Objective:To predict pre-treatment clinical parameters that are associated with poor response and prognosis to ursodeoxycholic acid (UDCA) in patients with primary biliary cholangitis (PBC) and to use second-line treatment drugs in the early stages to delay the progression of the disease so that patients can benefit from early-stage treatment.Methods:Patients diagnosed with PBC at the Second Affiliated Hospital of Kunming Medical University from 2013 to 2022 were collected. Two hundred fifty-seven cases were screened in accordance with the inclusion and exclusion criteria. The response and prognosis conditions one year after treatment were followed up in outpatient and inpatient departments, as well as through telephone calls. Statistical analyses were performed using t-tests, Mann-Whitney U test, χ2 test, Fisher's exact test, and logistic regression analysis according to different data. Results:A total of 257 PBC cases were included, with 223 females (86.80%) and 34 males (13.20%). Univariate and multivariate binary logistic regression analyses showed that baseline high albumin levels [odds ratio ( OR): 0.882, 95% confidence interval ( CI): 0.805～0.967, P=0.008] were a protective factor for PBC patients' response to UDCA treatment after adjusting for different confounding factors, while baseline high alkaline phosphatase ( OR: 1.012, 95% CI: 1.008～1.016, P<0.001) and baseline high neutrophil/lymphocyte ratio (NLR) level ( OR: 1.462, 95% CI:1.079～1.981, P=0.014) were risk factors for a poor response to UDCA. Trend analysis showed that the baseline NLR quantile was positively correlated with the risk of poor response to UDCA ( OR: 5.512, 95% CI: 1.040～29.216, P=0.045) in patients with PBC. Cox proportional hazards regression analysis identified that age [hazard ratio ( HR): 1.050, 95% CI: 1.019～1.082] and NLR value ( HR:1.089, 95% CI:1.021～1.161) were independent influencing risk factors for all-cause mortality in PBC patients ( P<0.05). Conclusion:Baseline high albumin levels are protective factors against a poor biochemical response to UDCA, while baseline high alkaline phosphatase levels and high NLR are risk factors for a poor biochemical response to UDCA in patients with PBC. Additionally, baseline high NLR values are positively correlated with poor biochemical response to UDCA treatment.

Objective:To predict pre-treatment clinical parameters that are associated with poor response and prognosis to ursodeoxycholic acid (UDCA) in patients with primary biliary cholangitis (PBC) and to use second-line treatment drugs in the early stages to delay the progression of the disease so that patients can benefit from early-stage treatment.Methods:Patients diagnosed with PBC at the Second Affiliated Hospital of Kunming Medical University from 2013 to 2022 were collected. Two hundred fifty-seven cases were screened in accordance with the inclusion and exclusion criteria. The response and prognosis conditions one year after treatment were followed up in outpatient and inpatient departments, as well as through telephone calls. Statistical analyses were performed using t-tests, Mann-Whitney U test, χ2 test, Fisher's exact test, and logistic regression analysis according to different data. Results:A total of 257 PBC cases were included, with 223 females (86.80%) and 34 males (13.20%). Univariate and multivariate binary logistic regression analyses showed that baseline high albumin levels [odds ratio ( OR): 0.882, 95% confidence interval ( CI): 0.805～0.967, P=0.008] were a protective factor for PBC patients' response to UDCA treatment after adjusting for different confounding factors, while baseline high alkaline phosphatase ( OR: 1.012, 95% CI: 1.008～1.016, P<0.001) and baseline high neutrophil/lymphocyte ratio (NLR) level ( OR: 1.462, 95% CI:1.079～1.981, P=0.014) were risk factors for a poor response to UDCA. Trend analysis showed that the baseline NLR quantile was positively correlated with the risk of poor response to UDCA ( OR: 5.512, 95% CI: 1.040～29.216, P=0.045) in patients with PBC. Cox proportional hazards regression analysis identified that age [hazard ratio ( HR): 1.050, 95% CI: 1.019～1.082] and NLR value ( HR:1.089, 95% CI:1.021～1.161) were independent influencing risk factors for all-cause mortality in PBC patients ( P<0.05). Conclusion:Baseline high albumin levels are protective factors against a poor biochemical response to UDCA, while baseline high alkaline phosphatase levels and high NLR are risk factors for a poor biochemical response to UDCA in patients with PBC. Additionally, baseline high NLR values are positively correlated with poor biochemical response to UDCA treatment.

A quadruplex RT-qPCR method was developed for rapid identification and diagnosis of transmissible gastroenteritis virus(TGEV)of swine,porcine epidemic diarrhea virus(PEDV),porcine deltacorona virus(PDCoV),and porcine rotavirus type A(PoRVA).The full-length sequences of PEDV(77 strains),TGEV(63 strains),PDCoV(17 strains)that are prevalent in China,as well as the 85 VP6 gene sequences of PoRVA,were downloaded from the NCBI database for homology analysis.Based on the relatively conserved sequences,the corresponding primers and probes for each virus were designed and used to establish the quadruplex RT-qPCR method.After optimization of the probes and the reaction conditions,the specificity,sensitivity,and repeatability were determined.Using the established method,109 clinical samples of diarrhea were tested and further compared with the results by standard method.The results showed that the quadruplex RT-qPCR method established in this experiment has good amplification effect,with the C,value linearly correlated with the copies of templates(R2＞0.99).Specificity assay demonstrated that the quadruplex RT-qPCR method can identify TGEV,PEDV,PDCoV,PRoVA strains,and do not de-tect African swine fever virus(ASFV),porcine circovirus type 2(PCV2),porcine reproductive and respiratory syndrome virus(PRRSV),classical swine fever virus(CSFV),pseudorabies virus(PRV)and other epidemic viruses.Sensitivity assay showed that the detection limits for TGEV,PEDV,PDCoV and PoRVA were 10,20,20 and 50 copies/μL,respectively.The method exhibits excellent reproducibility,with coefficients of variation(Cv)for both intra-and inter-assay repli-cates being less than 1％.Detection of 109 samples of diarrhea by this method yielded the coinci-dence rate of 100％with the industry standard,indicating high practical applicability.The devel-oped method possesses the advantages of strong strain compatibility,high sensitivity,strong speci-ficity,good repeatability and stability.It is suitable for virus diagnosis and large-scale clinical sam-ple testing,providing technical support for disease prevention and control as well as epidemiologi-cal investigation.

A quadruplex RT-qPCR method was developed for rapid identification and diagnosis of transmissible gastroenteritis virus(TGEV)of swine,porcine epidemic diarrhea virus(PEDV),porcine deltacorona virus(PDCoV),and porcine rotavirus type A(PoRVA).The full-length sequences of PEDV(77 strains),TGEV(63 strains),PDCoV(17 strains)that are prevalent in China,as well as the 85 VP6 gene sequences of PoRVA,were downloaded from the NCBI database for homology analysis.Based on the relatively conserved sequences,the corresponding primers and probes for each virus were designed and used to establish the quadruplex RT-qPCR method.After optimization of the probes and the reaction conditions,the specificity,sensitivity,and repeatability were determined.Using the established method,109 clinical samples of diarrhea were tested and further compared with the results by standard method.The results showed that the quadruplex RT-qPCR method established in this experiment has good amplification effect,with the C,value linearly correlated with the copies of templates(R2＞0.99).Specificity assay demonstrated that the quadruplex RT-qPCR method can identify TGEV,PEDV,PDCoV,PRoVA strains,and do not de-tect African swine fever virus(ASFV),porcine circovirus type 2(PCV2),porcine reproductive and respiratory syndrome virus(PRRSV),classical swine fever virus(CSFV),pseudorabies virus(PRV)and other epidemic viruses.Sensitivity assay showed that the detection limits for TGEV,PEDV,PDCoV and PoRVA were 10,20,20 and 50 copies/μL,respectively.The method exhibits excellent reproducibility,with coefficients of variation(Cv)for both intra-and inter-assay repli-cates being less than 1％.Detection of 109 samples of diarrhea by this method yielded the coinci-dence rate of 100％with the industry standard,indicating high practical applicability.The devel-oped method possesses the advantages of strong strain compatibility,high sensitivity,strong speci-ficity,good repeatability and stability.It is suitable for virus diagnosis and large-scale clinical sam-ple testing,providing technical support for disease prevention and control as well as epidemiologi-cal investigation.

Objective:To explore the correlation between gross tumor volume (GTV) and prognosis of patients with esophageal cancer undergoing radiotherapy.Methods:A retrospective cohort study was conducted. The clinical data of 130 newly diagnosed esophageal squamous cell carcinoma patients who received radiotherapy at Shanxi Province Cancer Hospital from February 2016 to June 2018 were analyzed. All patients underwent conformal intensity-modulated radiotherapy (IMRT) for esophageal lesions. Pinnacle planning system was used to calculate GTV, and GTV classification was performed: GTV ≤ 30 cm 3 was classified as grade Ⅰ, GTV > 30 cm 3 and ≤ 60 cm 3 was classified as grade Ⅱ, and GTV > 60 cm 3 was classified as grade Ⅲ. Kaplan-Meier method was used to analyze the progression free survival (PFS) and overall survival (OS) of patients, and the multivariate Cox proportional hazards model was used to analyze the independent influencing factors of poor PFS and OS. Results:The median age of 130 patients [ M ( Q1, Q3)] was 59 years old (56 years old, 69 years old), with 90 males and 40 females; Karnofsky performance scores were all ≥ 70 points; tumors were located in the neck in 10 cases, upper chest in 34 cases, middle chest in 55 cases, and lower chest in 31 cases; clinical staging for esophageal carcinoma treated with non-surgical methods: 3 cases in stage Ⅰ, 37 cases in stage Ⅱ, 79 cases in stage Ⅲ, and 11 cases in stage Ⅳ; 25 cases were classified as GTV grade Ⅰ, 62 cases as GTV grade Ⅱ, and 43 cases as GTV grade Ⅲ. The 1-year PFS rate of 130 patients was 55%, the 2-year PFS rate was 19%, and the median PFS time was 14 months; the 1-year OS rate was 76%, the 2-year OS rate was 32%, and the median OS time was 20 months. PFS and OS of patients in stages Ⅰ+Ⅱ, Ⅲ and Ⅳ deteriorated sequentially, and the differences between the three groups were statistically significant (both P < 0.001); the PFS and OS of patients with GTV grades Ⅰ, Ⅱ and Ⅲ deteriorated sequentially, and the differences in PFS and OS between the three groups were statistically significant (both P < 0.001); there were no statistically significant differences in PFS and OS among patients of different genders, ages, and tumor locations (all P > 0.05). The results of multivariate Cox regression analysis showed that high clinical staging (stage Ⅳ vs. stage Ⅰ, HR = 8.34, 95% CI: 3.88-17.94, P < 0.001) and high GTV grading (grade Ⅱ vs. grade Ⅰ: HR = 6.81, 95% CI: 3.39-13.67, P < 0.001; grade Ⅲ vs. grade Ⅰ: HR = 23.97, 95% CI: 10.81-53.14, P < 0.001) were independent risk factors for poor PFS; high clinical staging (stage Ⅳ vs. stage Ⅰ: HR = 9.94, 95% CI: 4.50-21.97, P < 0.001) and high GTV grading (grade Ⅱ vs. grade Ⅰ: HR = 13.55, 95% CI: 5.58-32.91, P < 0.001; grade Ⅲ vs. grade Ⅰ: HR = 35.01, 95% CI: 13.57-90.34, P < 0.001) were independent risk factors for poor OS. Conclusions:GTV is associated with the prognosis of patients with esophageal cancer undergoing radiotherapy.

OBJECTIVE To explore the mechanism of pomegranate peel in improving non-alcoholic steatohepatitis (NASH) based on network pharmacology and cell experiments verification. METHODS Using the Traditional Chinese Medicine System Pharmacology Database(TCMSP) to obtain the active components of pomegranate peel and their corresponding targets. NASH-related disease targets were obtained from five disease databases, including the Human Gene Database(GeneCards), etc. To screen the targets of pomegranate peel and NASH and obtain the common targets through Venn diagrams. The protein-protein interaction network of pomegranate peel-NASH was constructed using the protein interaction database(STRING), and the “pomegranate peel-component-target-NASH” network was established with Cytoscape 3.7.1. Gene ontology(GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway analysis were performed using Metascape software. Finally, the effect of the main active components in pomegranate peel on NASH was observed with human hepatoma cells(HepG2). RESULTS There were 7 active ingredients in pomegranate peel, 191 target genes, 1 818 NASH targets, and 98 intersection targets. Topological analysis showed that the core components of pomegranate peel in the treatment of NASH were quercetin, kaempferol, and luteolin, and the core targets were protein kinase B(Akt1), interleukin-1β(IL-1β), interleukin-6(IL-6), tumor necrosis factor(TNF). KEGG pathway analysis predicted that pomegranate peel treatment of NASH mainly involved phosphatidylinositol-3-kinase(PI3K)/Akt, nuclear factor kappa B(NF-κB), and other signaling pathways. The results of in vitro cell experiments showed that the expression levels of phosphorylated protein kinase B(p-Akt), IL-6 and other proteins were elevated in the model group compared with the control group(P<0.05). Compared with the model group, the active ingredients of pomegranate peel could significantly reduce the expression level of p-Akt and IL-6(P<0.05), as well as the mRNA expression level of IL-6 and TNF-α(P<0.05). CONCLUSION Pomegranate peel can exert anti-NASH effects through multiple components, multiple targets, and multiple pathways. The mechanism may be related to the active components quercetin, kaempferol, and luteolin in pomegranate peel affecting core targets such as Akt1 and regulating PI3K/Akt, NF-κB, and other signaling pathways, thereby inhibiting the expression of related inflammatory factors.

Objective To investigate whether Toll-like receptor 4 (TLR4) inhibition affects liver regeneration during acetaminophen (APAP)-induced liver injury in mice, as well as the mechanism of TLR4 involved in liver regeneration. Methods A total of 78 male CD-1 mice were divided into nine groups using a random number table, i.e., three control groups (normal control group, solvent control group, inhibitor control group) with 6 mice in each group and six experimental groups (APAP 24-hour group, TAK-242+APAP 24-hour group, APAP 48-hour group, TAK-242+APAP 48-hour group, APAP 72-hour group, TAK-242+APAP 72-hour group) with 10 mice in each group. The mice in the experimental groups were given a single dose of intraperitoneally injected APAP (300 mg/kg), and TAK-242 was intraperitoneally injected at a dose of 3 mg/kg at 3 hours before APAP administration. Serum and liver tissue samples were collected at different time points. The biochemical method was used to measure the serum level of alanine aminotransferase (ALT); HE staining was used to observe liver pathological changes; RT-PCR, Western blot, and immunohistochemistry were used to measure the expression levels of Cyclin D1, PCNA, Ki-67, STAT3, and p-STAT3. The t -test was used for comparison of normally distributed continuous data between two groups; a one-way analysis of variance was used for comparison between multiple groups, and the least significant difference t -test was used for further comparison between two groups. The Mann-Whitney U test was used for comparison of non-normally distributed continuous data between two groups, and the Kruskal-Wallis H test was used for comparison between multiple groups and further comparison between two groups. Results Compared with the normal control group, the APAP 24-hour and 48-hour groups had a significantly higher serum level of ALT (both P < 0.05), and the TAK-242+APAP 24-hour and 48-hour groups had a significantly higher serum level of ALT than the APAP group at the same time point (both P < 0.05). HE staining showed typical central lobular necrosis in the liver of APAP-treated mice, and the TAK-242+APAP 24-hour and 48-hour groups had a significantly larger necrotic area than the APAP group at the same time point (both P < 0.05). RT-PCR, Western blot, and immunohistochemistry showed that the TAK-242+APAP 24-hour, 48-hour, and 72-hour groups had significantly lower mRNA and protein expression levels of Cyclin D1 than the APAP group at the same time point (all P < 0.05); the TAK-242+APAP 24-hour, 48-hour, and 72-hour groups had a significantly lower mRNA expression level of PCNA than the APAP group at the same time point (all P < 0.05), and the TAK-242+APAP 24-hour and 48-hour groups had a significantly lower protein expression level of PCNA than the APAP group at the same time point (all P < 0.05); the TAK-242+APAP 24-hour and 72-hour groups had a significantly lower mRNA expression level of Ki-67 than the APAP group at the same time point (all P < 0.05), and the TAK-242+APAP 24-hour, 48-hour, and 72-hour groups had a significantly lower protein expression level of Ki-67 than the APAP group at the same time point (all P < 0.05). In addition, the TAK-242+APAP 24-hour and 48-hour groups had a significantly lower phosphorylation level of STAT3 than the APAP group at the same time point (both P < 0.05). Conclusion TLR4 may promote liver regeneration by increasing the phosphorylation level of STAT3 during APAP-induced liver injury in mice.

Objective : To used Toll⁃like receptor 4( TLR4) inhibitor ( TAK⁃242) investigate the role of TLR4 in the early stage of acetaminophen (APAP) Ⅳinduced acute liver injury in mice. Methods : Fifty⁃eight male institute of cancer research( ICR) mice were randomly divided into control group ( normal control group , solvent control group , inhibitor control group) and experimental group (APAP 4 ⁃ hour group , APAP + TAK⁃242 4 ⁃ hour group , APAP 12 ⁃ hour group , APAP + TAK⁃242 12 ⁃ hour group) . Mice in the experimental group were given a single dose of APAP (300 mg/kg) and TAK⁃242 (3 mg/kg) were given intraperitoneal injection 3 ⁃ hour before APAP injection. The serum alanine aminotransferase (ALT) level and liver index of mice in each group were compared ; HE staining showed pathological changes of liver tissue;the level of high mobility group box⁃1 (HMGB1) was determined by immunohistochemistry;the levels of TLR4 , HMGB1 and nuclear factor kappa B(NF⁃κB) protein were detected by Western blot;the levels of monocyte chemoattractant protein⁃1( MCP⁃1) , interleukin( IL) Ⅳ1β , tumor necrosis factor⁃α ( TNF⁃α ) and IL⁃6 genes were determined by real⁃time fluorescence quantitative PCR ( RT⁃qPCR) . The content of IL⁃6 in liver tissue was determined by enzyme linked immunosorbent assay (ELISA) . Results : HE staining showed liver tissue of mice obvious swelling and congestion of in APAP group at 4 ⁃hour and typical lobular central necrosis in APAP group at 12 ⁃ hour;the degree of liver necrosis in APAP + TAK⁃242 groups at 4 ⁃ hour and 12⁃ hour was less than that in APAP group at the same time point. Compared with the normal control group , serum ALT level , liver index , TLR4 , HMGB1 , NF⁃κB protein content , MCP⁃1 , IL⁃1β , TNF⁃α , IL⁃6 gene levels and IL⁃6 content in liver tissue of APAP 4 ⁃ hour group increased ; serum ALT level , liver index , HMGB1 protein content and IL⁃6 content in liver tissue increased in APAP 12 ⁃ hour group. Compared with APAP 4 ⁃ hour group , serum ALT level , liver index , TLR4 , HMGB1 , NF⁃κB protein content , MCP⁃1 , IL⁃1β , TNF⁃α , IL⁃6 gene level and IL⁃6 content in liver tissue decreased in APAP + TAK⁃242 4 ⁃ hour group. Compared with APAP 12 ⁃hour group , the levels of TLR4 , HMGB1 protein and MCP⁃1 , IL⁃1β , TNF⁃α genes in APAP + TAK⁃242 12 ⁃ hour group decreased. Conclusion nhibition of TLR4 may inhibit TLR4/HMGB1 pathway to reduce the inflammatory response in the early stage of acetaminophen⁃induced acute liver injury in mice.

OBJECTIVE: To assess the value of using flat-sided culture tubes for preparing chromosomes through chorionic villi (CV) and amniotic fluid (AF) cell cultures during prenatal diagnosis. METHODS: From February to March 2020, 157 CV samples and 147 AF samples subjected to prenatal diagnosis at the Maternal and Child Health Care Hospital of Guangxi Zhuang Autonomous Region were selected as the study subjects. For each sample, one flat-sided tube and one flask culture were set up by following the standard protocols. The methods were evaluated by comparing the cell growth, experimental process, quality of chromosome preparation and costs. RESULTS: The success rates for the culturing of CV and AF samples by the flat-sided culture tube method were 97.45% (153/157) and 97.96% (144/147), respectively. By contrast, the success rates for the conventional flask method were 98.72% (155/157) for CV and 98.64% (145/147) for AF samples. No significant difference was found between the two methods (P > 0.05). The average harvest time required by the flat-sided culture tube method was 8.45 days for CV and 9.43 days for AF cultures, whilst the average harvest time for conventional flask method was 9.05 days and 9.54 days, respectively. The flat-sided culture tube method for CV had required significantly shorter average harvest time than the conventional method (P < 0.001). No statistical significant difference was found in the average harvest time for AF by the two methods (P > 0.05). The conventional culturing method had required three containers with two sample transfers. By contrast, the flat-sided culture tube method was carried out in one tube without any sample transfer. The average total amount of medium used was 3.91 mL for each flat-sided culture tube and 6.26 mL for each conventional flask. CONCLUSION The flat-sided culture tube method can provide a simple, cost-effective and error-reducing procedure for the CV and AF samples culture during prenatal diagnosis.
Child ; Female ; Pregnancy ; Humans ; China ; Prenatal Diagnosis ; Chorionic Villi Sampling ; Amniotic Fluid ; Cell Proliferation