Cadmium is a non-essential,non-degradable heavy metal element,the accumulation of cadmium can cause kidney damage.The purpose of this study was to reveal the ferroptosis mecha-nism induced by cadmium exposure in porcine kidney PK-15 cells.First of all,cell viability of gradi-ent concentration of cadmium chloride(CdCl2)on PK-15 cells was detected by CCK-8 method to screen suitable work concentration of CdCl2.Secondly,PK-15 cells were treated with 10 μmol/L CdCl2 for 3,6 and 12 h.The contents of lactate dehydrogenase(LDH)malondialdehyde(MDA)were detected by colorimetry,the expression of ferroptosis related protein were detected by West-ern blot,and the content of ferrous ion(Fe2+)was detected by fluorescence probe.Finally,PK-15 cells were pretreated with 10 μmol/L HO-1 inhibitor zinc protoporphyrin(ZnPP)for 6 h,and then treated with 10 μmol/L CdCl2 for 12 h.The morphology of PK-15 cells was observed by phase contrast microscope and the expression of ferroptosis related proteins was detected by West-ern blot.The results showed that CdCl2 treatment caused a significant decrease in cell viability.The levels of LDH,MDA and Fe2+,the protein levels of Nrf2,HO-1 and ALOX5,and the expression level of FTH1 protein were significantly decreased in CdCl2 treatment cells(P＜0.01).ZnPP could significantly improve the ferroptosis of PK-15 cells induced by CdCl2 treatment.Compared with CdCl2 treatment cells,the cell morphology was significantly improved,the protein levels of Nrf2,HO-1 and ALOX5 protein were significantly decreased,and the protein levels of FTH1 protein were significantly increased in ZnPP pretreatment group(P＜0.01).The results showed that cad-mium induced iron overload and lipid peroxidation,which result in ferroptosis in PK-15 cells through Nrf2/HO-1 pathway.

Porcine kidney cell line(PK-15)with HO-1 gene knockout was constructed by CRISPR/Cas9 system to provide experimental materials for exploring the role and molecular mechanism of HO-1 in cadmium-induced ferroptosis in PK-15 cells.Three sgRNA targeting HO-1 gene were de-signed,synthesized and ligated to lentiviral vector Lenti CRISPRv2.The constructed lentiviral plas-mid was transfected into human embryonic cells(HEK293FT)to obtain recombinant lentivirus,which was used to infect PK-15 cells.The monoclonal cell line with HO-1 knockout was obtained by puromycin screening and limited dilution method.The knockout effect was analyzed by sequencing and Western blot detection.The HO-1 gene knockout cells were treated with 10 μmol/L cadmium chloride(CdCl2).The cell viability was detected by CCK-8 method,the cell morpholo-gy was observed by phase contrast microscope,and the content of ferrous ion(Fe2+)was detected by fluorescence probe.The results showed that sgRNA2 possessed the highest editing efficiency.The base insertion or deletion of HO-1 gene and the frameshift of the target gene occurred in all five knockout cells.Western blot results showed that no expression of HO-1 protein was detected,indicating that the PK-15 cell line with HO-1 gene knockout was successfully constructed.After CdCl2 treatment,compared with the control cells,the cell viability was significantly increased and the Fe2+content was significantly decreased in PK-15 cells with HO-1 gene deletion,indicating that HO-1 gene knockout could significantly alleviate the abnormal iron metabolism caused by cadmium treatment.In conclusion,this study successfully constructed a PK-15 cell line knocking out HO-1 gene by using CRISPR/Cas9 gene editing technique,and confirmed that HO-1 plays an important role in iron metabolism abnormality in PK-15 cells induced by cadmium treatment,which lays a foundation for further study on the regulatory mechanism of HO-1 in ferroptosis in PK-15 cells induced by cadmium exposure.

Objective:To analyze the prevalence and related factors of depression symptom among middle-aged and older adults belonging to Zhiguo ethnic minority groups,who experienced relative isolation prior to mod-emization and bypassed the transitional period associated with feudal monarchy in Yunnan Province.Methods:Using stratified cluster sampling,a total of 1 035 individuals from Zhiguo ethnic minority groups were investigated with the 9-item Patient Health Questionnaire(PHQ-9)to measure depression symptom scores(a total score≥5 was considered indicative of depression symptom).A self-administered questionnaire was utilized to investigate 24 cova-riates across five dimensions:sociodemographic,social-interpersonal network,mental health status,physical health status,and health behaviors.Results:Among the surveyed participants,231 individuals(22.32％)exhibited depres-sion symptoms.The logistic regression analysis showed that middle-aged and elderly people belonging to Zhiguo ethnic minority groups with occupations as laborers(OR=8.73),farmers(OR=7.34),and other professions(OR=7.13),those who reported feeling stressed or anxious in the last month(OR=5.43),those suffering from one(OR=2.81)or more chronic illnesses(OR=2.14),experiencing sleep problems(OR=3.74)/debilitating(OR=1.69)/mild-moderate pain(OR=2.23,4.37),and engaging in sedentary behavior(OR=2.27)were at higher risk for depressive symptoms.The risk of depressive symptoms was lower among middle-aged and elderly people be-longing to Zhiguo ethnic minority groups with higher social support scores(OR=0.96)and meaning in life scores(OR=0.96).Conclusion:The prevalence of depressive symptoms among middle-aged and elderly individuals of Zhiguo ethnic minorities is substantial,influenced by multiple factors.

Objective:To observe the effects of acupuncture and moxibustion on ubiquitin-like containing PHD and RING finger domains 1(UHRF1)and DNA methyltransferase 1(DNMT1)in ectopic endometrium of rats with endometriosis(EMS). Methods:Forty Sprague-Dawley rats were randomly divided into a sham operation group with 10 rats and a model-building group with 30 rats according to body mass.EMS rat models were established in the model-building group and then were divided into a model group,an acupuncture and moxibustion group,and a progesterone group,with 10 rats in each group.All rats were fixed by a fixator.The sham operation group and the model group were given normal saline by gavage.The acupuncture and moxibustion group received acupuncture at Xuehai(SP10)and Sanyinjiao(SP6),moxibustion at Guanyuan(CV4),and gavage of normal saline.The progesterone group was given the mixed liquid made of dydrogesterone and normal saline by gavage.After 28 d of treatments,the three diameters(length,width,and height)of EMS rats'ectopic cysts were measured,the cyst volumes were calculated,the volumes before intervention were subtracted,and the difference values were used to evaluate the growth of ectopic cysts.UHRF1 and DNMT1 mRNA and protein levels in normal endometrium,eutopic endometrium,and ectopic endometrium were detected by real-time polymerase chain reaction and immunohistochemistry. Results:There was no significant difference in the ectopic cyst volume difference between the acupuncture and moxibustion group and the progesterone group(P>0.05),but they were smaller than that of the model group(P<0.05).The levels of UHRF1 and DNMT1 mRNA and protein in the ectopic endometrium of the model group were lower than those in the normal endometrium(P<0.05).The levels of DNMT1 mRNA and UHRF1 protein in the eutopic endometrium of the model group were lower than those in the normal endometrium(P<0.05).The levels of UHRF1 mRNA and protein and the level of DNMT1 protein in the ectopic endometrium of the acupuncture and moxibustion group were higher than those in the model group(P<0.05),and the level of UHRF1 mRNA was higher than that in the progesterone group(P<0.05).The level of DNMT1 mRNA in the eutopic endometrium of the acupuncture and moxibustion group was higher than that in the model group(P<0.05).The levels of UHRF1 and DNMT1 mRNA and protein in the acupuncture and moxibustion group were insignificantly different from those in the normal endometrium(P>0.05). Conclusion:Acupuncture and moxibustion may up-regulate the levels of UHRF1 mRNA and UHRF1 and DNMT1 proteins in the ectopic endometrium to the normal level so as to reduce the volume of ectopic cysts and cure EMS in rats.

【Objective】 To identify the specificity of alloantibody against high-frequency antigens in one case suffering with severe hemolytic diseases of the fetus and newborn (HDFN) and to screen for matching blood for transfusion. 【Methods】 The HDFN test and the antibody serological identification tests in the mother were performed. Several common high frequency antigens of maternal red blood cells (RBCs) were determined. IgG subtype coated on the RBCs of the newborn was determined. The phagocytic efficiency of the antibody was tested using the monocyte phagocytosis of sensitized erythrocyte by flow cytometry in vitro. Sanger sequencing of DI gene was performed in the mother, father and mother’s brother. The diluted maternal plasma was used for large scale screening of matching blood using IAT in Coomb’s gel card. 【Results】 Di(b-) phenotype was identified in the mother of the newborn and anti-Di^b (titer: 512) related HDN was detected in the newborn. IgG1 and IgG2 subtypes of anti-Di^b were detected and the rate of monocyte phagocytosis was 88.83%(74.7/84.09). The compatible blood was not detected in the maternal relatives. Subsequently, the newborn received the matching RBCs of two Di(b-) donors identified from 5 520 blood donors and discharged from the hospital. We screened out 17 Di(b-) donors out of 51 334 blood donors, indicating that the distribution frequency of Di(b-) among blood donors in Guangzhou was about 0.033% (17/51 334). 【Conclusion】 By serology and molecular biology methods, the newborn was identified with HDFN caused by anti-Di^b, and an effective large-scale screening method for Di (b -) rare blood types was established to find matching blood, which supported the establishment of rare Di(b-) blood database.

Objective To elucidate the prediction ability of monocyte monolayer assay(MMA)used in hemolytic dis-ease of fetus and newborn(HDFN)caused by IgG anti-M.Methods Plasma from eight pregnant women containing IgG an-ti-M were collected,and were divided into two groups(4 cases with HDFN,with severe clinical symptoms such as fetal hy-drops,and 4 cases without HDFN)according to the clinical outcomes.M antigen positive cells were sensitized with dithioth-reitol(DTT)treated plasma from eight pregnant women respectively.MMA was performed by coincubation with monocytes and sensitized M cells,along with negative and positive control set up.T-test was conducted to compare the difference in phagocytic efficiency between two groups.Results The phagocytic efficiency in group with HDFN were 15.37%,13.05%,9.17%and 24.50%respectively,with the mean value of 15.52%,while the group without HDFN were 8.74%,11.07%,5.12%and 6.23%respectively,with the mean value of 7.79%.There was no significant difference in phagocytic efficiency between two groups(P>0.05).The mean values of both groups were not significantly different from the negative control(P>0.05),but both were significantly lower than positive control(P<0.05).Conclusion The low phagocytic efficiency couldn't convince that the MMA is an effective predictor for the HDFN caused by IgG anti-M,indicating that another mech-anism might be responsible for it rather than monocyte phagocytosis.The assessment of the peak systolic velocity in middle cerebral artery of the fetal should be considered in the management for pregnant women who produce IgG anti-M to estimate the situation of fetal anemia.

Objective:To explore the effect of clinical conventional fractionated dose radiation on the expression levels of immunogenic cell death (ICD) related proteins in patients with nasopharyngeal carcinoma (NPC).Methods:A total of 38 newly-treated NPC patients admitted to the Affiliated Cancer Hospital of Guizhou Medical University from November 2020 to December 2021 were enrolled, all of whom received induction chemotherapy and concurrent chemoradiotherapy, and another 20 healthy volunteers were selected as controls for a prospective study. The contents of ICD related proteins, namely calreticulin (CRT), high mobility group box 1 protein (HMGB-1) and heat shock protein 70 (HSP70) and the proportion of dendritic cell (DC) in the peripheral blood of patients were detected before treatment, after induction chemotherapy and after concurrent chemoradiotherapy, respectively. The correlation between the above indicators, general clinical data and short-term efficacy was analyzed by statistical methods such as t-test and analysis of variance (ANOVA). Results:The levels of HSP70 and HMGB-1 in peripheral blood of NPC patients before treatment were higher than those of healthy controls (both P<0.05). After concurrent chemoradiotherapy, the content of CRT was significantly higher than that before treatment ( P<0.05), whereas the difference before and after induction chemotherapy and the difference before and after concurrent chemoradiotherapy were not significantly correlated with the short-term efficacy of NPC patients. HSP70 level was significantly decreased after concurrent chemoradiotherapy ( P<0.001). There were no significant differences in the content of HMGB-1 after induction chemotherapy and concurrent chemoradiotherapy (both P>0.05). Conclusion:NPC patients receiving TPF regimen (docetaxel+cisplatin+fluorouracil) for induction chemotherapy and sequential cisplatin concurrent chemotherapy may induce ICD in NPC cells, and CRT has potential value in reflecting the clinical efficacy of NPC.

【Objective】 To solve the difficulty of RhD blood group typing in a patient with double population(DP) of red blood cells for RhD antigen by serological and genotyping analysis. 【Methods】 Separation of the two populations of red blood cells of the patient was performed using capillary centrifugation method. ABO, RhD and RhCE typing, direct anti-human globulin test (DAT), irregular antibody screening, antibody identification and blood crossmatching of the patient were conducted using the standard serological methods. The hybrid Rhesus zygosity analysis of the RHD gene was performed by PCR-RFLP method. RHD and RHCE genotype of the patients were identified by PCR-SSP method. 【Results】 The patient was B type but with DP of red blood cells for RhD, Rhc and RhE antigens. DAT of the patient was positive and the alloanti-D was detected in serum. The RHD zygosity was D-/D- homozygote. PCR-SSP testing showed the RHD gene deletion (RHD * 01N. 01/01N.01 genotype) and Ccee of RHCE genotype in the patient, which was consistent with RHD zygosity analysis. 【Conclusion】 This is a special case with D-negative phenotype which was wrongly detected as D-positive type after D-positive red blood cells transfusion in emergency. When the DP of red cells for D antigen encountered like this case, the RhD typing can be accurately determined by using RHD genotyping analysis to provide strong evidence to the clinical blood transfusion.

Autoimmune hepatitis (AIH) is a chronic progressive inflammatory disease of the liver caused by the attack of liver cells by the autoimmune system, with the features of positive serum autoantibodies, high IgG, and/or γ-globulinemia. Current studies on pregnancy in patients with AIH mainly focus on labor complications, and there is still a lack of systematic recommendations for the evaluation, treatment, and management of diseases in the progestational stage, during pregnancy, and after delivery. Although immunity is suppressed during pregnancy, poor disease control within one year before pregnancy and spontaneous drug withdrawal during pregnancy can significantly increase adverse pregnancy outcomes. Therefore, this article describes how to implement multidisciplinary collaboration and management of the whole cycle of pregnancy, so as to improve maternal and fetal safety.

Objective:To compare the differences in exploratory behaviors between infants with high-risk autism spectrum disorder (HR-ASD) and normal development (TD) infants, and to explore the relationship between exploratory behaviors and the severity of ASD symptoms.Methods:A total of 31 infants aged 6-23 months with HR-ASD in the Pediatric Clinic, the Affiliated Brain Hospital of Nanjing Medical University from January 2019 to August 2020 were retrospectively recruited in the HR-ASD group, and 37 TD infants aged from 6 to 23 months in Nanjing were selected as the TD group.The development level of the 2 groups was evaluated by Gesell development scale, and the depth and breadth of exploratory behaviors were measured in free play.Autism Diagnostic Observation Schedule (ADOS) was used to evaluate the severity of ASD symptoms in HR-ASD group.Differences in exploratory behaviors between the 2 groups were analyzed by the independent sample t-test and Chi- square test. Pearson′ s correlation analysis was performed to explore the correlation between exploratory behaviors and symptom severity in HR-ASD group.Binary Logistic regression analysis was used to explore the discrimination efficiency of sexual behavior in the 2 groups. Results:Compared with TD group, the depth and breadth of exploratory behaviors in HR-ASD group were significantly lower (55.06±25.73 vs.132.78±44.69, 4.42±2.20 vs.8.78±3.28)( t=-8.95, -6.53, all P<0.01), and atypical exploratory behavior and avoidance withdrawal behavior were significantly worse [18 cases(58.06%) vs.6 cases(16.22%), 10 cases(32.26%) vs.3 cases(8.11%)]( χ2=15.30, 6.36, all P<0.05). Significant differences in the frequency and duration of object exploration, environmental exploration and social exploration were detected between HR-ASD group and TD group (3 vs. 0 vs. 0, 45 vs. 0 vs. 0)(all P<0.01). Pearson correlation analysis showed that, the frequency of object exploratory behavior was positively correlated with the dimension of ADOS in HR-AD group ( r=0.40, P<0.05); while the duration of object exploratory behavior, the complexity of object explo-ratory behavior and the depth of exploratory behavior were negatively correlated with CARS score ( r=-0.45, -0.47, -0.42, all P<0.05). The depth of exploratory behavior was negatively correlated with the stereotyped dimension of ADOS ( r=-0.40, P<0.05). Binary Logistic regression analysis showed that the depth of exploratory behavior had a discrimination effect ( P<0.01). Conclusions:The characteristics of exploratory behaviors in HR-ASD infants are abnormal, especially the distinguishing effect on the depth of exploratory behaviors, which can be used as a candidate behavioral index for early screening of ASD.