BACKGROUND:Human periodontal ligament stem cells are potential functional cells for periodontal tissue engineering.However,long-term in vitro culture may lead to reduced stemness and replicative senescence of periodontal ligament stem cells,which may impair the therapeutic effect of human periodontal ligament stem cells. OBJECTIVE:To investigate the effect of oxymatrine on the stemness maintenance and osteogenic differentiation of periodontal ligament stem cells in vitro,and to explore the potential mechanism. METHODS:Periodontal ligament stem cells were isolated from human periodontal ligament tissues by tissue explant enzyme digestion and cultured.The surface markers of mesenchymal cells were identified by flow cytometry.Periodontal ligament stem cells were incubated with 0,2.5,5,and 10 μg/mL oxymatrine.The effect of oxymatrine on the proliferation activity of periodontal ligament stem cells was detected by CCK8 assay.The appropriate drug concentration for subsequent experiments was screened.Western blot assay was used to detect the expression of stem cell non-specific proteins SOX2 and OCT4 in periodontal ligament stem cells.qRT-PCR and western blot assay were used to detect the expression levels of related osteogenic genes and proteins in periodontal ligament stem cells. RESULTS AND CONCLUSION:(1)The results of CCK8 assay showed that 2.5 μg/mL oxymatrine significantly enhanced the proliferative activity of periodontal stem cells,and the subsequent experiment selected 2.5 μg/mL oxymatrine to intervene.(2)Compared with the blank control group,the protein expression level of SOX2,a stem marker of periodontal ligament stem cells in the oxymatrine group did not change significantly(P>0.05),and the expression of OCT4 was significantly up-regulated(P<0.05).(3)Compared with the osteogenic induction group,the osteogenic genes ALP,RUNX2 mRNA expression and their osteogenic associated protein ALP protein expression of periodontal ligament stem cells were significantly down-regulated in the oxymatrine+osteogenic induction group(P<0.05).(4)The oxymatrine up-regulated the expression of stemness markers of periodontal ligament stem cells and inhibited the bone differentiation of periodontal ligament stem cells,and the results of high-throughput sequencing showed that it may be associated with WNT2,WNT16,COMP,and BMP6.

Objective To use bioinformatics analysis to identify the key genes and signaling pathways involved in cardiac dysfunction after acute ischemic stroke.Methods The GSE102558 dataset was downloaded from the Gene Expression Omnibus(GEO)database,and genes with values of P<0.05 and|log2FC|>0.6 were identified as being differentially expressed.The MCODE plugin in Cytoscape software performed a functional module analysis of the protein-protein interaction(PPI)network,while the CytoHubba plugin screened for core genes.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analyses were also performed.A middle cerebral artery occlusion model was constructed to verify core gene expression using real-time PCR.Results Among the screened differential genes,385 were upregulated and 354 were downregulated.The top ten core genes were Col1a1,Col1a2,Col3a1,Fbn1,Postn,Col5a1,Mmp3,Eln,Acta2,and Timp3.The GO enrichment mainly involved the extracellular matrix,the collagen fiber tissue,vascular development,and protease binding.KEGG was mainly enriched in protein digestion and absorption,the relaxin pathway,advanced gly-cation end products-receptor for advanced glycation end products,and platelet activation.Real-time PCR verified that Col3a1and Postn expressions decreased in the heart tissue after acute ischemic stroke.Conclusion Col3a1and Postnexpressions may be closely associ-ated with cardiac dysfunction occurrence and development after acute ischemic stroke.

Objective:A recombinant adenoviral vector vaccine based on non-replicating human adenovirus type 5 (Ad5), encoding the conserved domain of respiratory syncytial virus G protein (RSV-G) was constructed. The immunogenicity and protective efficacy of this vaccine were subsequently evaluated in mice.Methods:The recombinant Ad5 vector plasmid (Ad5-Gbcc-Gacc) was constructed by inserted conserved domains of RSV A and RSV B. The recombinant adenovirus Ad5-Gbcc-Gacc was rescued in HEK293A cells. The genome of virus Ad5-Gbcc-Gacc was identified by multi-enzyme digestion, and the expression of Ad5-Gbcc-Gacc was verified by Western blot. Recombinant adenovirus was used to immunize BALB/c mice via intramuscular injection with signal dose, and then challenged with RSV Long strain at week 6. The levels of G specific IgG and antibody subtypes in serum were detected by enzyme-linked immunosorbent assay, the level of neutralizing antibodies was determined by micro-neutralization assay. After challenge, the mice′s weight was recorded daily, the copies of RSV virus in the lung and nasal tissues were detected. Pathological changes in lung tissue were also examined.Results:Western blot and multi-enzyme digestion identification confirmed the successful rescue of the recombinant adenovirus. Ad5-Gbcc-Gacc elicit high titers of specific IgG, robust neutralizing antibodies, and a balanced Th1/Th2 immune response in mice. In comparison to unimmunized controls, mice immunized with Ad5-Gbcc-Gacc reduced the viral copies in both lung and nasal tissue, and exhibited only minimal pathological damage of lung tissue following RSV challenge. In conclusion, Ad5-Gbcc-Gacc induced robust immunogenicity and offers protective effects against RSV infection in murine models.Conclusions:Ad5-Gbcc-Gacc induce robust immunogenicity and can protect mice from RSV challenge, which lays a foundation for further development of RSV vaccine based on G protein.

Objective To explore the application value of modified prophylactic ileostomy in natural orifice specimen extraction surgery(NOSES)for patients with mid-low rectal cancer.Methods We retrospectively analyzed 63 patients who received prophylactic ileostomy in NOSES for mid-low rectal cancer in our hospital from September 2017 to May 2023.The patients were divided into the observation group(those who received modified ileostomy,n=31)and the control group(those who received conventional loop ileostomy,n=32)according to different ostomy methods.The operation time of ostomy,operation time of ostomy reversal surgery,early-stage complications(stoma leakage,peristomal dermatitis,stoma pain,peristomal trocar hole infection,stoma bleeding,stoma ischaemic necrosis,stoma oedema,peristoma skin-mucosal separation and stoma proximal bowel obstruction)and long-stage complications(stoma stenosis,stoma retraction,stoma prolapse,parastomal hernia),tumor recurrence and death of the two groups were compared and analyzed.Results Both prophylactic ileostomy and ostomy reversal surgery were successfully completed in all the 63 cases.The operation time of ostomy in the observation group was 7(6-8)min,which was significantly shorter than that of 23(21-24)min in the control group(Z=-6.853,P=0.000),and the operation time of ostomy reversal surgery in the observation group was(63.2±5.7)min,which was significantly shorter than(93.5±4.7)min in the control group(t=-23.109,P=0.000).Neither stoma bleeding nor stoma ischaemic necrosis were observed in both groups.The incidence of stoma pain in the observation group was lower than that in the control group[6.4％(2/31)vs.65.6％(21/32),x2=21.766,P=0.000].The incidence of peristomal incision infection in the observation group was lower than that in the control group[0％(0/31)vs.53.1％(17/32),P=0.000].There was no stoma stenosis in both groups.There were 3 cases of parastomal hernia,1 case in the observation group and 2 cases in the control group,the difference of the incidence being not statistically significant(P=1.000).There was 1 case of stoma retraction and 1 case of stoma prolapse in the control group.All the 5 cases with complications received prompt treatment in the second ostomy reversal surgery.Follow-up visits for 6-60 months in the 63 cases showed no tumor recurrence or death.Conclusion Modified prophylactic ileostomy in NOSES for patients with mid-low rectal cancer is safe,feasible,and easy to operate,having certain practicality and promotion value.

Objective:To establish a rapid quantitative PCR (qPCR) technique for Mycobacterium marinum skin infections, and to analyze its clinical diagnostic efficiency. Methods:DNA was extracted from Mycobacterium marinum colonies and serially diluted (10 -1 to 10 -8). Twelve pairs of previously reported primers and probes, as well as 6 pairs of newly designed primers and probes in this study, were used for qPCR amplification to identify the most sensitive primers and probes for the detection of Mycobacterium marinum. Skin lesion tissues were collected from 72 patients with confirmed Mycobacterium marinum infections (experimental group) and 68 with other mycobacterial infections (control group) at Shandong Provincial Hospital for Skin Diseases & Shandong Provincial Institute of Dermatology and Venereology, Shandong First Medical University & Shandong Academy of Medical Sciences in 2021. These skin tissues were subjected to qPCR amplification, interferon-gamma release assay (IGRA), acid-fast staining, and tissue culture to evaluate the diagnostic efficacy. Results:The newly designed primers and probes targeting the mycobacterial enhanced infection locus 2 (Mel2) demonstrated the highest sensitivity, with a detection limit of 0.86 copies/μl (cycle threshold value = 37) ; the qPCR amplification with the Mel2 primers/probes did not yield positive results when used for the detection of other mycobacteria (including Mycobacterium leprae and Staphylococcus spp) . Among the 72 patients in the experimental group, 44 were positive for qPCR with a sensitivity of 61.1% (95% CI: 49.6% - 71.5%), and 47 were positive for culture with a sensitivity of 65.2% (95% CI: 53.8% - 75.3%) ; all the 68 controls were negative for both qPCR and culture, with their specificities both being 100%. Among 65 patients subjected to IGRA, 31 were positive with a sensitivity of 47.7% (95% CI: 36.0% - 59.6%), while 16 out of 25 controls were negative for IGRA with a specificity of 64.0% (95% CI: 44.5% - 79.8%). Among 58 patients subjected to acid-fast staining, 37 were positive with a sensitivity of 63.8% (95% CI: 50.9% - 74.9%), and 52 out of 66 controls were negative for acid-fast staining with a specificity of 78.8% (95% CI: 67.5% - 86.9%). The combination of qPCR and culture resulted in a sensitivity of 93% and a specificity of 100% for the detection of Mycobacterium marinum. Conclusion:In this study, a highly sensitive qPCR assay was developed for the detection of Mycobacterium marinum, and its combination with culture could further improve the detection sensitivity.

Dynamic changes in gut dysbiosis and metabolomic dysregulation are associated with immune-complex glomerulonephritis(ICGN).However,an in-depth study on this topic is currently lacking.Herein,we report an ICGN model to address this gap.ICGN was induced via the intravenous injection of cationized bovine serum albumin(c-BSA)into Sprague-Dawley(SD)rats for two weeks,after which mycophenolate mofetil(MMF)and losartan were administered orally.Two and six weeks after ICGN establishment,fecal samples were collected and 16S ribosomal DNA(rDNA)sequencing and untargeted metabolomic were conducted.Fecal microbiota transplantation(FMT)was conducted to determine whether gut normali-zation caused by MMF and losartan contributed to their renal protective effects.A gradual decline in microbial diversity and richness was accompanied by a loss of renal function.Approximately 18 genera were found to have significantly different relative abundances between the early and later stages,and Marvinbryantia and Allobaculum were markedly upregulated in both stages.Untargeted metabolomics indicated that the tryptophan metabolism was enhanced in ICGN,characterized by the overproduction of indole and kynurenic acid,while the serotonin pathway was reduced.Administration of losartan and MMF ameliorated microbial dysbiosis and reduced the accumulation of indoxyl conjugates in feces.FMT using feces from animals administered MMF and losartan improved gut dysbiosis by decreasing the Firmicutes/Bacteroidetes(F/B)ratio but did not improve renal function.These findings indicate that ICGN induces serous gut dysbiosis,wherein an altered tryptophan metabolism may contribute to its pro-gression.MMF and losartan significantly reversed the gut microbial and metabolomic dysbiosis,which partially contributed to their renoprotective effects.

BACKGROUND:Studies have found that glucagon-like peptide-1 and its analogues have a significant neuroprotective effect,and some drugs have been applied to the clinical stage Ⅲ study of Alzheimer's disease.However,the mechanism of its neuroprotective effect is still unclear,which needs to be further explored and clarified. OBJECTIVE:To screen out the genes related to the pathogenesis of Alzheimer's disease and the related targets of semaglutide for the treatment of Alzheimer's disease based on bioinformatics and network pharmacology analyses,to identify the potential target genes by comprehensive analysis of the two and to verify them at the cellular level. METHODS:Using DisGeNET database,differentially expressed genes between Alzheimer's disease patients and healthy population were screened out.The chemical structure formula and two-dimensional structure diagram of semaglutide were obtained using PubChem online database.GO/KEGG enrichment analysis was performed using DAVID online database.A protein-protein interaction network was constructed by using the STRING database.The HPA database was used to determine the distribution characteristics of the target proteins in various human tissues.Finally,western blot was used to detect relevant protein expression in HT22 cells after semaglutide intervention. RESULTS AND CONCLUSION:With the dataset in DisGeNET database,3 374 differentially expressed genes between Alzheimer's disease patients and healthy people were obtained,and meanwhile,101 target genes of semaglutide potential drugs were obtained.There were 23 intersection genes between them.Ten key genes were identified based on the protein-protein interaction network,which were silent information regulator 1(SIRT1),CASP9,CCND1,CASP1,KEAP1,DLG4,CASP4,GRB2,GRIA1,and EDNRA.The results of GO gene functional annotation analysis of key genes showed that the positive regulatory activity of cysteine endopeptidase,the positive regulation of proteolysis,and the positive regulation of cysteine endopeptidase involved the cytoplasmic part of the apoptotic activity process;AMPA glutamate receptor complex,inflammatory complex,CARD domain binding,cysteine endopeptidase activity,and cysteine endopeptidase activity were involved in the apoptotic process.The results of KEGG signaling pathway analysis indicated that colorectal cancer,non-small cell carcinoma,and endometrial carcinoma were related to immune infiltration,inflammation and autophagic apoptosis.In addition,according to the association ranking of key genes and their distribution in different tissues of HPA online database,SIRT1 was identified as the most significant differential gene.The expression level of SIRT1 protein was significantly down-regulated in HT22 cells after β-amyloid protein 1-42 treatment,but it could be significantly increased after being treated with semaglutide.To conclude,SIRT1 may be a target gene for semaglutide in the treatment of Alzheimer's disease.

BACKGROUND:With the rapid development of minimally invasive spinal surgery and enhanced recovery after surgery,endoscopic intervertebral fusion techniques have gradually emerged and been widely used in clinical practice in recent years. OBJECTIVE:To analyze the early clinical efficacy of uniaxial spinal endoscopic intervertebral fusion combined with posterior percutaneous pedicle screw fixation in the treatment of lumbar degenerative diseases. METHODS:135 patients with lumbar degenerative diseases treated by uniaxial spinal endoscopic intervertebral fusion combined with posterior percutaneous pedicle screw fixation in the Suining Central Hospital from October 2020 to December 2021 were enrolled in this study.There were 59 males and 76 females,aged 47-79 years.The lower limb and lumbar pain was evaluated by visual analog scale and lumbar function was assessed by Oswestry disability index before the operation,1 week,1,and 6 months after the operation,and at the end of follow-up.The overall pain recovery of patients was evaluated by the scoring criteria for low back pain surgery of Spine Group of Chinese Orthopedic Association and the lumbar physiological curvature and intervertebral fusion were evaluated on lumbar lateral X-ray preoperatively and at the end of follow-up. RESULTS AND CONCLUSION:(1)The 135 patients were followed up for(17.8±3.0)months after surgery.There was 1 case of endplate injury,1 case of cerebrospinal fluid leakage,1 case of nerve root injury,1 case of intervertebral cage subsidence and displacement,1 case of chronic infection,and 1 case of pedicle screw rupture.The complication rate was 5.2%.(2)The lumbar visual analog scale score and Oswestry disability index significantly decreased in the waist and lower limbs at various time points postoperatively compared with those preoperatively in 135 patients(P<0.05).The scoring criteria for low back pain surgery of the Spine Group of the Chinese Orthopedic Association were significantly better at the last follow-up than that preoperatively in 135 patients(P<0.05).(3)At the last follow-up,there was no significant difference in physiological curvature of lumbar vertebra as compared with that preoperatively in 135 patients(P>0.05),with a fusion rate of 95.8%.(4)It is concluded that uniaxial spinal endoscopic intervertebral fusion combined with posterior percutaneous pedicle screw fixation in the treatment of lumbar degenerative diseases has shown satisfactory early clinical results and is a highly safe minimally invasive spinal surgery mode.

AIM:To observe the anti-scarring effects and safety of triamcinolone acetonide(TA)-loaded hydrogel sustained-release sheeting on stab incision glaucoma surgery(SIGS)with “one-step tunnel method” in rabbit eyes.METHODS:A total of 48 healthy New Zealand white rabbits were randomly selected and divided into 4 groups(12 rabbits in each group), trabeculectomy(Trab)group, SIGS group, polyvinyl alcohol hydrogel(PVAH)sheeting was implanted under the conjunctiva flap during SIGS(PVAH group), and hydrogel sustained-release sheeting loaded with TA was implanted under the conjunctiva flap during SIGS(TA/PVAH group). On the 1, 2, 3, and 4 wk after surgery, the intraocular pressure, filtering bubble morphology, anterior chamber reaction, and other complications were observed and recorded in each group. Then animals were euthanized, and the surgery area tissues of right eye were taken for pathological tissue paraffin section. Masson staining, picric acid-Sirius rose red staining, as well as α-smooth muscle actin(α-SMA)and fibroblast growth factor 2(FGF2)immunohistochemistry staining was performed on every section. The infiltration of inflammatory cells, proliferation of fibroblasts and synthesis of type I and type III collagen fibers in local tissues were observed. The average positive area ratio of α-SMA and FGF2 antibody immunohistochemical staining in each group was calculated and compared.RESULTS: The TA/PVAH group maintained diffuse and elevated functional filtering blebs, while flat filtering blebs appeared in Trab, SIGS and PVAH groups at 2 wk after surgery. Functional filtering blebs were present in 1 eye(33%), 2 eyes(67%)in the PVAH and TA/PVAH group at 4 wk after surgery, respectively, while the other filtering blebs were flattened. Masson staining showed that the hydrogels in PVAH and TA/PVAH groups did not degrade at 4 wk after surgery. Compared with the Trab and SIGS groups, the filtration passages were more obvious, with less collagen fiber proliferation. Sirius red staining showed that the expression of type I collagen and type III collagen in the TA/PVAH group was less than that in the Trab group, SIGS group and PVAH group at 4 wk after surgery. Immunohistochemical staining showed that the α-SMA expression in the TA/PVAH group was significantly lower than that in the Trab and SIGS groups at 1 wk after surgery(P<0.01). The α-SMA expression was the highest in the Trab and SIGS groups at 2 wk after surgery, while the α-SMA expression in the PHAP and TA/PVAH groups was significantly lower than that in the first two groups(P<0.01). Compared with the Trab group, the expression of FGF2 in the PVAH and TA/PVAH group was significantly increased at 1, 2, 3 and 4 wk after surgery(P<0.05). Compared with the SIGS group, FGF2 expression in the TA/PVAH group was significantly increased at 4 wk after surgery(P<0.05).CONCLUSION:In SIGS surgery of rabbit eyes, implanting hydrogel sustained-release sheeting loaded with TA under conjunctival flap can effectively inhibit the scarring of the filtering bleb, which may be the interaction of the anti-scar effect of TA and the stent function of hydrogel.

Objective:To investigate the relationship between thyroid function abnormality-immune related adverse event (TFA-irAE) and treatment efficacy and survival in advanced cancer patients treated with programmed death-1 (PD-1) inhibitors.Methods:The clinical data of 90 patients with advanced cancer who received 6 cycles of PD-1 inhibitor treatment from January 2021 to June 2022 in Department of Oncology of Yuncheng Central Hospital Affiliated to Shanxi Medical University were collected. Serum levels of thyroid stimulating hormone (TSH), free thyroxine (FT 4), thyroid peroxidase antibody (TPOAb), and thyroglobulin antibody (TGAb) were measured by chemiluminescence immunoassay in patients after PD-1 inhibitor treatment, and the incidence of TFA-irAE was observed in the patients after 6 cycles of therapy. According to the occurrence of TFA-irAE, the patients were divided into TFA-irAE occurrence group ( n=40) and TFA-irAE non-occurrence group ( n=50), the therapeutic efficacy and survival of the two groups were calculated and compared. The thyroid function indexes of patients with different efficacy (33 cases in effective group and 57 cases in ineffective group) and patients with different prognosis (30 cases in survival group, 60 cases in death group) were compared, and the influencing factors of efficacy and survival were analyzed by multivariate logistic regression and Cox analysis. Kaplan-Meier survival curve was drawn, and the survival of TFA-irAE occurrence group and TFA-irAE non-occurrence group were compared by log-rank test. Results:One year after treatment, the treatment effective rate of TFA-irAE occurrence group and TFA-irAE non-occurrence group were 42.5% (17/40), 32.0% (16/50), respectively, with no statistically significant difference ( χ2=1.06, P=0.304). After 6 cycles of PD-1 inhibitor treatment, serum levels of TSH [ (2.56±0.41) mU/ml vs. (3.11±0.53) mU/ml], TPOAb [ (56.78±5.72) U/ml vs. (62.67±6.31) U/ml] and TGAb [ (81.57±8.23) U/ml vs. (92.34±9.31) U/ml] in the effective group were significantly lower than those in the ineffective group, with statistically significant differences ( t=4.45, P<0.001; t=3.89, P<0.001; t=5.29, P<0.001). The serum levels of TSH [ (2.69±0.46) mU/ml vs. (3.06±0.65) mU/ml], FT 4 [ (10.45±1.13) pmol/L vs. (11.50±1.36) pmol/L], TPOAb [ (56.27±5.61) U/ml vs. (62.47±6.34) U/ml] and TGAb [ (81.62±8.31) U/ml vs. (91.73±9.35) U/ml] in the survival group were significantly lower than those in the death group, with statistically significant differences ( t=2.27, P=0.025; t=3.02, P=0.003; t=3.79, P<0.001; t=4.19, P<0.001). Multivariate logistic regression analysis showed that TSH ( OR=1.52, 95% CI: 1.13-2.05, P=0.006), TPOAb ( OR=1.42, 95% CI: 1.13-1.78, P=0.002) and TGAb ( OR=1.35, 95% CI: 1.05-1.73, P=0.018) were all independent factors affecting the efficacy of patients with advanced cancer treated with PD-1 inhibitors. Multivariate Cox regression analysis showed that TSH ( HR=1.42, 95% CI: 1.06-1.92, P=0.030), TPOAb ( HR=1.31, 95% CI: 1.05-1.64, P=0.018), TGAb ( HR=1.41, 95% CI: 1.09-1.83, P=0.008) and FT 4 ( HR=1.36, 95% CI: 1.02-1.81, P=0.038) were all independent factors affecting the survival of patients with advanced cancer treated with PD-1 inhibitors. Survival analysis showed that the median overall survival in the TFA-irAE occurrence group and the TFA-irAE non-occurrence group were 10.8 and 8.0 months, respectively, with a statistically significant difference ( χ2=9.53, P=0.002) . Conclusion:Although the occurrence of TFA-irAE may have less effect on the efficacy of advanced tumor patients treated with PD-1 inhibitors, it may affect the survival of patients. TSH, TPOAb and TGAb are all independent influencing factors for the efficacy of patients with advanced tumors treated with PD-1 inhibitors, while TSH, TPOAb, TGAb and FT 4 are independent influencing factors for the survival of patients with advanced tumors treated with PD-1 inhibitors.