of NSCLC cell A549,inhibit the expression of PD-L1,activate CD8+T cells in tumor microenvironment,and then inhibit immune es-cape,which may play a role by targeting ERK2.

Objective:To reduce the immunogenicity of vaccinia virus vector by replacing the D8L region, which is a neutralizing antibody epitope in vaccinia virus, with an exogenous gene.Methods:A gene fragment encoding influenza virus hemagglutinin (HA) was inserted into the D8L region to replace it using homologous recombination technique. Then, a recombinant vaccinia virus influenza vaccine was constricted. A recombinant vaccinia virus vaccine with the TK region expressing HA was used as a control. The expression of HA was validated by Western blot. BALB/c mice were immunized with the vaccines and the serum antibody titers two weeks after each immunization were evaluated by ELISA and hemagglutination inhibition assay. The protective efficacy of the recombinant vaccinia virus was assessed through a challenge experiment.Results:Western blot confirmed the successful expression of HAD8L protein in the constructed recombinant vaccines. ELISA and hemagglutination inhibition assay showed that after the primary immunization, the anti-HA antibody titer induced by the recombinant vaccinia virus with D8L region mutation was slightly higher than that induced by the vaccine with TK region mutation, and the difference was statistically significant with the increase of immunization times ( P<0.05). The recombinant vaccinia virus with D8L region mutation showed significantly lower immunogenicity than the recombinant virus with TK region mutation after the primary immunization, but there was no significant difference between them with the increase of immunization times ( P>0.05). After H1N1pdm challenge, no virus was detected in the mice immunized with the recombinant vaccinia virus with D8L region mutation and the mice showed mild lung inflammation and less tissue damage. Conclusions:This study indicated that inserting exogenous genes into the D8L region of the neutralizing antibody epitope in the vaccinia virus vector could help to reduce the immunogenicity of the vector itself and enhance the immunogenicity of the exogenous genes. This provided a reference for the use of the vaccinia virus vector as a delivery tool in the field of vaccines or gene therapy.

Objective : High performance liquid chromatography⁃mass spectrometry (LC⁃MS/MS) system was used to accurately determine 22 bile acids in serum , liver, amniotic fluid and placenta of pregnant mice , and a LC⁃MS/ MS method was established for efficient detection and analysis of bile acids in serum , liver, amniotic fluid and placenta of mice. Methods : Pregnant mice serum , liver, amniotic fluid and placenta samples were processed , with 0. 1% glacial acetic acid in 4 mmol/L ammonium acetate aqueous solution as mobile phase A and pure methanol as mobile phase B , the flow rate was 0. 4 ml/min , a gradient elution program was used to elute with Phenomenex Gemini 3 μm NX⁃C18 110A ( 100 mm × 2. 0 mm) chromatographic column elution , and mass spectrometry detection system used an electrospray ion source for negative ion multiple reaction monitoring. Results : The linear relationship of 22 bile acids in the quantitative range was good. The RSD of inter⁃day and intra⁃day precision at low , medium and high concentrations was 0. 5% - 7. 4% , the matrix effect was 88% - 110% , and the extraction recovery was 84% - 108% . Conclusion In this experiment , LC⁃MS/MS was established to detect 22 bile acids in serum , liver, amniotic fluid and placenta of pregnant mice. The method not only has high sensitivity and selectivity , but also can stably detect a large number of samples.

Carcinogenesis ; Cell Transformation, Neoplastic ; Hepatoblastoma ; Humans ; Liver ; Liver Neoplasms ; Organoids ; YAP-Signaling Proteins

Bioengineering majors require students to acquire excellent abilities of thinking and analyzing complex problems and have high requirements for students' comprehensive practical skills. Because of the professional characteristics, it is necessary to develop students' abilities to solve complex problems via the teaching of a series of experiments. Therefore, it is particularly important to reform the traditional experiment teaching for students majoring in bioengineering to improve the teaching quality, which have great significance for the cultivation of comprehensive talents. In this study, with the advantages of geographical location and resources to cultivate application-oriented innovative talents, the course group of Comprehensive Experiment of Bioengineering has designed the course based on virtual simulation technology in Binzhou University. Taking the experiment of extraction and bioactivity analysis of Suaeda salsa (growing in the Yellow River Delta) polysaccharide in fermentation as a case, we studied the course design idea, experimental process, teaching method and result analysis, and have improved the teaching performance. This case analysis provides new ideas and content reference for the teaching reform of similar courses.
Bioengineering/education* ; Biomedical Engineering/education* ; Humans ; Students ; Technology ; Universities