Objective:To explore the regulation mechanism of the quorum sensing regulator AphA on the functional activity of type Ⅵ secretion system VflT6SS2 in Vibrio fluvialis. Methods:Western Blot analysis was used to detect the relative expression and secretion of VflT6SS2 signature component hemolysin-coregulated protein (Hcp) in wild type (WT), Δ aphA, and corresponding complementary strains. Quantitative reverse transcription PCR and luminescence activity assay of the promoter- lux fusion system was used to measure the mRNA expression levels and promoter activity of the VflT6SS2 core and accessory gene-cluster representative genes tssB2, hcp ( tssD2) and vgrG ( tssI2), and the quorum sensing regulator HapR in WT and Δ aphA strains. A point mutation experiment combined with a luminescence activity assay was used to verify the regulatory binding site of AphA in the tssD2b promoter region. Electrophoretic mobility shift assay (EMSA) was used to determine AphA binding to the hapR promoter. Results:The mRNA expression levels of tssB2, hcp( tssD2), vgrG ( tssI2), and hapR as well as the protein expression and secretion levels of Hcp in Δ aphA strain, were significantly higher than those in the WT strain. The promoter activities of the VflT6SS2 core cluster, tssD2a, tssI2a, and hapR were higher in Δ aphA strain than in the WT strain, while the promoter activity of tssD2b showed the opposite trend. The promoter sequence analysis of tssD2a and tssD2b found significant differences in the region from -335 bp to -229 bp, and two potential AphA binding sites on tssD2b. The promoter activity of tssD2b decreased significantly after the point mutation of the two potential AphA binding sites. EMSA results showed that AphA binds directly to the promoter region of hapR. Conclusions:AphA indirectly inhibits the regulation of the VflT6SS2 core and accessory gene clusters at the promoter level by directly repressing the expression of hapR. AphA showed opposite regulation patterns for tssD2a and tssD2b, and AphA could positively regulate the expression of tssD2b by directly binding to the tssD2b promoter region (-335 bp to -229 bp).

Objective It aims to construct an evaluation index system for the development level of intelligent health insurance,which can serve as a reference for health insurance management departments in assessing the develop-ment level of intelligent health insurance and the implementation of health insurance informatization.Methods Key events in intelligent health insurance were identified based on event system theory and text analysis.The evaluation index system was determined through a combination of expert interviews and Delphi expert consultations.The entro-py method was used to calculate the weights of each index,followed by the assessment of the current and ideal de-velopment levels.Results A total of 16 experts were consulted.After two rounds of Delphi expert consultation,two first-level indicators and 18 second-level indicators were finally included in the system.The current development level of intelligent health insurance in China is at the intelligent development stage(2.524 points),while the ideal de-velopment level is at the intelligent improvement stage(4.073 points).The positivity coefficient of both rounds of Del-phi expert consultation was 100%,with an authority coefficient of 0.842,and the degree of expert coordination im-proved with each round.Conclusion The constructed evaluation index system exhibits high scientificity,stability,and generalizability.It can provide an effective evaluation tool for the development of intelligent health insurance in various pooled areas.

Taking the reform of DRG payment methods as the background,it discusses how the medical in-surance department uses information technology to achieve refined monitoring and management of medical institu-tions,so as to improve the quality and efficiency of medical services and control the unreasonable growth of medical expenses.The three stages of"precision monitoring-refined supervision-precision governance"of medical insurance DRG based on"refined theory"are proposed;taking Nanjing's"medical insurance high-speed railway"as an example,a DRG refined supervision and governance model framework is constructed,and its analysis is carried out monitoring elements and governance elements,and finally put forward implementation suggestions,including hori-zontal collaboration led by medical insurance,establishing a service and cost evaluation mechanism that combines in-ternal and external services.

It dified framework of health system resilience analysis.The research integrates practical elements from the case of the online pandemic material procurement and allocation hall in Nanjing,categorizing the resilience-building of local health systems via informatization into two distinct dimensions:static foundation and dynamic endowment.It conducts an in-depth examination of the logical pathways that leverage informatization to bolster resilience,and further investigates the inherent advantages and potential areas for optimization within informatization.The findings suggest that the synergistic empowerment of both static foundation and dynamic endowment effectively amplifies the risk defense capability and resilience of local health systems.

Information collaboration is an important realisation path to deepen the reform of the medical and health system and to promote the collaborative development and governance of the"Three Medicine"during the"14th Five-Year Plan"period.It employs the SFIC model and makes appropriate modifications to it.The analytical framework comprises six elements:"initial conditions-external environment-catalytic leadership-institutional de-sign-collaborative process-results feedback".This framework is used to dissect the current collaborative dilemma in the"Three Medicine"information collaboration and governance.Based on this analysis,an optimized path for infor-mation collaborative governance is proposed:consolidating the foundation of"Three Medicine"information collabora-tion,enhancing the catalytic leadership capability of meta-governance,optimizing the institutional design of informa-tion life-cycle governance,reshaping the information collaboration process,and focusing on the evaluation feedback mechanism.

Digital transformation is driving the repositioning of government work and the reshaping of public ser-vice models.It uses TOE theory combined with a technology analysis framework as a theoretical perspective and a single-case study approach to explore the operational mechanism and optimization path of health insurance gover-nance modernization.The findings show that the digital transformation of health insurance is in line with the three-stage path of"structuring the enabling mechanism-forming digital service capacity-enabling value creation".The next stage is to promote the implementation of digital coding standards,accelerate the application of technology integration,respond to the needs of the insured,improve the supporting measures for the linkage of the three health care systems,and bring into play the effectiveness of modern governance of health care.It expands the scope of government governance modernisation research and has both theoretical and practical value.

Corneal refractive surgery and intraocular collamer lens(ICL)implantation are the mainstream refractive surgery methods at present. Many studies have proved that ICL implantation can effectively improve the postoperative visual acuity of patients. ICL implantation has gained favor among refractive doctors and patients because of its multiple advantages. Excellent postoperative visual acuity and visual quality are the key factors to improve patients' satisfaction. In order to evaluate the subjective and objective visual quality of patients after operation and avoid complications, this article reviews the visual quality and postoperative complications after ICL implantation.

Objective:To observe the expression levels of Toll-like receptor 4 (TLR4) signaling pathway-related proteins and their phosphorylation in the liver tissues of rats with inorganic arsenic poisoning, and to explore the role of TLR4-mediated inflammatory signaling pathway in arsenic-induced liver fibrosis injury.Methods:Eighteen healthy weanling SD rats were divided into 3 groups according to their body weight (80 - 100 g) using a random number table (6 rats in each group, half males and half females). The control group was given 10 ml/kg of normal saline by gavage. The sodium arsenite (NaAsO 2) exposure group was given 10 mg/kg of NaAsO 2 by gavage. The TAK-242 intervention group was given 10 mg/kg of NaAsO 2 by gavage, and 0.5 mg/kg of TAK-242 was also administered intraperitoneally to inhibit TLR4 after 12 weeks. All rats were administered 6 days a week for 36 weeks. At the end of the treatment, the liver tissues and serum of the rats in each group were collected. HE and Masson staining were used to observe the pathological and fibrotic changes of the liver tissues. Automatic biochemical analyzer was used to detect serum liver function indexes of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP). Western blot was used to detect the expression changes of rat liver fibrosis protein α-smooth muscle actin (α-SMA), transforming growth factor-β1 (TGF-β1), Vimentin and TLR4 signaling pathway-related proteins TLR4, nuclear factor κB (NF-κB)-p65 subunit (p65), NF-κB-p50 subunit (p50) and their phosphorylation p-p65 and p-p50 expression levels. Enzyme-linked immunosorbent assay (ELISA) was used to detect the secretion levels of inflammatory related factors interleukin (IL)-6, tumor necrosis factor-α (TNF-α) and IL-10. Results:HE and Masson staining results showed that compared with the control group, the NaAsO 2 exposure group showed significant inflammatory cell infiltration, hepatocyte necrosis and collagen fibrous deposition, while the TAK-242 intervention group showed improvement of the inflammatory cell infiltration and reduction of collagen fibrous deposition compared with the NaAsO 2 exposure group. The results of serum liver function indexes showed that ALT, AST and ALP in NaAsO 2 exposure group were increased compared with the control group, but the TAK-242 intervention group was significantly decreased compared with the NaAsO 2 exposure group ( P < 0.05). Western bolt results showed that in NaAsO 2 exposure group, the expression levels of fibrosis protein α-SMA, TGF-β1 and Vimentin (1.04 ± 0.19, 0.92 ± 0.14, 1.20 ± 0.21) and TLR4 signaling pathway-related proteins and their phosphorylation TLR4, p50, p-p50 and p-p65 (1.16 ± 0.21, 0.95 ± 0.16, 1.24 ± 0.23, 1.56 ± 0.25) were higher than the control group (0.44 ± 0.08, 0.42 ± 0.08, 0.72 ± 0.07, 0.69 ± 0.15, 0.71 ± 0.11, 0.46 ± 0.07, 0.54 ± 0.11, P < 0.05), and the TAK-242 intervention group (0.60 ± 0.13, 0.59 ± 0.16, 0.49 ± 0.11, 0.47 ± 0.08, 0.86 ± 0.09, 0.79 ± 0.14, 1.02 ± 0.17) were lower than the NaAsO 2 exposure group ( P < 0.05). There was no significant difference in the expression level of TLR4 signal pathway-related protein p65 among the three groups ( F = 14.29, P = 0.053). ELISA results showed that the secretion levels of IL-6 and TNF-α [(98.89 ± 4.58), (83.25 ± 4.57) ng/g] in rats liver tissues of the NaAsO 2 exposure group were higher than the control group [(27.30 ± 3.92), (27.77 ± 1.83) ng/g, P < 0.05], while the secretion level of IL-10 [(36.88 ± 3.86) ng/g] was lower than the control group [(77.96 ± 7.87) ng/g, P < 0.05]. In TAK-242 intervention group, IL-6 and TNF-α secretion levels [(44.32 ± 3.60), (36.51 ± 2.93) ng/g] were lower and IL-10 secretion level [(60.40 ± 4.94) ng/g] was higher compared with the NaAsO 2 exposure group ( P < 0.05). Conclusion:TLR4-mediated inflammatory signaling pathway-related proteins and their phosphorylation are highly expressed in the liver tissues of rats with inorganic arsenic poisoning, and inhibition of TLR4 signaling pathway could significantly reduce the degree of liver fibrosis injury caused by inorganic arsenic in rats.

Objective:To investigate the clinical efficacy of Yunkang oral liquid combined with dexamethasone and low-molecular-weight heparin in the treatment of recurrent spontaneous abortion (RSA) complicated with pre thrombotic state (PTS), and its effects on thromboelastogram and serum progesterone, β-human chorionic gonadotropin (β- hCG), and helper T cell (Th) 1/Th2 cytokine levels in patients with recurrent spontaneous abortion (RSA).Methods:A prospective selection of 94 RSA patients with PTS admitted to the Baoding Second Central Hospital from January 2015 to December 2020 was conducted. They were randomly divided into two groups using a random number table method, with 47 patients in each group. The control group was treated with dexamethasone combined with low molecular weight heparin, while the observation group was treated with Yunkang oral liquid in addition to the control group. Continuously treat until 12 weeks of pregnancy to evaluate the efficacy of both groups and observe the adverse reactions in both groups. Two sets of thromboelastogram parameters were compared before and after treatment, including clot formation time (K value), clot reaction time (R value), and clot formation rate (α angle), maximum clot strength (MA value), and coagulation comprehensive index (CI), as well as serum progesterone β- hCG, Th1/Th2 cytokine levels. The pregnancy outcomes of the two groups were followed up.Results:The total effective rate of the observation group [95.7%(45/47)] was significantly higher than that of the control group [83.0%(39/47), P<0.05]. After treatment, the K and R values of both groups were significantly prolonged compared to before treatment (all P<0.05), the angle α, MA value, and CI value were significantly reduced compared to those before treatment (all P<0.05); After treatment, the K and R values in the observation group were significantly longer than those in the control group (al P<0.05), The angleα, MA value, and CI value were significantly lower than those of the control group (all P<0.05). After treatment, the levels of β- hCG serum progesterone of the two groups were significantly higher than those before treatment (all P<0.05), and The β-hCG levels of the serum progesterone were all higher than those in the control group (all P<0.05). After treatment, The levels serum Th1 cytokines [interferon-γ (INF- γ), tumor necrosis factor- α (TNF- α)] of both groups were significantly decreased compared to those before treatment (all P<0.05), and the levels of serum Th2 cytokines [interleukin-4, IL-10] were significantly increased compared to those before treatment (all P<0.05); After treatment, the observation group's serum IFN- γ, TNF- α were significantly lower than those of the control group (all P<0.05), and The levels of serum IL-4 and IL-10 were significantly higher than those of the control group all P<0.05). The live birth rate of the observation group was 87.2%(41/47), which was higher than that of the control group [70.2%(33/47)] ( P<0.05). There was no statistically significant difference in the adverse reaction rate between the observation group [6.4%(3/47)] and the control group [4.3%(2/47)] ( P>0.05). Conclusions:The treatment of RSA combined with PTS with Yunkang oral liquid, dexmedroxyprogesterone, and low molecular weight heparin can significantly alleviate blood hypercoagulability, increase reproductive endocrine hormone levels, regulate Th1/Th2 cytokine balance, improve live birth rate and clinical efficacy, and is safe and reliable.

OBJECTIVE: To determine the expression level of Sonic hedgehog (Shh) in the passage of hair follicle stem cells (HFSCs), analyze the effect of Shh overexpression on the proliferation activity of HFSCs, and explore the survival of HFSCs after Shh overexpression and its effect on hair follicle regeneration. METHODS: Hair follicles from the normal area (H1 group) and alopecia area (H2 group) of the scalp donated by 20 female alopecia patients aged 40-50 years old were taken, and the middle part of the hair follicle was cut under the microscope to culture, and the primary HFSCs were obtained and passaged; the positive markers (CD29, CD71) and negative marker (CD34) on the surface of the fourth generation HFSCs were identified by flow cytometry. The two groups of HFSCs were transfected with Shh-overexpressed lentivirus. Flow cytometry and cell counting kit 8 assay were used to detect the cell cycle changes and cell proliferation of HFSCs before and after transfection, respectively. Then the HFSCs transfected with Shh lentivirus were transplanted subcutaneously into the back of nude mice as the experimental group, and the same amount of saline was injected as the control group. At 5 weeks after cell transplantation, the expression of Shh protein in the back skin tissue of nude mice was detected by Western blot. HE staining and immunofluorescence staining were used to compare the number of hair follicles and the survival of HFSCs between groups. RESULTS: The isolated and cultured cells were fusiform and firmly attached to the wall; flow cytometry showed that CD29 and CD71 were highly expressed on the surface of the cells, while CD34 was lowly expressed, suggesting that the cultured cells were HFSCs. The results of real-time fluorescence quantitative PCR and Western blot showed that the expression levels of Shh protein and gene in the 4th, 7th, and 10th passages of cells in H1 and H2 groups decreased gradually with the prolongation of culture time in vitro. After overexpression of Shh, the proliferation activity of HFSCs in the two groups was significantly higher than that in the blank group (not transfected with lentivirus) and the negative control group (transfected with negative control lentivirus), and the proliferation activity of HFSCs in H1 group was significantly higher than that in H2 group before and after transfection, showing significant differences ( P<0.05). At 5 weeks after cell transplantation, Shh protein was stably expressed in the dorsal skin of each experimental group; the number of hair follicles and the expression levels of HFSCs markers (CD71, cytokeratin 15) in each experimental group were significantly higher than those in the control group, and the number of hair follicles and the expression levels of HFSCs markers in H1 group were significantly higher than those in H2 group, and the differences were significant ( P<0.05). CONCLUSION Lentivirus-mediated Shh can be successfully transfected into HFSCs, the proliferation activity of HFSCs significantly increase after overexpression of Shh, which can secrete and express Shh continuously and stably, and promote hair follicle regeneration by combining the advantages of stem cells and Shh.
Animals ; Female ; Mice ; Alopecia/surgery* ; Hair Follicle ; Hedgehog Proteins/genetics* ; Mice, Nude ; Regeneration ; Stem Cells