基于TLR4/MyD88/NF-κB信号通路探讨针药联合治疗病毒感染后嗅觉障碍的疗效及调控机制

目的探讨针药联合治疗病毒感染后嗅觉障碍的临床效果及其潜在调控机制。方法将104例病毒感染后嗅觉障碍患者随机分成对照组和联合组,每组52例。其中,对照组给予常规鼻用激素糠酸莫米松鼻喷剂治疗,联合组在对照组基础上鼻内镜下针刺双侧内迎香穴和鼻丘穴。两组患者治疗前和治疗4周后行Sniffin Sticks嗅棒测试和T&T嗅觉计测试;采用副鼻窦CT扫描评估嗅裂通气情况;采集两组患者静脉血,采用Western blotting检测髓样分化因子88(MyD88)、Toll样受体4(TLR4)和核转录因子-κB(NF-κB)p65蛋白相对表达量,采用流式细胞术检测CD4^(+)CD25^(+)T细胞和CD4^(+)CD25^(high)T细胞百分率。结果两组治疗4周后和治疗前TDI评分差值比较,差异有统计学意义(P<0.05),联合组差值高于对照组。两组治疗4周后和治疗前T&T测试差值比较,差异有统计学意义(P<0.05),联合组差值高于对照组。两组治愈率和改善率比较,差异均有统计学意义(P<0.05),联合组高于对照组。两组患者静脉血MyD88、TLR4和NF-κB p65蛋白相对表达量治疗前与治疗4周后比较,差异有统计学意义(P<0.05),治疗4周后均低于治疗前;两组治疗4周后静脉血MyD88、TLR4和NF-κB p65蛋白相对表达量比较,差异有统计学意义(P<0.05),联合组低于对照组。两组患者静脉血CD4^(+)CD25^(+)T细胞百分率和CD4^(+)CD25^(high)T细胞百分率治疗前与治疗4周后比较,差异有统计学意义(P<0.05),治疗4周后均高于治疗前;两组患者治疗4周后静脉血CD4^(+)CD25^(+)T细胞百分率和CD4^(+)CD25^(high)T细胞百分率比较,差异有统计学意义(P<0.05),联合组高于对照组。结论针药联合可恢复病毒感染后嗅觉障碍患者的嗅觉功能并减轻炎症,且疗效优于单纯的药物治疗,其机制与抑制TLR4/MyD88/NF-κB信号通路有关。

Effect of Yuchang suppository on PPAR γ/NF-KB signaling pathway in rats with ulcerative colitis

Objective: To investigate the therapeutic effect of Yuchang suppository on experimental ulcerative colitis rats and the effect of PPARγ/NF-KB signaling pathway. Methods: The UC rat model was replicated by 2.4.6-trinitrobenzenesulfonic acid (TNBS)/ethanol method and randomly divided into normal control group, model group, high-dose group of Yuchang suppository, middle dose group , Low-dose group and sulfasalazine group, 8 rats in each group. After 14 days of treatment, the general condition of the rats was observed and the disease activity index was scored. The changes of serum TNF-α, IL-1β and IL-6 levels were detected by ELISA. The colonic mucosal injury index (CMDI) score and colon were observed by colon specimens. Histopathological changes;RT-PCR and Western blot were used to detect the expression of PPARγ and NF-KB genes and proteins in colon tissue. Results: Compared with the normal control group, the DAI and CMDI scores in the model group and the levels of serum TNF-α, IL-1β, IL-6, NF-κB p65 mRNA and protein in colon tissue were significantly increased, PPAR. - γ mRNA and protein expression levels were significantly reduced. The levels of IL-1β, IL-6, TNF-α and the expression of NF-κB p65 mRNA and protein in colon tissue were lower than those in the model group, and the expression levels of PPARγ mRNA and protein were high. In the model group. In the improvement of NF-κBp65 and PPAR-γ gene and protein expression in rat colonic mucosa, the high-dose group and the middle-dose group were significantly better than the low-dose group. Conclusion:Yuchang suppository can significantly improve the symptoms and histopathology of UC model rats. The mechanism may be through activation of PPARγ, blocking the activation of NF-κB signaling pathway, and down-regulating inflammatory factors such as TNF-α and IL-1β. The expression is such that the inflammatory response is alleviated or eliminated, thereby achieving the therapeutic effect of UC.

Establishment and evaluation of Type 2 Diabetes Mellitus andits complications induced by low dose of multiple streptozotocin

Objective: To explore a simple, economical and feasible method for establishing type 2 diabetes mellitus and its complication model. Method: SD rats were randomly divided into two groups: normal group (15 rats) normal diet, model group (20 rats) with high glucose and high fat diet. After 2 weeks of feeding, model group was injected low dose(30mg/Kg) streptozotocin in the abdominal cavity, once a day for 6 days, normal group was injected with the same dose of citric acid buffer. Observe the changes of rat diet, body, hair, spirit, urine volume, and monitor weight, blood glucose. After 6 weeks, fast insulin, blood lipid, serum calcium, phosphorus and other biochemical indexes of rats, after HE staining observe morphologic variations changes of pancreas and kidney using microscope, and the changes of femoral bone in SD rats. Results: Compared with the normal group, the model group showed obvious symptoms, drink more, eat more, polyuria, emaciation, hyperglycemia, low calcium, low phosphorus, hyperlipidemia and other metabolic abnormalities, pancreatic atrophy, tissue fibrosis, glomerular area increased, basement membrane thickening, osteoporosis. Conclusion:High glucose and high fat diet combined with repeated intraperitoneal injecti on of low dose STZ can be successfully established the model of type 2 diabetes, and its high successful rate, low mortality rate, relatively simple, economical and feasible, has a certain significance of research on diabetes and its complications.

Effect and mechanism of high glucose on apoptosis and cell cycle arrest of islet β cells via p27 pathway

Objective:To study the effect of mechanism of high glucose on apoptosis and cell cycle arrest of isletβcells via p27 pathway.Methods:Islet INS-1 cells were cultured and divided into groups.The control group was treated with ordinary medium,the high glucose group was treated with high glucose medium containing 25mmol/L glucose,the high glucose+si-NC group was treated with high glucose medium and transfected with NC siRNA,and the high glucose+si-P27 group was treated with high glucose medium and transfected with p27 siRNA.After 24 hours treatment,MTS assay was used to detect the cell viability A490,TUNEL assay was used to detect apoptosis rate,flow cytometry was used to detect the cell cycle distribution and western blot was used to detect the expression levels of P27,caspase-8 and cyclinD1.Results:Compared with those in the control group,the A490,the ratio of S phase and G2/M phase as well as the expression level of CyclinD1 decreased,while the apoptosis rate,the ratio of G0/G1 phase as well as the expression levels of P27 and caspase-8 increased in the high glucose group(P<0.05);compared with those in the high glucose group,the A490,cell cycle as well as the expression levels of P27,caspase-8 and cyclinD1 were not different from those in the high glucose+si-NC group(P>0.05);compared with those in the high glucose group and high glucose+si-NC group,the A490,the ratio of S phase and G2/M phase as well as the expression levels of cyclinD1 increased,while the apoptosis rate,the ratio of G0/G1 phase as well as the expression levels of p27 and caspase-8 decreased in the high glucose+si-P27 group(P<0.05).Conclusion:The apoptosis and cell cycle arrest induced by high glucose are related to P27 pathway activation.

Beneficial Effect of Moderately Increasing Hypothermic Machine Perfusion Pressure on Donor after Cardiac Death Renal Transplantation

Background:Vascular resistance and flow rate during hypotherrnic machine perfusion (HMP)of kidneys is correlated with graft function. We aimed to determine the effects of increasing HMP pressure versus maintaining the initial pressure on kidney transplantation outcomes. Methods:We retrospectively reviewed the data of 76 primary transplantation patients who received HMP-preserved kidneys from 48 donors after cardiac death between September 1,2013,and August 31,2015.HMP pressure was increased from 30 to 40mmHg (1mmHg =0.133kPa)in kidneys with poor flow and/or vascular resistance (increased pressure [IP]group;36 patients);otherwise,the initial pressure was maintained (constant pressure group;40 patients).Finally,the clinical characteristics and transplantation outcomes in both groups were assessed. Results:Delayed graft function (DGF)incidence,1-year allograft,patient survival,kidney function recovery time,and serum creatinine level on day 30 were similar in both groups,with improved flow and resistance in the IP group.Among patients with DGF,kidney function recovery time and DGF duration were ameliorated in the IP group.Multivariate logistic regression analysis revealed that donor hypertension (odds ratio [OR]:1.43,95%confidence interval [CI]:1.02-2.06,P =0.035),donor terminal serum creatinine (OR:1.27,95%C7:1.06-1.62,P =0.023),warm ischemic time (OR:3.45,95%CI:1.97-6.37,P =0.002),and terminal resistance (OR:3.12,95%CI:1.76-6.09,P =0.012)were independent predictors of DGF.Cox proportional hazards analysis showed that terminal resistance (hazard ratio:2.06,95%C1:1.32-5.16,P =0.032)significantly affected graft survival. Conclusion:Increased HMP pressure improves graft perfusion but does not affect DGF incidence or 1-year graft survival.

Predictive Score Model for Delayed Graft Function Based on Hypothermic Machine Perfusion Variables in Kidney Transplantation

Background: Hypothermic machine perfusion(HMP) is being used more often in cardiac death kidney transplantation; however, the significance of assessing organ quality and predicting delayed graft function(DGF) by HMP parameters is still controversial. Therefore,we used a readily available HMP variable to design a scoring model that can identify the highest risk of DGF and provide the guidance and advice for organ allocation and DCD kidney assessment.Methods: From September 1, 2012 to August 31, 2016, 366 qualified kidneys were randomly assigned to the development and validation cohorts in a 2:1 distribution. The HMP variables of the development cohort served as candidate univariate predictors for DGF. The independent predictors of DGF were identified by multivariate logistic regression analysis with a P < 0.05. According to the odds ratios(ORs) value, each HMP variable was assigned a weighted integer, and the sum of the integers indicated the total risk score for each kidney. The validation cohort was used to verify the accuracy and reliability of the scoring model.Results: HMP duration(OR = 1.165, 95% confidence interval [CI ]: 1.008–1.360, P = 0.043), resistance(OR = 2.190, 95%CI: 1.032–10.20, P < 0.001), and flow rate(OR = 0.931, 95% CI: 0.894–0.967, P = 0.011) were the independent predictors of identified DGF. The HMP predictive score ranged from 0 to 14, and there was a clear increase in the incidence of DGF, from the low predictive score group to the very high predictive score group. We formed four increasingly serious risk categories(scores 0–3, 4–7, 8–11, and 12–14)according to the frequency associated with the different risk scores of DGF. The HMP predictive score indicates good discriminative power with a c?statistic of 0.706 in the validation cohort, and it had significantly better prediction value for DGF compared to both terminal flow(P = 0.012) and resistance(P = 0.006).Conclusion: The HMP predictive score is a good noninvasive tool for assessing the quality of DCD kidneys, and it is potentially useful for physicians in making optimal decisions about the organs donated.

Predictive Score Model for Delayed Graft Function Based on Easily Available Variables before Kidney Donation after Cardiac Death

Background： How to evaluate the quality of donation after cardiac transplantation in China. Hence, the aim of this study was to develop kidneys before DCD. death （DCD） kidneys has become a critical problem in kidney a simple donor risk score model to evaluate the quality of DCD Methods： A total of 543 qualified kidneys were randomized in a 2：1 manner to create the development and validation cohorts. The donor variables in the development cohort were considered as candidate univariate predictors of delayed graft function （DGF）. Multivariate logistic regression was then used to identify independent predictors of DGF with P 〈 0.05. Date from validation cohort were used to validate the donor scoring model. Results： Based on the odds ratios, eight identified variables were assigned a weighted integer; the sum of the integer was the total risk score for each kidney. The donor risk score, ranging from 0 to 28, demonstrated good discriminative power with a C-statistic of 0.790. Similar results were obtained from validation cohort with C-statistic of 0.783. Based on the obtained frequencies of DGF in relation to different risk scores, we formed tour risk categories of increasing severity （scores 04, 5 9, 10-14, and 15 28）. Conclusions： The scoring model might be a good noninvasive tool for assessing the quality of DCD kidneys before donation and potentially useful for physicians to make optimal decisions about donor organ offers.

Comparison of the characteristics of macrophages derived from murine spleen, peritoneal cavity, and bone marrow

Macrophages have a diverse set of functions based upon their activation states. The activation states, including resting（M0） and polarizing（M1 and M2） states, of macrophages derived from the mouse bone marrow, spleen, and peritoneal cavity（BMs, SPMs, and PCMs, respectively） were compared. We evaluated the macrophage yield per mouse and compared the surface markers major histocompatibility complex（MHC） Ⅱ and CD86 by flow cytometry. The relative mRNA levels of tumor necrosis factor-α（TNF-α）, interleukin（IL）-1β, mannose receptor（MR）, and Ym1 in the M0, M1, and M2 states were also compared using real-time polymerase chain reaction（PCR） analysis. Bone marrow yielded the most macrophages with the best homogeneity, but they were polarized toward the M2 phenotype. All three types of macrophages had the capacity to polarize into the M1 and M2 states, but SPMs had a stronger capacity to polarize into M1. The three types of macrophages showed no differences in their capacity to polarize into the M2 state. Therefore, the three types of macrophages have distinct characteristics regardless of their resting or polarizing states. Although bone marrow can get large amounts of homogeneous macrophages, the macrophages cannot replace tissue-derived macrophages.

Polyglycolic Acid Fibrous Scaffold Improving Endothelial Cell Coating and Vascularization of Islet

Background：Improving islet graft revascularization has become a crucial task for prolonging islet graft survival.Endothelial cells （ECs） are the basis of new microvessels in an isolated islet,and EC coating has been demonstrated to improve the vascularization and survival of an islet.However,the traditional method of EC coating of islets has low efficiency in vitro.This study was conducted to evaluate the effect of a polyglycolic acid （PGA） scaffold on the efficiency of islet coating by ECs and the angiogenesis in the coated islet graft.Methods：A PGA fibrous scaffold was used for EC coating of islet culture and was evaluated for its efficiency of EC coating on islets and islet graft angiogenesis.Results：In in vitro experiments,we found that apoptosis index of ECs-coating islet in PGA group （27% ± 8%） was significantly lower than that in control group （83% ± 20%,P 〈 0.05） after 7 days culture.Stimulation index was significantly greater in the PGA group than in the control group at day 7 after ECs-coating （2.07 ± 0.31 vs.1.80 ± 0.23,P 〈 0.05）.vascular endothelial growth factor （VEGF） level in the PGA group was significantly higher than the coating in the control group after 7 days culture （52.10 ± 13.50 ng/ml vs.16.30 ± 8.10 ng/ml,P 〈 0.05）.Because of a tight,circumvallated,adhesive and three-dimensional growth microenvironment,islet cultured in a PGA scaffold had higher coating efficiency showing stronger staining intensity of enzyme than those in the control group after 14 days of culture following ECs-coating.For in vivo study,PGA scaffold significantly prolonged the average survival time of EC-coated islet graft after transplantation compared with control group （15.30 ± 5.60 days vs.8.30 ± 2.45 days,P 〈 0.05）.The angiogenesis and area of survived grafts were more in the PGA group compared with the control group by measuring the mean microvessel density （8.60 ± 1.21/mm2 vs.5.20 ± 0.87/mm2,P 〈 0.05）.In addition,expression of VEGF and tyrosin-protein kinase receptor （Tie-2） gene increased in PGA scaffold group than that in control group by real-time reverse transcription-polymerase chain reaction analysis.Conclusions：These results demonstrate that the efficiency of EC coating of islets was successfully increased by culturing ECs on a PGA scaffold.This method enhances the function,survival,and vascularization of isolated islets in vitro and in vivo.

Early Immunosuppressive Exposure of Enteric-Coated-Mycophenolate Sodium Plus Tacrolimus Associated with Acute Rejection in Expanded Criteria Donor Kidney Transplantation

Background： lmmunosuppressive agents are still inefficient in preventing biopsy-proven acute rejection （BPAR） alter expanded criteria donor （ECD） kidney transplantation. The aim of this study was to investigate the relationships between early imrnunosuppressive exposure and the development of BPAR. Methods： We performed a retrospective study of 58 recipients of ECD kidney transplantation treated with enteric-coated-mycophenolate sodium, tacrolirnus （Tac）, and prednisone. The levels of mycophenolic acid-area under the curve （MPA-AUC）0-12h and Tac C0 were measured at the 1st week and the 1st month posttransplant, respectively. The correlation was assessed by multivariate logistic regression. Results： The occurrence rates of BPAR and antibody-mediated rejection were 24.1% and 10.3%, respectively. A low level of MPA-AUC0-12h at the 1st week posttransplant was found in BPAR recipients （38.42 ± 8.37 vs. 50.64 ± 13.22, P 〈 0.01）. In addition, the incidence of BPAR was significantly high （P 〈 0.05） when the MPA-AUC0-12h level was 〈30 mg·h-1·L-1 at the 1st week （ 15.0% vs. 44.4%） or the Tac C0 was 〈4 ng/ml at the 1 st month posttransplant （33.3% vs. 21.6%）. Multivariable logistic regression analysis showed that the MPA-AUC 0-12 h at the 1st week （OR： 0.842, 95% CI： 0.784-0.903） and the Tac C0 at the 1st month （OR： 0.904, 95% C7： 0.822-0.986） had significant inverse correlation with BPA R （ P 〈 0.05 ）. Conclusions： Low-level exposure of MPA and Tac C0 in the early weeks posttransplant reflects an increased acute rejection risk, which suggested that MPA-AUC0-12h 〈30 mg·h-1·L -1 and Tac C0 〈4 ng/ml should be avoided in the first few weeks alter transplantation.

Allograft rejection-related gene expression in the endothelial cells of renal transplantation recipients after cytomegalovirus infection

Objective： To explore the effects of cytomegalovirus （CMV） infection on rejection-related gene expression in the endothelial cells of renal transplantation recipients. Methods： Endothelial cells （ECs） were cultured and stimulated by a variety of factors： A, normal control group; B, inactivated human cytomegalovirus （HCMV） infection group; C, HCMV infection group; D, HCMV supematant infection group; and E, ganciclovir HCMV group. Expression of intercellular adhesion molecule-1 （ICAM-1） and major histocompability complex （MHC） class I and class II antigens was detected by flow cytometry （FCM） and immuno- histochemistry. Results： We found characteristic CMV-infected ECs in this study. There were no significant differences among groups A, B and D （P〉0.05）. Although the expression levels of ICAM-1 were not significantly different between groups C and E （P〉0.05）, the ICAM-1 expression in these two groups was significantly higher than that in group A （P〈0.05）. ICAM-1 expression was detected in groups C and E, while there was no expression in groups A, B and D. Furthermore, there was no significant difference of ICAM-1 mRNA expression between groups C and E （P〉0.05）. Human leucocyte antigen （HLA）-ABC expression was detected in all the groups, while HLA-DR expression was only detected in groups C and E. There were no significant dif- ferences of HLA-ABC and HLA-DR expression among groups A, B and D （P〉0.05）. However, the HLA-ABC and HLA-DR expression levels in groups C and D were higher than those of the remaining groups previously reported （P〈0.05）. Meanwhile, the HLA-ABC and HLA-DR expression levels in group E were lower than those of group C （P〈0.05）. Conclusion： CMV could up-regulate the expression levels of ICAM-1 and MHC antigens, which was closely related to allograft rejection.

Donor-specific antibodies,glomerulitis,and human leukocyte antigen B eplet mismatch are risk factors for peritubular capillary C4d deposition in renal allografts

Background:The complement system plays an important role in the immune response to transplantation,and the diagnostic significance of peritubular capillary(PTC)C4d deposition(C4d+)in grafts is controversial.The study aimed to fully investigate the risk factors for PTC C4d+and analyze its significance in biopsy pathology of kidney transplantation.Methods:This retrospective study included 124 cases of kidney transplant with graft biopsy and donor-specific antibody(DSA)testing from January 2017 to December 2019 in a single center.The effects of recipient pathological indicators,eplet mismatch(MM),and DSAs on PTC C4d+were examined using univariate and multivariate logistic regression analyses.Results:In total,35/124(28%)were PTC C4d+,including 21 with antibody-mediated rejection(AMR),eight with renal tubular injury,three with T cell-mediated rejection,one with glomerular disease,and two others.Univariate analysis revealed that DSAs(P<0.001),glomerulitis(P<0.001),peritubular capillaritis(P<0.001),and human leukocyte antigen(HLA)B eplet MM(P=0.010)were the influencing factors of PTC C4d+.According to multivariate analysis,DSAs(odds ratio[OR]:9.608,95%confidence interval[CI]:2.742–33.668,P<0.001),glomerulitis(OR:3.581,95%CI:1.246–10.289,P=0.018),and HLA B eplet MM(OR:1.166,95%CI:1.005–1.353,P=0.042)were the independent risk factors for PTC C4d+.In receiver operating characteristic curve analysis,the area under the curve was increased to 0.831 for predicting PTC C4d+when considering glomerulitis,DSAs,and HLA B eplet MM.The proportions of HLA I DSAs and PTC C4d+in active antibody-mediated rejection were 12/17 and 15/17,respectively;the proportions of HLA class II DSAs and PTC C4d+in chronic AMR were 8/12 and 7/12,respectively.Furthermore,the higher the PTC C4d+score was,the more serious the urinary occult blood and proteinuria of recipients at the time of biopsy.Conclusions:PTC C4d+was mainly observed in AMR cases.DSAs,glomerulitis,and HLA B eplet MM are the independent risk factors for PTC C4d+.