Objective:To discover a novel and intuitive anterograde tracing method for observing the course of sensory nerve fibers in dorsal root ganglia (DRG).Methods:From July 2022 to August 2023, studies were conducted in the Department of Plastic Surgery of the First Affiliated Hospital of Sun Yat-sen University. Three healthy 1-month old female SD rats, weighed approximately 180-220 g, were randomly selected. An adeno-associated virus carrying a neuron-specific promoter and red fluorescent protein (AAV- syp-mCherry) was prepared. The virus was injected into the spinal roots outside L 4-L 6 DRG through sterile surgery. After 1 month of breeding, L 4-L 6 spinal cord tissue of immunofluorescent staining of the side injected with viral tracer, DRG, sciatic nerve, and plantar skin of the SD rats were collected for immunofluorescent staining of neurofilament protein (NF) and microtubule associated protein 2 (MAP2). Distribution characteristics of AAV-positive nerve fibers were observed using a confocal microscope by average optical density (AOD) . Results:One month after injection of the viral tracer, red markers were visible in the dorsal horn of the L 4-L 6 spinal cord (AOD=0.5419±0.0245), DRG (AOD=0.4917±0.0087), sciatic nerve (AOD=0.6237±0.0634), dermis of plantar skin and skin appendages on the injected side (AOD=0.4943±0.0165). The red fluorescent clusters were found co-labeled with NF-positive nerve fibers or MAP2-positive neurons, it suggested that AAV- syp-mCherry could be transported anterogradely within sensory axons to distant target organs (skin) and offered a long-term image. Conclusion:The anterograde tracing method by adeno-associated virus can effectively show the complete trace of the central and peripheral processes of primary sensory neurons, and can serve as an effective methodology in observation of the pathways of peripheral sensory nerve fibers.

Objective To explore the mechanism of curcumin in enhancing gemcitabine sensitivity by regulating pancreatic cancer cells.Methods In order to verify the effect of curcumin on p53 and NF-κB p65 nuclear translocation in pancreatic cancer cell PANC1,the pancreatic cancer PANC1 cells were treated with 0,10,20,30 μmol/L curcumin to obtain the blank group,10 μmol/L curcumin group,20 μmol/L curcumin group and 30 μmol/L curcumin group.The intracellular distribution of NF-κB p65 was determined by immunofluo-rescence staining under fluorescence microscope.The expression level of p53 protein in cells was determined by Western blot.The expression level of p53 mRNA was determined by reverse transcription-real-time fluo-rescence quantitative PCR.The combined use of miRstar and JASPAR softwares predicted to seek microRNA (miRNA) potentially regulated by NF-κB p65 nuclear translocation.The double luciferase reporting assay was used to respectively verify the relationship between miRNA and p53.In order to verify whether curcumin in-fect the sensitivity of PANC1 cell through NF-κB p65/miR-266-5p/p53 axis,the gemcitabine group was ob-tained by 10 μmol/L gemcitabine treating cells,and the curcumin combined gemcitabine group was obtained by 20 μmol/L curcumin and 10 μmol/L gemcitabine treating cells.After the cells were treated with curcumin (20 μmol/L) and gemcitabine (10 μmol/L),oligonucleotides mimic NC and miR-26b-5p mimic were trans-fected into cells,and the mimic NC group and miR-26b-5p group were obtained.pcDNA3.1 no-load plasmid and pcDNA3.1-p53 overexpression plasmid were transfected into cells,respectively,and the pcDNA3.1 group and p53 group were obtained.The cell activity was detected by MTT assay.The cellular apoptosis level was determined by Annexin V/PI double staining.Results Compared with the blank group,the intracellular p53 mRNA and protein expression levels in the various curcumin treated groups (10,20,30 μmol/L curcumin groups) all were increased.Compared with the blank group,the expression level of NF-κB p65 in the nucleus of 20μmol/L curcumin group was decreased.The miRstar and JASPAR prediction software found 8 miRNA that may be regulated by NF-κB p65 nuclear translocation,in which 3 miRNA had potential to target p53 genes,especially miR-26b-5p effect was most significant.The double luciferase report experiment confirmed that miR-26b-5p did interact with p53.Compared with the mimic NC group,the expression levels of p53 mR-NA and protein in the mimic miR-26b-5p group were decreased.Compared with the gemcitabine group,the cell viability in the curcumin combined gemcitabine group was decreased and the apoptosis rate was increased.Compared with the mimic NC group,the cell activity in the mimic miR-26b-5p group was increased and the apoptosis level was decreased.Moreover compared with the pcDNA3.1 group,the cell activity in the pcDNA3.1-p53 group was decreased and the apoptosis level was increased.Conclusion Curcumin reduces the expression of miR-26b-5p gene by inhibiting the nuclear translocation of NF-κB p65 protein,and then increase the p53 gene and protein expression,and enhance the sensitivity of pancreatic cancer cells to gemcitabine by the NF-κB p65/miR-26b-5p/p53 axis.

Objective:To discover a novel and intuitive anterograde tracing method for observing the course of sensory nerve fibers in dorsal root ganglia (DRG).Methods:From July 2022 to August 2023, studies were conducted in the Department of Plastic Surgery of the First Affiliated Hospital of Sun Yat-sen University. Three healthy 1-month old female SD rats, weighed approximately 180-220 g, were randomly selected. An adeno-associated virus carrying a neuron-specific promoter and red fluorescent protein (AAV- syp-mCherry) was prepared. The virus was injected into the spinal roots outside L 4-L 6 DRG through sterile surgery. After 1 month of breeding, L 4-L 6 spinal cord tissue of immunofluorescent staining of the side injected with viral tracer, DRG, sciatic nerve, and plantar skin of the SD rats were collected for immunofluorescent staining of neurofilament protein (NF) and microtubule associated protein 2 (MAP2). Distribution characteristics of AAV-positive nerve fibers were observed using a confocal microscope by average optical density (AOD) . Results:One month after injection of the viral tracer, red markers were visible in the dorsal horn of the L 4-L 6 spinal cord (AOD=0.5419±0.0245), DRG (AOD=0.4917±0.0087), sciatic nerve (AOD=0.6237±0.0634), dermis of plantar skin and skin appendages on the injected side (AOD=0.4943±0.0165). The red fluorescent clusters were found co-labeled with NF-positive nerve fibers or MAP2-positive neurons, it suggested that AAV- syp-mCherry could be transported anterogradely within sensory axons to distant target organs (skin) and offered a long-term image. Conclusion:The anterograde tracing method by adeno-associated virus can effectively show the complete trace of the central and peripheral processes of primary sensory neurons, and can serve as an effective methodology in observation of the pathways of peripheral sensory nerve fibers.

Objective:To investigate the role of IL-17A in the regulation of inflammatory factors and autophagy genes of PBMCs in pSS patients.Methods:Thirty patients fulfilled the diagnosis of pSS were selected, 20 mL of peripheral blood was drawn, PBMCs were isolated and divided into the PBMCs group, IL-17A stimulant group and IL-17A inhibitor group. After warm incubation 48 h of immunofluorescence was applied to detect microtubule-associated protein l light chain 3 (LC3), and the ELISA method was used to detect the expression of the inflammatory factors IL-4, IFN-γ, IL-13 expression. Real-time fluorescence polymerase chain reaction (qRT-PCR) was used to detect the expression of autophagy-inducing genes Ambra-1, Bif-1 and apoptosis genes Bcl-2 and Bcl-XL mRNA, and immunoprotein blotting was used to detect the expression of Beclin1 and LC3 protein. ANOVA was used to compare the differences between groups, and t-test was used for two-by-two comparisons. Results:The immunofluorescence results showed a significant increase in LC3 autophagic vesicles in the IL-17A inhibited group compared with the IL-17A stimulator group. The ELISA results showed that, compared with the PBMCs group [IL-4: (13.39±0.32) pg/ml, IFN-γ: (14.4±0.4) pg/ml, and IL-13: (854±36) pg/ml], IL-4 secretion in the IL-17A stimulated group (11.54±0.30) was decreased ( t=12.83, P=0.024), IFN-γ and IL-13 secretion [(17.6±0.4), (908±51) pg/ml] were increased ( t=19.35, P=0.033; t=2.55, P=0.020); compared with IL-17A inhibitor group [IL-4: (15.65±0.26) pg/ml, IFN-γ: (13.6±0.3) pg/ml, and IL-13: (792±57) pg/ml]. Compared with the IL-17A stimulator group, IL-4 secretion was decreased ( t=21.31, P=0.006), and IFN-γ and IL-13 expression was increased ( t=17.34, P=0.015; t=5.14, P=0.007). The PCR results showed that, compared with Ambra-1, Bif-1, Bcl-2, and Bcl-XL mRNA expression (5.61±0.33, 5.04±0.60, 1.28±0.09, 1.56±0.03) in the PBMCs group, Ambra-1, Bif-1 mRNA in the IL-17A-stimulated group expression (3.76±0.24, 4.68±0.41) were down-regulated ( t=14.30, P=0.007; t=15.02, P=0.012), and Ambra-1, Bif-1 mRNA expression (7.91±1.17, 9.30±0.25) were increased in the IL-17A inhibition group, ( t=13.59, P=0.025; t=11.54, P=0.031), anti-apoptotic proteins Bcl-2, Bcl-XL mRNA expression (1.75±0.06, 2.43±0.16) was up-regulated in IL-17A stimulated group ( t=19.92, P=0.006; t=21.04, P=0.007) were up-regulated and Bcl-2, Bcl-XL mRNA expression (0.48±0.03, 0.83±0.10) were down-regulated in the IL-17A inhibition group ( t=29.44, P=0.027; t=16.31, P=0.023). The results of protein blotting assay showed that, Beclin-1 and LC3 protein expression (0.51±0.10, 0.559±0.010) were decreased in IL-17A stimulated group compared with Beclin-1, LC3 protein expression (0.72±0.09, 0.635±0.017) in PBMCs group ( t=14.38, P=0.034; t=17.99, P=0.014); BecLin-1 and LC3 protein expression (0.83±0.11, 0.737±0.025) increased in the IL-17A inhibition group ( t=9.72, P=0.027; t=22.35, P=0.007). Conclusion:IL-17A plays a role in pSS by regulating the expression of inflammatory factors IL-4, IFN-γ, IL-13 and autophagy related genes Beclin1 and LC3.

Objective : To investigate the changes of intestinal flora in patients with rheumatoid arthritis (RA) before and after treatment with technetium[ 99 Tc] methylenediphosphonate injection ( 99 Tc⁃MDP) . Methods : Thirty⁃two patients with RA were collected the fresh fecal specimens before and after they treated with 99 Tc⁃MDP. The genome DNA of fecal specimens was extracted and the V3 ⁃V4 regions of 16S rRNA were sequenced by Illumina Miseq platform. The sequencing results were analyzed by bioinformatics methodology. Results : The Chao , observed species , Shannon and Simpson index between the two groups in Alpha diversity analysis had no statistical difference (P > 0. 05 ) , but the value in post⁃treatment group were increased compared with the pre⁃treatment group. Compared with the pre⁃treatment of RA , there was no statistical difference in the higher proportion relative abundance on Phylum level of the intestinal microbes of RA patients , and the relative abundance of Tenericutes decreased ( P < 0. 05 ) . At the genus level , the relative abundance of Ruminococcaceae , Butyricicoccus , Christensenellaceae_R ⁃7_group , Holdemania decreased after treatment ( P < 0. 05 ) . And the relative abundances of Pasteurella , Lachnoanaerobaculum and Stomatobaculum increased (P < 0. 05) . Conclusion The abundance and species of gut microbiota in RA patients have some changes after treatment with 99Tc⁃MDP , and the diversity increases after treatment.

Objective:To compare the clinical efficacy and safety of ledipasvir/sofosbuvir (LDV/SOF) and elbasvir/grazoprevir (EBR/GZR) in treatment of patients with chronic hepatitis C (CHC).Methods:The clinical data of 143 patients with genotype 1b CHC treated in Huzhou Central Hospital from January 2020 to December 2021 were retrospectively analyzed, including 74 cases treated with LDV/SOF and 69 cases treated with EBR/GZR. The virological response after 4 and 12 weeks of treatment and 12wk after drug withdrawal was determined; and the serological and liver inflammation indexes before and after treatment in two groups were compared. SPSS 25.0 software was used for statistical analysis of the data.Results:The virological response rates of the LDV/SOF group and EBR/GZR group were 97.30% and 98.55%, 98.65% and 100.00%, 97.30% and 98.55% after 4 and 12 weeks of treatment and 12 weeks after the end of treatment, respectively (all P > 0.05). At the end of treatment, the liver inflammation indexes ALT, AST and GGT in the two groups were significantly lower than the baseline levels ( Z=-7.470 and -6.974, -9.757 and -6.832, -3.578 and -4.054, P<0.01). Adverse reactions in both groups were mild, and no serious adverse events occurred. Conclusion:Both LDV/SOF and EBR/GZR have good clinical efficacy in the treatment of genotype 1b CHC patients. And the patients are well tolerated.

Objective To investigate the clinical significance of heart fatty acid binding protein (H-FABP) and pregnancy associ-ated plasma protein A(PAPP-A) in acute coronary syndrome(ACS) .Methods A case-control study was conducted in 60 patients with ACS ,45 patients with stable angina pectoris (SAP) and50 patients without coronary heart diseases (control group) .All plasma samples were tested H-FABP and PAPP-A .Results Concentrations of H-FABP and PAPP-A were significantly different among the 3 groups(P<0 .01) .H-FABP and PAPP-A in ACS group were significant higher than those of SAP group and control group (P<0 .01) ,however there were no significant differences between SAP and control group (P>0 .05) .The sensitivity and specificity of H-FABP were 91 .7% and 78 .0% respectively analyzed by ROC curve .Similarly ,the sensitivity and specificity of PAPP-A were 48 .3% and 98 .0% respectively .The correlation of H-FABP and PAPP-A was high(r=0 .835 ,P<0 .01) according to the analysis by Pearson correlation analysis .Conclusion Concentrations of plasma H-FABP and PAPP-A had close relationship with ACS ,the sensitivity of H-FABP was much higher ,both of which could be the potential biomarkers and contributed to the diagnosis of exist-ence and progress of ACS .

Melkersson-Rosenthal syndrome (MRS) is an uncommon granulomatous disease characterized by the triad of relapsing facial paralysis, orofacial swelling, and fissured tongue. Genital swelling in MRS is rarely reported. We presented the first case of complete MRS with genital swelling in a child. Biopsy examinations of both the child's lower lip and penis showed noncaseating granuloma and intralymphatic granuloma infiltration. No symptoms or signs of other systemic disease (Crohn's disease or sarcoidosis) were observed after 2 years of follow-up. Genetic screening for CARD15/NOD2 in this patient showed negative, which further confirmed the diagnosis of MRS. Eleven other cases of suspected complete or incomplete MRS with genitalia involved were reviewed. Our case emphasizes the specific clinical feature of MRS with genitalia involved, which was genetically different from Crohn's disease and could be an independent entity. Lymphatic obstruction is responsible for localized edema in MRS.
Biopsy ; Child* ; Crohn Disease ; Diagnosis ; Edema ; Facial Paralysis ; Follow-Up Studies ; Genetic Testing ; Genitalia* ; Granuloma ; Humans ; Lip ; Lymphatic Vessels ; Male* ; Melkersson-Rosenthal Syndrome* ; Penis ; Tongue, Fissured

Melkersson-Rosenthal syndrome (MRS) is an uncommon granulomatous disease characterized by the triad of relapsing facial paralysis, orofacial swelling, and fissured tongue. Genital swelling in MRS is rarely reported. We presented the first case of complete MRS with genital swelling in a child. Biopsy examinations of both the child's lower lip and penis showed noncaseating granuloma and intralymphatic granuloma infiltration. No symptoms or signs of other systemic disease (Crohn's disease or sarcoidosis) were observed after 2 years of follow-up. Genetic screening for CARD15/NOD2 in this patient showed negative, which further confirmed the diagnosis of MRS. Eleven other cases of suspected complete or incomplete MRS with genitalia involved were reviewed. Our case emphasizes the specific clinical feature of MRS with genitalia involved, which was genetically different from Crohn's disease and could be an independent entity. Lymphatic obstruction is responsible for localized edema in MRS.
Biopsy ; Child* ; Crohn Disease ; Diagnosis ; Edema ; Facial Paralysis ; Follow-Up Studies ; Genetic Testing ; Genitalia* ; Granuloma ; Humans ; Lip ; Lymphatic Vessels ; Male* ; Melkersson-Rosenthal Syndrome* ; Penis ; Tongue, Fissured

Objective: To establish an enzyme linked immunosorbent assay(ELISA)for erythropoietin(EPO) in serum, and observe its clinical application value thereof. Methods: Prepare the EPO polyclonal antibody, wash the plate with isopropyl alcohol, and then choose the suitable concentration of the antibody, enzyme labeled antibody, and antigen. After the reaction, check the sensitivity, recovery, specificity and stability of the method. The serum samples of anaemia and breast carcinoma individuals were detected. The results of radioimmunodetection were compared with that of normal control group. Results: The immo-assay plate showed strong adherence to proteins. The optimal concentrations of the antibody, enzyme labelled antibody and antigen were 1∶1 000, 1∶6 000 and 1∶800 separately. The sensitivity was 0.46 U/L. The cross-reaction with growth hormone and ferritin was low. The mean recoveries of samples with high and low concentrations were 96.3%, 97.3% respectively. The coefficients of variation of intra-assay and inter-assay were just 8.31% and 7.82%, and the stability was good. The EPO levels were higher in anaemia and breast carcinoma groups than that of normal group. There was no significant difference between the results of the radioimmunodetection and ELISA. Conclusion: The double-antibody sandwich ELISA method was established for EPO in serum, which had certain clinical application value.