Aim To investigate the effects of harpagide on cerebral ischemia and the mitochondria mediated Caspase dependent apoptotic signaling pathway in mice.Methods The MCAO was employed to establish MCAO model.When the models were established, the mice were given harpagide (4, 8, 12 mg·kg-1) and edaravone (3.2 mg·kg-1) [0.1 ml·(10 g)-1] by tail vein injection after MCAO immediately.And the model and control mice were given equivalent normal saline by the same way.After MCAO for 6 h, the behavior, volume of cerebral ischemia and pathological changes in the brain were observed.Westernblot was employed to determine the contents of Cyt C in mitochondrion and pro-caspase-3 in endochylema.Results Compared with the model group, harpagide (4, 8, 12 mg·kg-1) could significantly decrease the increased nerve functional score, brain index, brain water content and volume of cerebral ischemia induced by cerebral ischemia.Harpagide (4, 8, 12 mg·kg-1) could reduce the contents of Cyt C in mitochondrion and pro-caspase-3 in endochylema.Conclusion Harpagide may have protective effect on the cerebral ischemia injury in mice, which might be related to the inhibition of the cerebral mitochondria mediated Caspase dependent apoptotic signaling pathway.

Objective To report a simple formula to estimate phosphate removal by standard four-hour hemodialysis in Chinese patients.Methods A total of 165 MHD patients in Huashan Hospital were enrolled.Effluent dialysate samples were collected during treatment to estimate the total amount of phosphate removal.Pre-dialysis levels of serum phosphate,potassium (K+),hematocrit(Hct),parathyroid hormone(iPTH),carbon dioxide combining power(CO2CP),alkaline phosphatase (AKP),Kt/V,and ultrafiltration volume,age,gender,dry body weight,blood flow,phosphate clearance of dialyser,phosphate concentration of dialysate at 60 min after the start of HD were obtained.80％ observations were randomly selected for formula building by backward stepwise and the remaining 20％ observations were used to validate the formula.Results The formula was described as Tpo4 =88.6 ×C60-0.03 ×Age + 1.07 ×Gender +0.06 ×Clearance-4.59,where C60 was phosphate concentration in dialysate measured 60 min into HD and Clearance was the phosphate clearance of dialyser.Formula validation further suggested good predictive ability.Conclusion This study derives an approach to quantify phosphate removal by a simple formula,which will be helpful for clinicians to treat patient individually.

Objective:To establish an in vitro culture system of small intestinal organoid in normal mice and perform functional identification, and to provide an in vitro research tool for material transport in the intestine under chronic kidney disease. Methods:The small intestinal crypts of C57BL/6J mice were isolated, extracted and cultured in an in vitro three dimension culture system. The formation of small intestinal organoid was observed with inverted microscope. The tissue structure of the small intestinal organoid was observed by hematoxylin and eosin staining. The cellular composition of the small intestinal organoid was identified by immunofluorescence. The expression of substance absorption-related transporters in the small intestinal organoid was detected by real time fluorescence quantitative PCR. Results:The small intestinal crypts were successfully extracted. The organoids of small intestine and different intestinal segments were successfully constructed. The cultured organoids had vigorous proliferation ability and maintained proliferation ability after passing through generations. Immunofluorescence results showed that the small intestinal organoids expressed mucin2, chromogranin A, oflm4 and lysozyme, which were different types of intestinal cell biomarkers. The PCR results showed that small intestinal organoids expressed calcium, phosphate and sodium absorption-related transporters, and the mRNA expression levels of major transporters for sodium and phosphate absorption in different intestinal segments-like organs were consistent with those in vivo, which was consistent with the characteristics of small intestinal segmental absorption. Conclusions:The successful construction of small intestine and different intestinal segments organoids, and the first observation of the expression of substance absorption-related transporters in such organoid, provide a stable and convenient in vitro research tool for the development of intestinal substance transport in chronic kidney disease.

Objectives: . We aimed to develop a new calculation model for calcium requirements in dialysis patients following parathyroidectomy. Methods: . A total of 98 patients with secondary hyperparathyroidism receiving parathyroidectomy from January 2014 to January 2022 were enrolled in this study. Among these patients, 78 were randomly selected for construction of the calcium requirement calculation model, and the remaining 20 patients were selected for model validation. The calcium requirement model estimated the total calcium supplementation for 1 week after surgery using variables with significant relationships in the derivation group by stepwise multiple linear regression analysis. Bias, precision, and accuracy were measured in the validation group to determine the performance of the model. Results: . The model was as follows: calcium requirement for 1 week after surgery=33.798–8.929×immediate postoperative calcium+0.190×C-reactive protein–0.125×age+0.002×preoperative intact parathyroid hormone+0.003×preoperative alkaline phosphatase (R2=0.8). The model was successfully validated. Conclusion . We generated a novel model to guide calcium supplementation. This model can assist in stabilizing the serum calcium levels of patients during the early postoperative period. Furthermore, it contributes to the individualized and precise treatment of hypocalcemia in patients following parathyroidectomy.

Objective To investigate the influencing factors of peripheral infections of knee joint tumor prosthesis as well as the value of serum D-D and TLR2 in predicting the infection risks so as to provide a reference for early diagnosis of tumorous periprosthetic infection(PJI)of knee joint.Methods The patients who were treated and followed up in our department from January 2008 to June 2020 were selected.According to the inclusion and exclusion criteria,136 of the patients were selected.The data including age,gender,BMI,history of diabetes,smoking history,tumor location,stage of malignant tumor,operation time,osteotomy length,intraoperative bleed-ing,and the percentage of neutrophils,leukocytes,serum D-dimer,and serum TLR value 3 days after operation were collected.The risk factors of PJI and the diagnostic value of serum D-dimer and serum TLR were analyzed.Results The incidence of PJI was 11.76%.Postoperative chemotherapy and operation time≥180 min were the risk factors of PJI(P<0.05).The area under curve(AUC)of the combination of two indicators,serum D-dimer and serum TLR2 were 0.917,0.894 and 0.778,respectively.The AUC of TLR2 was lower than that of the combina-tion of two indicators(P<0.05);The sensitivity was 0.975,0.908 and 0.708,respectively,and the specificity was 0.75,0.75,and 0.812,respectively.Conclusion Postoperative chemotherapy and operation time≥180 min are the risk factors of PJI.The combination of D-dimer and TLR2 has good diagnostic value.

Objective To observe the effects of three treatment methods on renal anemia in maintenance hemodialysis patients with hyperparathyroidism secondary to uremia and analyze the influencing factors of erythropoietin (EPO) dosage. Methods A total of 55 maintenance hemodialysis patients with secondary hyperparathyroidism at the hemodialysis center of Huashan Hospital affiliated to Fudan University from January 2015 to December 2016 were retrospectively divided into three groups according to different treatment methods, parathyroidectomy +forearm transplantation group (surgery group, n=16), cinacalcet treatment group (n=6), and calcitriol treatment group (n=33), respectively. The hemoglobin level and erythropoietin dosage were measured before treatment and in the 3rd month, the 6th month and the 12th month after treatment. The changes of hemoglobin and erythropoietin dosage in the three groups before and after treatment were observed, and the mixed effect model was used to analyze the difference of the change of hemoglobin and erythropoietin dosage among three groups. Multiple linear regression analysis was used to analyze the influencing factors of EPO dosage after one year. Results The levels of intact parathyroid hormone (iPTH) in the surgery group and the cinacalcet group before treatment were significantly higher than that in the calcitriol group (both P<0.05). In the 12th month after treatment, the levels of iPTH decreased significantly in the patients of surgery group and the cinacalcet group compared with those before treatment (both P<0.05). The levels of serum alkaline phosphatase, serum calcium and serum phosphorus in the surgery group also decreased significantly compared with those before treatment (all P<0.05). The mixed effect model analysis showed that the hemoglobin level of surgery group was on an upward trend after the treatment, and the overall level was significantly higher than cinacalcet and calcitriol treatment group (P=0.007). There was no significant difference in the dosage change of erythropoietin (EPO) in the three groups over time. However, the intra-group comparison of the mixed effect model showed that the dosage of EPO in the 12th month was significantly lower than that of before the treatment in surgery group (P=0.007). Multiple linear regression analysis showed that dialysis vintage (B=-0.064, P=0.012) and ferritin ≥ 500 μg/L (B=0.645, P=0.032) were independent influencing factors of EPO dosage. The longer the dialysis vintage, the less EPO dosage, and more EPO dosage were observed in patients with ferritin ≥ 500 μg/L. Conclusions Parathyroidectomy and forearm transplantation is more effective in reducing EPO dosage and improving renal anemia in maintenance hemodialysis patients with secondary hyperparathyroidism. Dialysis vintage and ferritin are independent influencing factors for the dosage of EPO.

Objective:To investigate the risk factor of renal anemia in maintenance hemodialysis patients (MHD) and the association of N-terminal pro-brain natriuretic (NT-proBNP) level with renal anemia.Methods:Patients who received MHD for more than 3 months at Huashan Hospital affiliated to Fudan University from August 2018 to November 2018 were selected as the subjects. The patients were divided into anemia group and non-anemia group according to the hemoglobin level. The patients' general data, the laboratory examination and dialysis related data during the observation period were collected. Pearson correlation analysis was used to analyze the correlation between anemia indicators, dialysis-related indicators and blood NT-proBNP levels. Stepwise multiple linear regression analysis was used to analyze the risk factors for anemia in MHD patients.Results:A total of 160 patients with MHD were included in this study, aged (63.11±11.35) years. There were 79 males (49.4%) and 81 females (50.6%). The dialysis age was (118.01±82.32) months, hemoglobin was (110.09±13.48) g/L, and the median NT-proBNP was 3 985 ng/L. There were 73 cases (45.6%) in anemia group and 87 cases (54.4%) in non-anemic group, and NT-proBNP levels were significantly higher in anemia group than that in the non-anemia group ( t=-3.714, P<0.001). Hemoglobin levels were positively correlated with weekly dialysis time ( r=0.228) and albumin ( r=0.349), and negatively correlated with NT-proBNP levels ( r=-0.318). Hematocrit was positively correlated with weekly dialysis time ( r=0.283), serum calcium ( r=0.317), phosphorus ( r=0.264) and albumin ( r=0.513) with significance (all P<0.05). Univariate regression analysis showed that the level of ln (NT-proBNP) was negatively correlated with hemoglobin ( P<0.001). Stepwise multiple linear regression results showed that low albumin level and high NT-proBNP level were independent risk factors for renal anemia in MHD patients. Conclusions:The increase level of NT-proBNP in MHD patients is independently associated with the decrease level of hemoglobin. Low albumin level and high NT-proBNP level are risk factors for renal anemia, suggesting that the treatment of renal anemia needs to consider improving the factors such as malnutrition and high volume.

Objective To investigate the expression level of receptor-interacting protein serine threonine kinase (RIP) 3 in macrophages/monocytes activation in autoimmune hepatitis (AIH) and its regulation on inflammatory cytokines.Methods The degree of macrophage infiltration and the expression of RIP3 in liver tissues from patients with AIH or hepatic cysts by double-immunofluorescence.After 24 hours treated with different concentrations (0,1,3,6,10 μg/mL) of lipopolysaccharide (LPS),Necrostatin-1,the specific inhibitor of RIP3 signaling pathway,and 6-thioguanine,the active metabolite of azathioprine,the expression levels of RIP1 (RIP3 upstream signal molecule),RIP3 and mixed-lineage kinase domain-like protein (MLKL,RIP3 downstream substrate) in RAW264.7 macrophages were detected by Western blotting.The expression of macrophage-associated cytokine at mRNA level of each treatment group was determined by real time quantitative polymerase chain reaction (PCR).Student's t test and sum rank test were performed for statistical analysis.Spearman analysis was performed for the correlation analysis.Results Compared with hepatic cysts adjacent liver tissues,the infiltration of CD68 positive macrophages in liver tissue of AIH patients was significantly increased (4.75 ± 0.96 vs 28.86 ± 6.23),and the difference was statistically significant (t =7.80,P＜0.05),and the expression level of RIP3 also was significantly increased (15,11 to 22 vs 0,0 to 1),and the difference was statistically significant (Z=-2.66,P＜0.05).In vitro,compared with those of control group,the expression levels of RIP1,RIP3 and MLKL stimulated by LPS at 0,1,3,6,10 μg/mL were significantly increased,and the differences were statistically significant (t=4.00,4.90,6.40,10.30;3.80,9.30,9.80,9.00;4.90,9.90,9.30 and 7.70;all P＜0.05),and were dose-dependent (r=0.91,0.86 and 0.79,all P＜0.05).Furthermore,compared with those of LPS-stimulated group,the expressions of RIP1,RIP3,MLKL of LPS+Necrostatin-1 group were significantly decreased (0.73±0.11 vs 0.47±0.13,0.60±0.07 vs 0.37 ± 0.05,0.65 ± 0.22 vs 0.38 ± 0.04,respectively),and the differences were statistically significant (t=2.60,4.50 and 2.10,all P＜0.05).And the expressions of interleukin (IL)-1β,IL-6and IL-10 at mRNA levels were also decreased (810.3±200.8 vs 463.7±118.1,1 504.4±482.7 vs 290.4±106.9,1 358.6 ± 559.2 vs 677.8 ± 297.6,respectively),and the differences were statistically significant (t=5.40,12.52,5.70,all P＜0.05).However,the expressions of IL-4 and TGF-β at mRNA levels up-regulated (0.3±0.2 vs 0.6±0.3,0.4±0.1 vs 0.9±0.4,respectively),and the differences were statistically significant (t=4.60 and 6.10,both P＜0.05).Compared with those of the LPS-stimulated group,the expressions of IL-1β,IL-6 and IL-10 at mRNA levels of LPS and 6-thiopurine stimulated group significantly down-regulated (810.3±200.8 vs 283.4±65.5,1 504.4±482.7 vs 354.4±73.8,1 358.6± 559.2 vs 625.6±336.3),and the differences were statistically significant (t=4.30,10.60 and 3.50,all P＜0.05);however,the expressions of IL-4 and TGF-β at mRNA levels significantly up-regulated (0.3±0.2 vs 0.6±0.1 and 0.4±0.1 vs 0.5±0.1),and the differences were statistically significant (t=5.20and 12.50,P＜0.05).Conclusions The regulation effects of 6-thiopurine on RIP3 signaling pathway and related cytokines are similar to those of Necrostatin-1.And the expression of RIP3 signaling protein increasing in activated macrophages of liver tissues from AIH patients is closely related to the regulation of IL-6.The RIP3 mediated inflammatory signaling pathway in macrophage may be involved in the genesis and development of AIH and may be a potential therapeutic target.

Objective:To explore the effects of dietary phosphate restriction education on serum phosphorus level, dietary phosphate intake and the knowledge of hyperphosphatemia in maintenance hemodialysis (MHD) patients.Methods:This study was a retrospective cohort study. A total of 116 hemodialysis patients in Huashan Hospital, Huadong Hospital and Tongji Hospital from October 2019 to December 2020 were enrolled in this study. They were divided into short-term group (84 cases) and long-term group (32 cases). The short-term group did not receive education or received education≤60 minutes. Meanwhile, the long-term group received education>60 minutes. Serum phosphorus level, dietary phosphate intake and knowledge of hyperphosphatemia were compared between the two groups after 4 weeks.Results:At baseline, age [64(56, 69) years old vs 65(60, 73) years old, Z=-1.493, P=0.136], the proportion of males [58.3%(49/84) vs 56.3%(18/32), χ2=0.041, P=0.839], dialysis age [55(26, 130) months vs 53(20, 132) months, Z=-0.062, P=0.951], body mass index, diabetes history, single-pool Kt/V, proportion of calctriol used, blood calcium, blood phosphorus, intact parathyroid hormone and dietary protein, dietary phosphorus and dietary phosphorus protein ratio had no statistical significance between short-term group and long-term group (all P>0.05). Adequate dietary phosphate restriction education reduced dietary phosphate intake [777.98(653.81, 943.16) mg/d vs 896.56(801.51, 1 015.51) mg/d, Z=-2.903, P=0.004], phosphate/protein ratio [13.16(11.52, 14.21) mg/g vs 15.27(13.31, 17.48) mg/g, Z=-3.929, P<0.001] and serum phosphorus level [(1.42±0.37) mmol/L vs (1.85±0.44) mmol/L, t=4.984, P<0.001]. Meanwhile, such education significantly improved achievement rate of serum phosphorus (62.5% vs 41.7%, χ2=4.034, P=0.045). In addition, patients in long-term group answered more questions correctly (completely correct plus partially correct) about the causes (93.8% vs 72.6%, χ2=6.120, P=0.013), poor prognosis (78.1% vs 52.4%, χ2=6.372, P=0.012) of hyperphosphatemia as well as the types of food with high phosphate (65.6% vs 52.4%, χ2=1.650, P=0.199). Conclusion:Adequate dietary phosphate restriction education reduces serum phosphorus level and dietary phosphate intake, and improves the knowledge of hyperphosphatemia in MHD patients.

Objective:To screen and identify H-2 d-restricted T cell epitopes in fusion (F) and attachment (G) glycoproteins of Nipah virus (NiV) in mice. Methods:The complete peptides (single peptide contains 15 amino acids, and 10 amino acids were repeated in the front and back peptides) derived from F and G antigens were mixed into peptide libraries. BALB/c mice were immunized with DNA vaccines expressing NiV F and G proteins alone and in combination. The full sequence peptide libraries of F and G antigens were mixed into peptide pools by matrix design, and spleen cells of immunized mice were collected and analyzed by IFN-γ ELISPOT assay to detect the dominant H-2 d-restricted epitope peptides. Results:Twelve dominant H-2 d-restricted peptides were screened from the F protein-specific peptide library and the 56th peptide produced the strongest reaction. Four dominant peptides were screened from the G protein-specific peptide library and the 72nd peptide produced the strongest reaction. Conclusions:In this study, 12 F antigen-specific and 4 G antigen-specific H-2 d restricted dominant T cell epitopes of NiV were screened and identified by IFN-γ ELISPOT, which could provide reference for immunological analysis of NiV and vaccine research.