ObjectiveTo investigate how Xiaoyao Shukun decoction （XYSKD） regulates the formation and release of neutrophil extracellular traps （NETs） via the phosphatidylinositol 3-kinase （PI3K）/protein kinase B （Akt）/mammalian target of rapamycin （mTOR） signaling pathway， thereby reducing inflammation， inhibiting the excessive proliferation of fibroblasts in pelvic adhesion tissue， decreasing adhesion and fibrosis， and repairing the tissue damage in sequelae of pelvic inflammatory disease （SPID）. MethodsA total of 84 Wistar rats were randomly allocated into seven groups： blank， model， XYSKD （8 mg·kg^-1）， mTOR agonist （10 mg·kg^-1）， mTOR agonist + XYSKD （10 mg·kg^-1+8 mg·kg^-1）， mTOR inhibitor （2 mg·kg^-1）， and mTOR inhibitor + XYSKD （2 mg·kg^-1+8 mg·kg^-1）. The rat model of SPID was constructed by starvation， fatigue， and ascending Escherichia coli infection. After 14 days of drug intervention， the ultrastructure of fibroblasts in the pelvic adhesion tissue was observed by transmission electron microscopy. The general morphology of the uterus， fallopian tube， and ovary was observed by laparotomy. The levels of interleukin-1β （IL-1β）， interleukin-17 （IL-17）， and tumor necrosis factor-α （TNF-α） in the peritoneal flushing fluid were determined by enzyme-linked immunosorbent assay （ELISA）. The expression of myeloperoxidase （MPO） and citrullinated histone 3 （H3） in the fallopian tube was detected by immunofluorescence. Western blot and Real-time quantitative polymerase chain reaction （Real-time PCR） were employed to determine the relative protein and mRNA levels， respectively， of neutrophil elastase （NE）， intercellular adhesion molecule-1 （CD54）， α-smooth muscle actin （α-SMA）， H3， PI3K， and Akt. ResultsCompared with the blank group， the model group presented a large number of collagen fibers in bundles， numerous cytoplasmic folds of fibroblasts， reduced or absent mitochondrial cristae， and disordered and expanded endoplasmic reticulum. By laparotomy， extensive pelvic congestion， connective tissue hyperplasia， thickening and hardening of the tubal end near the uterus， and tubal and ovarian adhesion or cyst were observed in the model group. In addition， the model group showed raised levels of IL-1β， IL-17， and TNF-α in the peritoneal flushing fluid （P<0.01）， increased average fluorescence intensities of MPO and H3 （P<0.01）， and up-regulated protein and mRNA levels of NE， H3， CD54， PI3K， and Akt （P<0.01）. Compared with the model group， the mTOR agonist group showed increased fibroblasts and cytoplasmic folds， absence of mitochondrial cristae， endoplasmic reticulum dilation， and evident collagen fiber hyperplasia. Pelvic adhesions were observed to cause aggravated damage to the uterine， fallopian tube， and ovarian tissues. The levels of IL-1β， IL-17， and TNF-α in the peritoneal lavage fluid elevated （P<0.01） and the average fluorescence intensities of MPO and H3 enhanced （P<0.01） in the mTOR agonist group. In contrast， the XYSKD group and the mTOR inhibitor group showcased decreased fibroblasts and collagen fibers， alleviated mitochondrial crista loss and endoplasmic reticulum dilation， improved morphology and appearance of the uterine， fallopian tube， and ovarian tissues， lowered levels of IL-1β， IL-17， and TNF-α in the peritoneal lavage fluid （P<0.01）， decreased average fluorescence intensities of MPO and H3 （P<0.01）， and down-regulated protein and mRNA levels of NE， H3， CD54， PI3K， and Akt （P<0.05）. Compared with the mTOR agonist group， the mTOR agonist + XYSKD group showed alleviated pathological changes in the pelvic tissue， declined levels of IL-1β， IL-17， and TNF-α （P<0.01）， decreased average fluorescence intensities of MPO and H3 （P<0.01）， and down-regulated protein levels of NE， H3， CD54， α-SMA， p-PI3K/PI3K， and p-Akt/Akt （P<0.01） and mRNA levels of NE， H3， CD54， α-SMA， PI3K， and Akt （P<0.01）. Compared with the mTOR inhibitor group， the mTOR inhibitor + XYSKD group demonstrated reduced pathological severity of the pelvic tissue， reduced levels of IL-1β， IL-17， and TNF-α （P<0.01）， decreased average fluorescence intensities of MPO and H3 （P<0.01）， and down-regulated protein and mRNA levels of NE and CD54 （P<0.05）. ConclusionXYSKD can inhibit the excessive formation and release of NETs via PI3K/Akt/mTOR to ameliorate the inflammatory environment and reduce fibrosis and adhesion of the pelvic tissue， thereby playing a role in the treatment of SPID. It may exert the effects by lowering the levels of IL-1β， IL-17， and TNF-α and down-regulating the expression of NE， H3， CD54， α-SMA， PI3K， and Akt in the pelvic adhesion tissue.

Background::Hyperglycemia frequently induces apoptosis in endothelial cells and ultimately contributes to microvascular dysfunction in patients with diabetes mellitus (DM). Previous research reported that the expression of integrins as well as their ligands was elevated in the diseased vessels of DM patients. However, the association between integrins and hyperglycemia-induced cell death is still unclear. This research was designed to investigate the role played by integrin subunit β5 (ITGB5) in hyperglycemia-induced endothelial cell apoptosis.Methods::We used leptin receptor knockout (Lepr-KO) ( db/ db) mice as spontaneous diabetes animal model. Selective deletion of ITGB5 in endothelial cell was achieved by injecting vascular targeted adeno-associated virus via tail vein. Besides, we also applied small interfering RNA in vitro to study the mechanism of ITGB5 in regulating high glucose-induced cell apoptosis. Results::ITGB5 and its ligand, fibronectin, were both upregulated after exposure to high glucose in vivo and in vitro. ITGB5 knockdown alleviated hyperglycemia-induced vascular endothelial cell apoptosis and microvascular rarefaction in vivo. In vitro analysis revealed that knockdown of either ITGB5 or fibronectin ameliorated high glucose-induced apoptosis in human umbilical vascular endothelial cells (HUVECs). In addition, knockdown of ITGB5 inhibited fibronectin-induced HUVEC apoptosis, which indicated that the fibronectin-ITGB5 interaction participated in high glucose-induced endothelial cell apoptosis. By using RNA-sequencing technology and bioinformatic analysis, we identified Forkhead Box Protein O1 (FoxO1) as an important downstream target regulated by ITGB5. Moreover, we demonstrated that the excessive macroautophagy induced by high glucose can contribute to HUVEC apoptosis, which was regulated by the ITGB5-FoxO1 axis. Conclusion::The study revealed that high glucose-induced endothelial cell apoptosis was positively regulated by ITGB5, which suggested that ITGB5 could potentially be used to predict and treat DM-related vascular complications.

Objective To review the characteristics of animal models of sequelae of pelvic inflammatory disease(SPID)to provide a reference for standardizing the modeling process and improving the modeling rate.Methods Literature relevant to animal models of SPID from the past 40 years was searched,and animal species,modeling method,modeling cycles,modeling substances,positive control drugs,and evaluation indexes were summarized and analyzed.Results A total of 243 study manuscripts were included,most of which induced the SPID model in rats via the phenol paste or microbial infection method.The modeling cycles were typically between 14 and 16 days,and the success of the models was mostly determined by pelvic tissue morphology observation and pathological HE staining.Few research reports have focused on the traditional Chinese medicine(TCM)disease combination model.Conclusions No consistent criteria have been established for SPID animal model modeling,and thus it is recommended that researchers evaluate changes to animal behavior,pelvic histomorphology,and pathology.TCM syndrome modeling lacks effective method and evaluation standards,which need further research and development.Finally,the selection and use of positive-control drugs need to be further explored and perfected.

Objective We aimed to explore the mechanism by which Bushen Yangjing Soup improves elderly infertility based on oocyte mitochondrion.Methods C57BL/6J mice were used,including 15 young females(6-8 weeks old),45 elderly females(10 months old),and ten young males(6-8 weeks old).The female 6-8-week-old young mice were assigned to the young group,and the female 10-month-old elderly mice were randomly divided into the model group,the dehydroepiandrosterone(DHEA)group,and the Bushen Yangjing Soup group.Mice in the DHEA and Bushen Yangjing Soup groups were given DHEA[11.26 mg/(kg·d)]and Bushen Yangjing Soup[11.41 g/(kg·d)].Mice in the other groups were given normal saline.After 45 days,oocytes were collected,the number of oocytes and the proportion of mature oocytes were recorded,and in vitro fertilization(IVF)was carried out,followed by calculation of the successful fertilization rate.Real-time PCR was used to determine the copy number of mitochondrial DNA in oocytes,mito-tracker green was used to observe the distribution of mitochondria,and the mitochondrial membrane potential(MMP)of oocytes was determined using the JC-1 kit.The mRNA and protein levels of mitofusin 2(Mfn2)in oocytes were determined by real-time PCR and Western blotting,respectively.Results Compared with the young group,the number of oocytes,the proportion of mature oocytes,and the IVF rate were significantly reduced(P＜0.01),the copy number of mitochondrial DNA in oocytes was decreased,the rate of abnormal mitochondrial distribution was increased,the MMP was decreased(P＜0.01),and the mRNA and protein levels of Mfn2 were decreased(P＜0.01)in the elderly model mice.Compared with the elderly model group,the number of oocytes,the proportion of mature oocytes,and the IVF rate were significantly increased(P＜0.05),the copy number of mitochondrial DNA in oocytes was increased(P＜0.05),the rate of abnormal mitochondrial distribution was decreased,the MMP was increased(P＜0.05),and the mRNA and protein levels of Mfn2 were increased(P＜0.01)in the Bushen Yangjing Soup group.Compared with the DHEA group,the number of oocytes,the proportion of mature oocytes,and the IVF rate were increased,the copy number of mitochondrial DNA in oocytes was increased,the rate of abnormal mitochondrial distribution was decreased,the MMP was increased,the mRNA and protein levels of Mfn2 were increased in the Bushen Yangjing Soup group,and there was a significant difference in the number of oocytes and the expression of Mfn2 protein(P＜0.05).Conclusion Bushen Yangjing Soup may improve the quantity,distribution,and function of oocyte mitochondria by increasing the mRNA and protein levels of Mfn2,thereby improving oocyte quality.This is one of the possible mechanisms for clinical application of Bushen Yangjing Soup to improve ovarian function and treat elderly infertility.

ObjectiveTo observe the regulation of Qigongwan on the expression of proliferation and apoptosis-related factors programmed cell death 4 （PDCD4） and proliferating cell nuclear antigen （PCNA） in ovarian granulosa cells （GCs） in patients with polycystie ovarian syndrome （PCOS） infertility with phlegm-dampness syndrome， and to explore the effect of Qigongwan on the quality of oocytes and embryonic development potential. MethodSixty-six patients with PCOS with phlegm-dampness syndrome who underwent in vitro fertilization and embryo transfer （IVF-ET） were randomly selected and divided into an observation group （Qigongwan + western medicine） and a control group （western medicine）， with 33 patients in each group. Antagonist regimen was used to promote ovulation in the two groups. The observation group was given Qigongwan one cycle before IVF based on the treatment of conventional western medicine， while the control group was not given Chinese medicine. The improvement of phlegm and dampness syndrome， the dosage and the number of days of using gonadotropins （Gn）， the levels of luteinizing hormone （LH）， estradiol （E₂）， and progesterone （P） on the day of human chorionic gonadotropin（HCG） injection， the 2PN fertilization rate， and the high-quality embryo rate of patients in the two groups were compared. Real-time polymerase chain reaction （Real-time PCR） and Western blot technology were used to detect the expression of PCNA and PDCD4 in GCs. ResultAs compared with groups before treatment， the score of phlegm-dampness syndrome in both groups was significantly lower （P<0.01）. The score of phlegm and dampness syndrome in the observation group was significantly lower than that of the control group （P<0.01）. As compared with the control group， the levels of LH， E₂， and P in the observation group was higher， but only the difference in the level of E₂ was statistically significant （P<0.01）. The 2PN fertilization rate ［82.25% （556/676） vs 69.92% （365/522）， χ²=25.172， P<0.01］ and high-quality embryo rate ［44.19% （190/430） vs 34.23% （102/298）， χ²=7.266， P<0.01］ in the observation group were significantly higher than that of the control group （P<0.01）. As compared with the control group， the mRNA and protein expression of PDCD4 in ovarian GCs was down-regulated in the observation group and that of PCNA was up-regulated （P<0.05）. ConclusionBy down-regulating the expression of PDCD4 and up-regulating the expression of PCNA， Qigongwan may interfere with follicle development， adjust hormone levels， improve the symptomatic manifestations of patients with PCOS with phlegm-dampness syndrome， inhibit the apoptosis of GCs， and promote growth， thus improving the quality of oocytes and embryonic development potential.

Objective:To investigate the risk factors of diabetic kidney disease (DKD) in type 2 diabetes mellitus (T2DM) patients in plain-sand areas and loess hilly areas of Gansu province.Methods:A total of 1 599 T2DM patients who participated in chronic disease and risk factors monitoring and basic public health service management were selected by multi-stage stratified random sampling method in the sandy plain areas and loess hilly areas of Gansu province. Questionnaire survey, physical measurement and laboratory tests were performed. Multivariate binary logistic model was used to analyze the influencing factors.Results:The prevalence of DKD was 22.1% (174/787) among T2DM patients in the sandy plain areas and 19.1%(155/812) in the loess hilly area, respectively. Hypertension ( OR=3.022), hyperuricemia ( OR=2.114) and HbA1c≥7%( OR=2.231) were the risk factors for DKD in the plain-sand areas, and the risk of DKD increased with age. In the loess hilly areas, female sex ( OR=0.379) was the protective factor for DKD; while duration of disease≥10 years ( OR=2.476), hyperuricemia ( OR=1.907), HbA1c≥7% ( OR=1.927) were the risk factors for DKD; and the risk of DKD increased with the increase of age, and decreased with the increase of per capita monthly income. Conclusions:The prevalence of DKD and its influencing factors are different between sandy plain areas and loess hilly areas in Gansu province. The prevention and treatment of hypertension should be given more attention in sandy plain areas. In addition, the screening of DKD should be conducted among T2DM patients, particularly for those with old age, hyperuricemia and HbA1c≥7% in both areas of the province.

Objective:To compare the therapeutic outcomes between use of antibiotic cement versus non-use of antibiotic cement in the treatment of tibial osteomyelitis after radical debridement.Methods:A retrospective analysis was made of the 68 patients with local tibial osteomyelitis of Cierny-Mader Type Ⅳ who had been treated at Department of Orthopaedic Trauma, The Second People’s Hospital of Shenzhen from January 2010 to June 2015. The dead space was filled with antibiotic-impregnated bone cement beans after radical debridement of the infected bone in 32 of them (cement group) but was not in 36 of them (no-cement group). The operations for both groups were performed by the same surgical team who filled the bone defects after excision of infected bone using Ilizarov bone transport. The 2 groups were compared in terms of Paley functional scores of bone and limb, external fixation index (EFI), infection recurrence rate, total hospital costs and other complications.Results:The 2 groups were comparable because there was no significant difference between them in the preoperative general data ( P>0.05). The cement group was followed up for (71.2±8.9) months and the no-cement group for (71.6±9.7) months, showing no significant difference ( P>0.05). By the Paley functional scores, the good to excellent rate for bone was 100% for both groups (32/32 versus 36/36) while the good to excellent rate for limb was 93.8% (30/32) for the cement group and 94.4% (34/36) for the no-cement group, showing no significant differences between them ( P>0.05). The EFI was (49.0±10.5) d/cm for the cement group and (49.5±11.4) d/cm for the no-cement group, showing no significant differences between them ( P>0.05). The infection recurrence rate at the final follow-up was 3.12% (1/32) for the cement group and 2.78% (1/36) for the no-cement group, showing no significant differences between them ( P>0.05). The total hospital cost was (70,944.1 ± 1,135.5) Yuan RMB for the cement group and (55,205.2 ± 897.3) Yuan RMB for the no-cement group, showing a significant difference ( P<0.05). No serious complications with sequelae were found in either of the 2 groups. Conclusion:In the treatment of local tibial osteomyelitis of Cierny-Mader Type Ⅳ, it is not necessary to fill the dead space with antibiotic cement when radical debridement is achieved.

AIM:To investigate the role of B cells in CD45RB antibody-induced transplantation immune toler-ance.METHODS:Single cell suspension was made from the spleen of BALB/c nude mice disposed by CD45RB antibod-y, then mixed cultured with T cells of BALB/c mice and spleen cells of C57BL/6 mice.The Th1, Th2, Treg and Tm cells were monitored by flow cytometry during the culture process .The skin graft model was set up with B 6.μMT-/-mice as re-ceptors and BALB/c mice as donors.CD45RB antibody was intraperitoneally injected into the receptors after transplantation and then CD3+CD45RBhi cells were detected by flow cytometry .In another mixed lymphocyte culture , CD45RB antibody was added, and then B cells were isolated and injected into B6.μMT-/-mice through the tail vein.The heart transplanta-tion model was established with B 6.μMT-/-mice as receptors and BALB/c mice as donors, and then the survival and the migration of B cells to the thymus were observed .RESULTS:When T lymphocytes were co-cultured with B lymphocytes treated with anti-CD45RB monoclonal antibody (mAb) in vivo, the percentages of Th2 and Treg cells were up-regulated and Th1 cells were down-regulated, but Tm cells were not altered as compared with the control .In vivo without B lympho-cytes, anti-CD45RB mAb also down-regulated the expression of CD45RB in T lymphocytes.The reduction was faster and the percentage of CD3 +CD45RBhi T cells was not altered as compared with the control .The B lymphocytes treated with an-ti-CD45RB mAb in vitro prolonged the lifetime of receptor in heart transplantation model but failed to induce complete toler -ance.After recieving B cells treated with anti-CD45RB mAb and allogeneic heart transplantation , B cells migrated to the thymus in B6.μMT-/-mice.CONCLUSION:B lymphocytes play a definite role in the transplantation immune tolerance induced by anti-CD45RB mAb through their affection on T-cell subgroups and also in the central tolerance .However, the induction of immune tolerance can not only rely on B cells .

Objective To analyze the causes of misdiagnosis and mistreatment of Dravet syndrome. Methods Patients with Dravet syndrome diagnosed according to clinical features and SCN1A gene mutation detection were recruited within recent 3 years. The patients were grouped into correct diagnosis-treatment group and misdiagnosis-mistreatment group according to whether the patients had ever been misdiagnosed and mistreated by sodium channel blockers. The clinical features were compared between two groups. Results Thirty-five cases with Dravet syndrome were collected and the rate of misdiagnosis reached 40%, Nine cases were misdiagnosed as symptomatic focal epilepsy, 4 as Lennox-Gastaut syndrome and 1 as Doose syndrome. The average age of onset in misdiagnosis-mistreatment group was (5.50 ± 3.56) months,and the age of confirmed diagnosis was (83.57 ± 105.62) months. The percentage of abnormal EEG, onset seizure with partial seizure, the seizure frequency within the first year from onset, onset with afebrile seizure, patients with status epilepticus or cluster seizures was higher in misdiagnosis-mistreatment group but it showed no significant statistical significance when compared with that of correct diagnosis-treatment group. The percentage of patients with mental retardation and focal neurological signs was significantly higher in misdiagnosis-mistreatment group (P=0.005 and 0.002, respectively). Conclusions Dravet syndrome is frequently misdiagnosed as symptomatic focal epilepsy. The appearance of focal neurological signs and mental retardation before confirmed diagnosis are important factors for misdiagnosis. Gene mutation screening will be helpful for differential diagnosis of Dravet syndrome.