BACKGROUND:Degeneration of nucleus pulposus cells is a key component of intervertebral disc degeneration.Ferroptosis,a novel form of programmed cell death,is closely associated with the onset and progression of intervertebral disc degeneration;however,its precise mechanisms remain unclear.OBJECTIVE:To establish an oxidative stress model in vitro by inducing ferroptosis in nucleus pulposus cells using tert-butyl hydroperoxide and to investigate the mechanisms of tert-butyl hydroperoxide-induced ferroptosis in nucleus pulposus cells,thereby elucidating the role of ferroptosis in the pathogenesis of intervertebral disc degeneration.METHODS:Nucleus pulposus cells were treated with varying concentrations of tert-butyl hydroperoxide(0,25,50,100,and 200 μmol/L),and cell morphology and viability were assessed using fluorescence microscopy and the cell counting kit-8 assay.Interventions with 100 μmol/L tert-butyl hydroperoxide,10 μmol/L RSL3,or dimethylsulfoxide were applied to nucleus pulposus cells,and cell proliferation was evaluated using the EdU assay.The expression levels of ferroptosis-related proteins(glutathione peroxidase 4,ferritin heavy chain 1,PTGS2,and ACSL4)and intervertebral disc degeneration marker proteins(matrix metalloproteinase 13 and Col2A)were analyzed via western blot and immunofluorescence.Additionally,reactive oxygen species and lipid peroxidation levels were quantified using the reactive oxygen species detection kit and C11-BODIPY probe.Mitochondrial morphological changes were observed under transmission electron microscopy.RESULTS AND CONCLUSION:(1)tert-Butyl hydroperoxide treatment significantly reduced the viability and proliferation of nucleus pulposus cells.(2)tert-Butyl hydroperoxide induced typical ferroptosis-related morphological changes in nucleus pulposus cells.(3)tert-Butyl hydroperoxide exposure led to a decrease in the expression of ferroptosis-suppressing proteins glutathione peroxidase 4 and ferritin heavy chain 1,while increasing the expression of ferroptosis-promoting factors ACSL4 and PTGS2.(4)tert-Butyl hydroperoxide elevated intracellular reactive oxygen species production and lipid peroxidation levels in nucleus pulposus cells.(5)Transmission electron microscopy revealed ferroptosis-specific mitochondrial changes in nucleus pulposus cells treated with tert-butyl hydroperoxide,including contraction,reduced cristae,and increased membrane density.(6)tert-Butyl hydroperoxide treatment also resulted in the increased expression of matrix metalloproteinase 13 and decreased expression of Col2A in nucleus pulposus cells.In conclusion,tert-butyl hydroperoxide induces ferroptosis in nucleus pulposus cells,contributing to the development of intervertebral disc degeneration.This process may represent a key pathological mechanism in intervertebral disc degeneration and offers potential targets for developing novel therapeutic strategies.

BACKGROUND:Degeneration of nucleus pulposus cells is a key component of intervertebral disc degeneration.Ferroptosis,a novel form of programmed cell death,is closely associated with the onset and progression of intervertebral disc degeneration;however,its precise mechanisms remain unclear.OBJECTIVE:To establish an oxidative stress model in vitro by inducing ferroptosis in nucleus pulposus cells using tert-butyl hydroperoxide and to investigate the mechanisms of tert-butyl hydroperoxide-induced ferroptosis in nucleus pulposus cells,thereby elucidating the role of ferroptosis in the pathogenesis of intervertebral disc degeneration.METHODS:Nucleus pulposus cells were treated with varying concentrations of tert-butyl hydroperoxide(0,25,50,100,and 200 μmol/L),and cell morphology and viability were assessed using fluorescence microscopy and the cell counting kit-8 assay.Interventions with 100 μmol/L tert-butyl hydroperoxide,10 μmol/L RSL3,or dimethylsulfoxide were applied to nucleus pulposus cells,and cell proliferation was evaluated using the EdU assay.The expression levels of ferroptosis-related proteins(glutathione peroxidase 4,ferritin heavy chain 1,PTGS2,and ACSL4)and intervertebral disc degeneration marker proteins(matrix metalloproteinase 13 and Col2A)were analyzed via western blot and immunofluorescence.Additionally,reactive oxygen species and lipid peroxidation levels were quantified using the reactive oxygen species detection kit and C11-BODIPY probe.Mitochondrial morphological changes were observed under transmission electron microscopy.RESULTS AND CONCLUSION:(1)tert-Butyl hydroperoxide treatment significantly reduced the viability and proliferation of nucleus pulposus cells.(2)tert-Butyl hydroperoxide induced typical ferroptosis-related morphological changes in nucleus pulposus cells.(3)tert-Butyl hydroperoxide exposure led to a decrease in the expression of ferroptosis-suppressing proteins glutathione peroxidase 4 and ferritin heavy chain 1,while increasing the expression of ferroptosis-promoting factors ACSL4 and PTGS2.(4)tert-Butyl hydroperoxide elevated intracellular reactive oxygen species production and lipid peroxidation levels in nucleus pulposus cells.(5)Transmission electron microscopy revealed ferroptosis-specific mitochondrial changes in nucleus pulposus cells treated with tert-butyl hydroperoxide,including contraction,reduced cristae,and increased membrane density.(6)tert-Butyl hydroperoxide treatment also resulted in the increased expression of matrix metalloproteinase 13 and decreased expression of Col2A in nucleus pulposus cells.In conclusion,tert-butyl hydroperoxide induces ferroptosis in nucleus pulposus cells,contributing to the development of intervertebral disc degeneration.This process may represent a key pathological mechanism in intervertebral disc degeneration and offers potential targets for developing novel therapeutic strategies.

Objective To explore the classification and changes of macrophage subsets in liver transplant rejection.Methods Rat liver transplantation model were established and divided into immune tolerance group(B-B),where the liver of BN rat donors was transplanted to BN rat recipients,and immune rejection group(L-B),in which the liver of Lewis rat donors was transplanted to BN rat recipients.Single-cell RNA sequencing and high-throughput RNA sequencing were used to distinguish the macrophage subsets of rat liver transplantation,and to find differential gene in rejection reactions.Immunohistochemistry was used to determine the changes and distribu-tion of protein expression and cell subsets.Results CD68 positive macrophages were higher in the rejection group than that in the tolerance group(P<0.05),and macrophages could be divided into 9 subsets.During the rejection reaction,the CXC chemokine ligand 9(CXCL9)in the 8th subsets of macrophages was significantly increased,while the gene for white blood cell differentiation antigen 74(CD74)in the 5th subsets was significantly increased(P<0.05).CD74 ranked first in the differential gene synthesis of macrophages during rejection,followed by CXCL9.Compared with the tolerance group,a large number of CD74 positive macrophages were observed in the hepatic portal area of the rejection group,and the infiltration of CD74 positive macrophages in the hepatic sinuses was also significantly increased(P<0.05),while a large number of CXCL9 positive macrophages were observed in the hepatic portal area and hepatic sinuses of the rejection group,especially in the portal area(P<0.05),and CD14 positive cells were significantly increased(P<0.05).Conclusions The CD74 positive macrophage subsets and CXCL9 positive macrophage subsets may be key subgroups in promoting liver transplant rejection,improving the mechanism of macrophage action in liver transplant rejection.

Objective To observe the effect of telephone visit for discharged patients with peritoneal dialysis catheter after surgery .Methods Sixty-seven patients with peritoneal dialysis catheter after surgery were chosen and were divided into the intervention group ( n=34 ) and the control group ( n=33 ) according to the random table .The control group received the routine health education at discharge , and the intervention group received the telephone visit ( 5 times ) after discharge .The effect of telephone visit was evaluated by the peritoneal dialysis knowledge assessment questionnaire , Nottingham health questionnarres and patient satisfaction questionnaire.Results The percent of the excellent, good, bad degrees of mastering the knowledge were respectively 52.9%, 38.2%, 8.8% in the intervention group, and were 24.2%, 30.3%, 45.5% in the control group, and the difference was statistically significant (Z=-3.252,P=0.001).The scores of sleep, physical activity, energy, pain, emotional reactions, social loneliness were respectively (10.6 ±6.3), (8.9 ±5.2), (22.3 ±0.5), (18.6 ±7.3), (13.6 ±9.3), (15.7 ±7.6) in the intervention group, and were lower than those in the control group , and the differences were statistically significant ( t =5.436,6.532,7.674, 8.213,6.567,7.324, respectively; P<0.01).The patient satisfaction in the intervention group was higher than that of the control group , and the satisfaction of director of department , the chief nurse , the attending doctor and the ordinary nurses were increased (P<0.05).Conclusions The telephone visit and guide for discharged patients with peritoneal dialysis catheter after surgery can increase their related knowledge and skill of peritoneal dialysis , and play a positive role in the life quality of patient and satisfaction with doctors and nurses .

Objective To investigate the effects of the rat serum with chronic renal failure (CRF) on ubiquitin-proteasome pathway,histone acetyltransferase p300 and activation of activating transcription factor 4(ATF4) of rat arterial vascular smooth muscle cells(VSMCs) cultured in vitro,and explore the possible mechanism.Methods To establish the rat model of CRF by 5/6 nephrectomy,VSMCs were incubated in the media with the 10％ of CRF serum or control serum in vitro.The mRNA expressions of ubiquitin(Ub),ubiquitin activating enzyme(E1),ubiquitin ligases enzymes (β-transducin repeat containing protein 1,β-TrCP1),p300 and ATF4 in the rat VSMCs were examined by using realtime PCR.Expressions of E1,β-TrCP1,p300 and ATF4 proteins in response to the CRF serum in VSMCs were determined by Western blotting analysis.The enzyme activities of 20S proteasomes in the total protein were examined by using three special fluorogenic peptide substrates.Results The CRF serum significantly promoted the mRNA expressions of Ub,E1,β-TrCP1,p300 and ATF4 in VSMCs in a time dependent manner.Compared with that in control serum group,the mRNA levels of Ub,E1,β-TrCP1,p300 and ATF4 in CRF serum group increased significantly (P ＜ 0.01).The CRF serum also increased the protein expressions of E1,β-TrCP1 and p300 in a time dependent manner.The expression of ATF4 was decreased,but the difference was not significant (P ＞ 0.05).Compared with that in control serum group,the protein expressions of E1,β-TrCP1,p300 and ATF4 in CRF serum group increased significantly (P ＜ 0.01).The activities of 20S proteasomes in the CRF serum group were significantly increased in a time dependent manner.Compared with that in control serum group,the activities of 20S proteasomes in the CRF serum group increased significantly (P ＜ 0.01).Conclusions The serum of CRF rat can effectively active the ubiquitin-proteasome pathway,but ATF4 ubiquitinylated degradation is blocked.The latter may be associated with increased expression of p300.

Objective To investigate the effects of chronic renal failure rabbit serum on proliferation and nuclear factor kappa B (NF-κB) activation of rabbit arterial smooth muscle cells (ASMCs) and to explore the possible mechanism. Methods Rabbit model of chronic renal failure was established by the ligation of renal arterial branches. ASMCs were incubated in the media with various concentrations of chronic renal failure serum cultured in vitro. Cell proliferation was assessed by MTT. Cell apoptosis was detected by Hoechst33342 staining. NF-κB p65 nuclear translocation was analyzed by immunofluorescence. Expression of proliferating cell nuclear antigen (PCNA) and NF-κB p65 proteins in response to chronic renal failure serum in ASMCs was determined by Western blotting. Results Lower concentrations of chronic renal failure serum (≤ 10%) could significantly promot the proliferation of ASMCs in a dose- and time-dependent manner. Higher concentrations of chronic renal failure serum (＞10%) could significantly inhibit the proliferation and induce apoptosis of ASMCs compared to the normal control (P＜0.05). Under the stimulation of lower concentrations of chronic renal failure serum, the expression of PCNA and NF-κB p65 increased significantly compared to the normal control (P＜0.01), while decreased markedly under the stimulation of higher concentrations of chronic renal failure serum compared to the normal control (P＜0.01). Under the stimulation of 10% chronic renal failure serum, nuclear translocation of NF-κB p65 in ASMCs was found. Conclusions Different concentrations of chronic renal failure rabbit serum can effectively induce ASMCs proliferation or apoptosis. The mechanism of promoting proliferation may be mediated by activating NF-κB, which will be useful for the treatment of accelerated atherosclerosis in chronic renal failure.

Objective To investigate the effects of the serum from chronic renal failure (CRF) rats on endoplasmic reticulum stress (ERS) and activation of activating transcription factor 4 (ATF4) of rat arterial smooth muscle cells (ASMCs) cultured in vitro and explore the possible mechanism. Methods The rat model of CRF was established by 5/6 nephrectomy. ASMCs were incubated in the media with 10% or 20% serum of CRF rats cultured in vitro. ATF4 nuclear translocation were analyzed by immunofluorescence. GRP78 (glucose regulated protein 78), p-PERK (pancreatic ER kinase) and ATF4 proteins in response to the CRF serum in ASMCs were determined by Western blot. The ATF4-DNA binding activity was analyzed by electrophoretic mobility shift assay (EMSA). Results The CRF serum could significantly promote the expression of GRP78 of ASMCs in dose-dependent manner. Under the stimulation of 20% CRF serum, nuclear translocation of ATF4 in ASMCs was found. Compared with control serum group, the expression of ATF4 and p-PERK of CRF serum group was increased significantly (P<0.01). The CRF serum could increase the ATF4-DNA binding activity. Conclusion The CRF rat serum can effectively induce ERS of ASMCs and activate the pathway of PERK-ATF4.

Objective To investigate the activation of NF-κB and regulation by ubiquitin (Ub)-proteasome pathway in the aorta of rats with chronic renal failure(CRF).Methods The CRF rat model was established by right nephreetomy and left branch renal artery ligation.The CRF rats were were randomly divided into simple CRF group(n=20)and CRF+M used as control group(CON).The NF-κB and the Ub mRNA expression were detected by RT-PCR,and its protein expression was analyzed by immunohistochemistry method.The activity of NF-κBwas mesured by EMSA method.The concentration of IL-1 and TNF-α was detected by ELISA.Results Compared with the CON group,the concentration of serum IL-1 and TNF-α was increased significantly in CRF group [IL-1:(9.02±1.29) vs (2.74±0.96)mg/L,P＜0.01;TNF-α:(50.02±9.52) vs (14.04±1.29)mg/L,P＜0.01]at month 4 after operation.The mRNA expression of NF-κB and Ub in the aorta of CRF group was 1.38 and 1.29 times as that of CON group(P＜0.01).and the protein expression of NF-κB and Ub was 3.75 and 20.5 times as that of CON group(P＜0.01).Compared with the CON group,the activity of NF-κB in the aorta of rats of CRF group was elevated markedly at month 4 after operation(P＜0.01).All the indices were further increased at month 6 after operation.Compared with CRF group,the concentrations of serum IL-1and TNF-α were decreased significantly in CRF+M group[IL-1:(2.94±0.33)mg/L,P＜0.01;TNF-α:(12.80±2.12)mg/L,P＜0.01].The mRNA and protein expression of NF-κB and Ub were also decreased markedly(P＜0.01),and the activity of NF-κB was decreased significantly at month 4 to 6 after operation(P＜0.01).But the amount of ubiquitnative protein was increased significantly in the aorta of CRF+M group as compared to CRF group(P＜0.01). Conclusion The inflammatory signal pathway of ubquitin-proteasome-NF-κB pathway was activated in the aorta of CRF rats,and the proteasome was probablely an important pharmacological intervention target to regulate the activation of NF-κB.

Objective To investigate the role of PTEN protein in secretion of collagen Ⅳ and fibronectin after stimulation of transforming growth factor beta 1(TGF-?1) from renal fibroblasts of rat in vitro.Methods The cultured rat renal fibroblasts were transfected with the reconstructed adenovirus containing PTEN or adenovirus only containing green fluorescence protein(GFP).The fibroblsts were treated in four manners:control group with no added treatment,TGF-?1 group with TGF-?1 stimulation,PTEN+ TGF-?1 group with TGF-?1 stimulation after Ad-PTEN transfection,and GFP+ TGF-?1 group with TGF-?1 stimulation after Ad-GFP transfection.Invert fluorescent microscope was used to detect the GFP expression,meanwhile the PTEN mRNA was determined by RT-PCR method.36h after the transfection,TGF-?1 was added into the culture medium in a concentration of 10ng/ml.After another 24h,ELISA method was used to evaluate the level of collagen Ⅳ and fibronectin.Results The expressions of both GFP and PTEN mRNA increased obviously after the rats' renal fibroblasts were transfected with adenovirus.The secretion of collagen Ⅳ and fibronectin increased significantly in both TGF-?1 group and GFP+ TGF-?1 group compared with that in control group,and decreased markedly in PTEN+ TGF-?1 group compared with that in both TGF-?1 group and GFP+ TGF-?1 group(P

Objective To explore the role of bone morphorgenetic protein-7(BMP-7) in the renal tubulointerstitial lesions induced by unilateral ureteral obstruction (UUO).Methods After dividing into normal control, sham operation and unilateral ureteral obstruction (UUO) groups, sixty rats were sacrificed at postoperative day 1, 3, 7, 14. The levels of BMP-7 and TGF-?1 mRNA were examined by RT-PCR. The sites and levels of protein expression of BMP-7, TGF-?1 and ?-SMA were detected by immunohistochemistry staining. Results Compared to control groups, BMP-7 mRNA was significantly decreased, but TGF-?1 mRNA was significantly increased in UUO rats. Immunohistochemistry staining studies indicated that BMP-7 mainly expressed in renal tubule and interstitium, rarely in glomeruli. In UUO rats, the protein expression of BMP-7 decreased, but the expression of TGF-?1 and ?-SMA increased in varying degrees according to the obstructed days. The renal tubulointerstitial expression of BMP-7 was significantly negatively correlated with the expression of TGF-?1 and ?-SMA. Conclusion The loss of BMP-7 is explored in the early phase of fibrotic process and may play a very important role in mediating the tubulointerstital lesions.