ObjectiveTo investigate the alleviating effect of calcium cyanamide （CaCN₂） soil fumigation on replanting problems of Rehmannia glutinosa. MethodsNewly soil （NP） was used as the control group， while three treatment groups were established： replanted soil （RP）， newly soil treated with CaCN₂ （120 g·m²， tillage depth 25 cm） （NPCC）， and replanted soil treated with CaCN₂ （RPCC）. R. glutinosa was cultivated in all groups. At harvest， the tuber agronomic traits （number of enlarged roots， maximum root diameter， fresh weight， dry weight） were measured. The content of catalpol and rehmannioside D was quantified by ultra-high-performance liquid chromatography （UPLC） to evaluate medicinal quality. Rhizosphere soil available nutrients and enzyme activities were analyzed by assay kits. The community structure and composition of fungi and bacteria in rhizosphere soil were assessed via internal transcribed spacer 2 （ITS2） sequencing and 16S rDNA sequencing， respectively. ResultsCompared with NP， the RP group showed obviously reduced in tuber agronomic traits and quality indicators （P0.05）. However， the RPCC group showed significant improvement in agronomic traits and a notable increase in rehmannioside D content compared to RP （P0.05）. The contents of available phosphorus and potassium in RPCC and NP groups were obviously lower than those in RP （P0.05）. The polyphenol oxidase soil （S-PPO） activity in RP was obviously lower than in NP （P0.05）， while sucrose soil （S-SC）， acid phosphatase soil （S-ACP）， and S-PPO activities in RPCC were obviously higher than in RP （P0.05）. Microbial richness and diversity in RP were obviously higher than in NP （P0.05）， whereas no significant differences were observed between the RPCC and NP. The relative abundances of fungal genera Nectria， Myrothecium， Tomentella， and bacterial genus Skermanella were obviousl lower in RPCC and NP than in RP （P0.05）. Correlation analysis that S-ACP activity was positively correlated with the content of rehmannioside D （P0.05）. Fungal genera Engyodontium and Alternaria， and bacterial genera Pir4 lineage， Pirellula， Methyloversatilis， Brevundimonas， Ralstonia， and Acidibacter were obviously positively correlated with tuber dry weight （P0.05）. Conversely， fungal genera Pseudaleuria， Nectria， Haematonectria， Ceratobasidium， and bacterial genera Streptomyces， Skermanella， RB41， Gemmatimonas， and Bacillus were obviously negatively correlated with dry weight （P0.05）. The fungal genus Alternaria and bacterial genera Brevundimonas， Ralstonia， Acidibacter， and Dongia showed positive correlations with medicinal quality of R.glutinosa tuber， while fungal genera Pseudaleuria， Nectria， Stachybotrys， Fusarium， Gibberella， Ceratobasidium， and bacterial genera Sphingomonas， Skermanella， RB41， Gemmatimonas， and Bacillus were obviously negatively correlated （P0.05）. ConclusionCaCN₂ soil fumigation can significantly improve enzyme activities in replanted Rehmannia rhizosphere soil， enhance the utilization of available nutrients， reshape microbial community structure of replanted R.glutinosa at the family and genus level， and notably improve tuber agronomic traits and medicinal quality. This study provides a novel approach to alleviating replanting problems and offers insights for the integrated development of standardized cultivation techniques， including soil disinfection， nutrient-targeted regulation， and microbial inoculant application.

OBJECTIVES: To investigate the mechanism mediating the regulatory effect of miR-155-5p on proliferation of human submandibular gland epithelial cells (HSGECs) in primary Sjogren's syndrome (pSS). METHODS: Dual luciferase reporter assay was used to verify the targeting relationship between miR-155-5p and the PI3K/AKT pathway. In a HSGEC model of pSS induced by simulation with TRAIL and INF-γ, the effects of miR-155-inhibitor-NC or miR-155 inhibitor on cell viability, cell cycle, apoptosis and proliferation were evaluated using CKK8 assay, flow cytometry and colony formation assay. ELISA and RT-PCR were used to detect the expressions of inflammatory cytokines and miR-155-5p mRNA in the cells; Western blotting was performed to detect the expressions of proteins in the PI3K/AKT signaling pathway. RESULTS: Dual luciferase assay showed that miR-155-5p targets the PI3K/AKT pathway via PIK3R1 mRNA. The HSGEC model of pSS showed significantly decreased cell viability, cell clone formation ability and expressions IL-10 and IL-4 and increased cell apoptosis, cell percentage in G2 phase, expressions of TNF‑α, IL-6, miR-155-5p and PIK3R1 mRNA, p-PI3K/PI3K ratio, p-Akt/AKT ratio, and PIK3R1 protein expression. Treatment of the cell models with miR-155 inhibitor significantly increased the cell viability, G1 phase cell percentage, colony formation ability, and expressions of IL-10 and IL-4 levels, and obviously reduced cell apoptosis rate, G2 phase cell percentage, expressions of TNF-α, IL-6, miR-155-5p and PIK3R1 mRNA, p-PI3K/PI3K ratio, p-AKT/AKT ratio, and PIK3R1 protein expression. CONCLUSIONS In HSGEC model of pSS, inhibition of miR-155-5p can promote cell proliferation and reduced cell apoptosis by targeting PI3K1 mRNA to negatively regulate the overexpression of PI3K/AKT signaling pathway.
Humans ; MicroRNAs/genetics* ; Cell Proliferation ; Signal Transduction ; Proto-Oncogene Proteins c-akt/metabolism* ; Sjogren's Syndrome/pathology* ; Epithelial Cells/cytology* ; Submandibular Gland/cytology* ; Phosphatidylinositol 3-Kinases/metabolism* ; Apoptosis ; Class Ia Phosphatidylinositol 3-Kinase ; Cells, Cultured

Progressive fibrosing interstitial lung disease （PF-ILD） has a complex etiology， and is classified as lung impediment stage， impediment-atrophy combination stage， and lung atrophy stage according to the different clinical manifestations during the progression of disease. Based on the theory of opening-closing-pivoting to analyse the characteristics of yin and yang disease mechanism and the idea of prescriptions in the three stages. For lung impediment stage， main as three-Yang fail to keep inside， disharmony between Ying qi （营气） and Wei qi （卫气）， shaoyin impairment， treatment should use Mahuang （Ephedra sinica） and Guizhi （Neolitsea cassia） flexibly to form a formula， or choose pungent-dispersing formulas like Baidu Powder （败毒散） to move qi and save yang， and diffuse and disperse impediment pathogen， meanwhile combining saving-shaoyin medicinals like Fuzi （Aconitum carmichaelii） and Shudihuang （Radix Rehmanniae Praeparata） to reinforce healthy qi and dispel pathogen； for impediment-atrophy combination stage， rooted as yangming impairment and progressed by over-movement of qi， treatment should use Mahuang Shengma Decoction （麻黄升麻汤） to resolve and decrease over-activities， emphasis on both opening and closing， and improve impediment and atrophy； for lung atrophy stage with three-Yin in a bad condition simultaneously and poor prognosis， treatment should use modified Jinshui Liujun Decoction （金水六君煎） to consolidate qi and save yin， disperse phlegm and stasis， to improve the quality of life for patients with PF-ILD.

Specific tumor-targeted gene delivery remains an unsolved therapeutic issue due to aberrant vascularization in tumor microenvironment (TME). Some bacteria exhibit spontaneous chemotaxis toward the anaerobic and immune-suppressive TME, which makes them ideal natural vehicles for cancer gene therapy. Here, we conjugated ZIF-8 metal-organic frameworks encapsulating eukaryotic murine interleukin 2 (Il2) expression plasmid onto the surface of VNP20009, an attenuated Salmonella typhimurium strain with well-documented anti-cancer activity, and constructed a TME-targeted Il2 delivery system named Il2/ZIF-8@Salmonella. Both in vitro and in vivo experiments demonstrated that Il2/ZIF-8@Salmonella maintained the tumor-targeting feature of bacteria, and could be effectively phagocytosed by intratumoral macrophages, thus leading to the expression and secretion of IL2 in TME. The detailed analysis of tumor immune microenvironment (TIME) showed that one dose of combinatorial Il2/ZIF-8@Salmonella achieved synergistic actions on a potent remodeling of TIME, marked by the activation of cytotoxic T cells and M1-polarization of macrophages in TME, thus leading to significant anti-tumor effects in melanoma, orthotopic hepatocellular carcinoma, and pulmonary metastasis models. More importantly, Il2/ZIF-8@Salmonella exhibited high safety to major organs and hematopoietic systems. Taken together, we report a novel plasmid/ZIF-8@Salmonella system that simultaneously achieves effective TME-targeted delivery of therapeutic gene, as well as synergistic re-activation of TIME.

Objective:To prepare a fluorescent probe Cetuximab-IRDye800CW targeting epidermal growth factor receptor (EGFR) and investigate its application value in surgical navigation of glioblastoma (GBM).Methods:The fluorescence properties of Cetuximab-IRDye800CW were determined by fluorescence spectrophotometer. The specificity of Cetuximab-IRDye800CW bound to GBM cells was verified by Western blot. The competitive binding method of enzyme-linked immunosorbent assay (ELISA) was used to prove whether the probe could achieve tumor targeting by binding to EGFR. Subcutaneous models of 6 nude mice of GBM were divided into experimental group ( n=3; injected with Cetuximab-IRDye800CW) and control group ( n=3; injected with IRDye800CW), and images were obtained at 5 min, 24 h, 48 h and 72 h after injection. Differences of mean fluorescence intensity (MFI) and tumor to background ratio (TBR) between experimental group and control group were compared. In situ models of GBM nude mice were established ( n=6), and MRI and intraoperative navigation were conducted, which were compared with pathological distribution. Independent-sample t test was used to analyze the data. Results:The maximum emission wavelength of Cetuximab-IRDye800CW was 820 nm, which could be received by near infrared fluorescence imaging equipment. Western blot showed that Cetuximab-IRDye800CW was only bound to GBM cells. The competitive binding of ELISA showed that Cetuximab-IRdye800CW could achieve tumor targeting by binding with EGFR. At 5 min, 24 h, 48 h and 72 h after injection of fluorescent materials, the MFI values of experimental group were 109.00±3.81, 73.36±9.93, 55.24±8.82, 37.71±6.11, which were higher than those of control group (91.32±4.17, 42.91±5.39, 25.08±6.05, 8.33±1.00; t values: 4.36-9.40, P values: 0.011-0.049). The TBR of experimental group was higher than that of control group at 24 h and 48 h after injection (24 h: 2.40±0.28 vs 1.57±0.07, t=4.94, P=0.039; 48 h: 2.07±0.12 vs 1.22±0.08, t=9.85, P=0.010). GBM in situ model was successfully constructed and verified by MRI, and the tumor was visualized under the fluorescence device navigation. Pathological distribution of the tumor with HE staining was consistent with fluorescence imaging. Conclusion:Cetuximab-IRDye800CW has fluorescence imaging capability and can identify tumor boundaries in intraoperative navigation of GBM, which has potential clinical application value.

Objective:To investigate the predictive value of plasma atherosclerosis index (AIP) on metabolic associated fatty liver disease (MAFLD) in physical examination population.Methods:It was a cross-sectional study. Total of 97 076 people who completed physical examination in the Health Management Center of the First Affiliated Hospital of Anhui Medical University from January to December 2021 and met the integrity of the study were selected as study subjects. Of the subjects, 31 176 people who met the diagnostic criteria of MAFLD were set as the MAFLD group, and the other 65 900 people were set as the non-MAFLD group. Laboratory indexes, height, weight, blood pressure, liver ultrasound and other indicators in the two groups were collected, and the AIP was calculated. The t-test was used for measurement data and chi-square test was used for counting data to compare the differences between the two groups. Univariate and multivariate logistic regression were used to analyze the influencing factors of MAFLD. The two groups were grouped further according to gender and age, and the difference of AIP prediction efficiency in different groups was evaluated by receiver operating characteristic (ROC) curve. Results:The average age (47.3 years, Z=-31.734), male proportion (76.9%, χ2=7 837.54) and the average value of AIP (0.23, Z=-155.089) in MAFLD group were all higher than those in non-MAFLD group (all P<0.001). After stratified by age, gender, body mass index (BMI), hypertensive or not, hemoglobin A1c (HbA 1c), triglyceride (TG), fasting blood glucose (FBG), the difference of AIP between the two groups was still statistically significant (all P<0.001). Multifactorial regression analysis showed that alanine aminotransferase (ALT) ( OR=1.024), aspartate aminotransferase (AST) ( OR=0.974), serum creatinine (sCr) ( OR=0.975), serum uric acid ( OR=1.004), HbA 1c ( OR=1.231), hemoglobin (HB) ( OR=1.011), platelet(PLT) ( OR=1.002), FBG ( OR=1.131), BMI ( OR=1.419), AIP ( OR=11.318), systolic blood pressure ( OR=1.002), and diastolic blood pressure ( OR=1.012) were independent risk factors for MAFLD (all P<0.001). In the overall population, AIP had an area under the ROC curve (AUC) of 0.808, a cut-off value of 2.045, a sensitivity of 74.5% and a specificity of 72.4%; in the gender subgroup, the AUC was greater in women than in men (0.815 vs 0.764), and the cut-off values, sensitivities and specificities in the two groups were -0.044 vs 0.091, 75.6% vs 72.2%, 73.3% vs 67.6%, respectively; in the age sub-group, the largest AUC (0.848), cut-off value (0.034), sensitivity (79.1%) and specificity (75.3%) were found in the 18-44 years group; the differences were statistically significantin the ROC curve analysis of each group ( P<0.001). Conclusion:AIP is an independent risk factor for MAFLD, which has good predictive value for the occurrence of the disease, and has better predictive effect in women and young groups (18-44 years old).

Objective:To examine the efficacy of robot-assisted laparoscopic modified ventral onlay lingual mucosal graft for complex ureteral stricture.Methods:The clinical data of 8 patients with ureteral stricture admitted to the Department of Urology, General Hospital of Southern Theater Command from May to October 2022 were retrospectively analyzed. There were 6 males and 2 females, aged (45.1±10.2) years (range: 34 to 64 years), body mass index (24.6±2.0) kg/m 2 (range: 20.7 to 26.6 kg/m 2). Five cases on the left side, 3 cases on the right side, the length of the ureteral structure was (3.1±0.7) cm (range: 2.2 to 4.5 cm). The value of preoperative serum creatinine was (113.8±22.3) μmol/L (range: 96 to 15 μmol/L). Before excising the structure segment, the titched anastomosed part of the dorsal wall of the ureter, and then the posteriorly augmented anastomotic, the remaining ventral side was augmented with a onlay lingual mucosa graft, then the omentum flap was used to wrap the reconstructed ureteral segment. The lingual mucosa graft with a length of 2.5 to 5.0 cm and a width of 1.0 to 1.5 cm was cut according to the actual structure. The surgery information of the patient, complications, and recent follow-up were recorded. Results:The operation under robot-assisted laparoscopy was performed successfully in the 8 patients without conversion to open surgery. The duration of the operation was (226.9±22.8) minutes (range: 210 to 255 minutes), estimated blood loss was (93.8±25.9) ml (range: 75 to 150 ml), the retention time of the postoperative drainage tube was (4.8±1.3) days (range: 3 to 7 days), and the duration of postoperative hospitalization was (11.1±3.6) days (range: 9 to 14 days). One week after the operation, the patient could pronounce correctly, enunciate clearly, and eat normally. Double J tubes were removed 4 to 8 weeks after the operation. The follow-up time in this group was 3 to 9 months, the follow-up patients underwent imaging and other examinations, which showed a significant improvement in hydronephrosis on the affected side, and the value of renal pelvic separation on the affected side was (1.4±0.8) cm (range: 0 to 2.3 cm). The serum creatinine value was (100.1±24.9) μmol/L (range: 76 to 155 μmol/L). Three months after the operation, the ureteroscopy showed that the ureter was smooth and the mucosa was normal.Conclusions:Robot-assisted laparoscopic ureteroplasty with a lingual mucosal graft is a safe and feasible operation for complex ureteral stricture without serious complications, which provides a surgical option for repairing ureteral stricture.

Objective:To examine the efficacy of robot-assisted laparoscopic modified ventral onlay lingual mucosal graft for complex ureteral stricture.Methods:The clinical data of 8 patients with ureteral stricture admitted to the Department of Urology, General Hospital of Southern Theater Command from May to October 2022 were retrospectively analyzed. There were 6 males and 2 females, aged (45.1±10.2) years (range: 34 to 64 years), body mass index (24.6±2.0) kg/m 2 (range: 20.7 to 26.6 kg/m 2). Five cases on the left side, 3 cases on the right side, the length of the ureteral structure was (3.1±0.7) cm (range: 2.2 to 4.5 cm). The value of preoperative serum creatinine was (113.8±22.3) μmol/L (range: 96 to 15 μmol/L). Before excising the structure segment, the titched anastomosed part of the dorsal wall of the ureter, and then the posteriorly augmented anastomotic, the remaining ventral side was augmented with a onlay lingual mucosa graft, then the omentum flap was used to wrap the reconstructed ureteral segment. The lingual mucosa graft with a length of 2.5 to 5.0 cm and a width of 1.0 to 1.5 cm was cut according to the actual structure. The surgery information of the patient, complications, and recent follow-up were recorded. Results:The operation under robot-assisted laparoscopy was performed successfully in the 8 patients without conversion to open surgery. The duration of the operation was (226.9±22.8) minutes (range: 210 to 255 minutes), estimated blood loss was (93.8±25.9) ml (range: 75 to 150 ml), the retention time of the postoperative drainage tube was (4.8±1.3) days (range: 3 to 7 days), and the duration of postoperative hospitalization was (11.1±3.6) days (range: 9 to 14 days). One week after the operation, the patient could pronounce correctly, enunciate clearly, and eat normally. Double J tubes were removed 4 to 8 weeks after the operation. The follow-up time in this group was 3 to 9 months, the follow-up patients underwent imaging and other examinations, which showed a significant improvement in hydronephrosis on the affected side, and the value of renal pelvic separation on the affected side was (1.4±0.8) cm (range: 0 to 2.3 cm). The serum creatinine value was (100.1±24.9) μmol/L (range: 76 to 155 μmol/L). Three months after the operation, the ureteroscopy showed that the ureter was smooth and the mucosa was normal.Conclusions:Robot-assisted laparoscopic ureteroplasty with a lingual mucosal graft is a safe and feasible operation for complex ureteral stricture without serious complications, which provides a surgical option for repairing ureteral stricture.

Objective: To construct and identify an overexpression recombinant adenovirus vector carrying the mouse PTG gene(NM＿016854), and to lay a foundation for in-depth study of the function of PTG. Methods: The coding sequence of the mouse PTG gene was chemically synthesized, amplified by polymerase chain reaction(PCR), digested with restriction enzymes, and inserted into the GV314 vector(CMV-MCS-3 FLAG-SV40-EGFP) to obtain the recombinant shuttle plasmid pGV314-PTG. BamHⅠ/AgeⅠ double enzyme digestion was further carried out, and the product was transferred into linearized expression vector pDC315 to construct recombinant adenovirus Ad-PTG, which was transfected into HEK293 T cells and packaged into recombinant virus particles. After repeated amplification of several generations of HEK293 T cells, the recombinant adenovirus was purified and titer detected. Finally, PCR, Western blot and sequencing were used to verify the recombinant adenovirus. Results: After PCR, Western blot and sequencing, the results showed that the pGV314-CMV-MCS-3 FLAG-SV40-EGFP-PTG overexpression adenovirus vector(Ad-PTG) was successfully constructed, and the virus titer measured by end-point dilution method was 4×1010PFU/ml, Western blot and RT-qPCR showed that the protein and mRNA expression levels of PTG increased significantly. Conclusion The recombinant adenovirus vector carrying mouse PTG gene is successfully constructed, and the expression of PTG gene in hepatocytes is effectively up regulated.