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 observe the effects of Fufang Lishao Pills (FFLSP) on the proinflammatory factors, pain-relatedproteins and JAK2/STAT3 signaling pathways in nitroglycerin-induced chronic migraine model, and exploring thepharmacological target and mechanism of FFLSP on chronic migraine rats. Methods: Male Sprague-Dawley rats wererandomly divided into 6 groups: Control, Migraine, FFLSP-L (420 mg·kg-1), FFLSP-M (840 mg·kg-1), FFLSP-H (1680mg·kg-1) and Flunarizine Hydrochloride group (FH, 1 mg·kg-1) . Chronic migraine model was made by subcutaneousinjection of nitroglycerin (10 mg·kg-1) once every 3 days for 5 times. The rats were treated with FFLSP by intragastricadministration once a day for 30 days. Western blot was used to detect the protein expression of iNOS, COX-2, CGRPand c-Fos and the phosphorylation levels of JAK2 and STAT3 in cortex of rats. The production of IL-1β and TNF-αwere detected by enzyme linked immunosorbent assay (ELISA) . Results: Compared with Control rats, and the level ofiNOS, COX-2, CGRP, c-Fos expression (P ＜ 0.01) and IL-1β, TNF-α production remarkably up-regulated (P ＜ 0.05), and the phosphorylation of JAK2 and STAT3 also significantly increased in cotex of Migrainerats (P ＜ 0.01) . However, compared with Migrainerats, the levels of iNOS, COX-2, CGRP, c-Fos expression and IL-1β, TNF-α productionobviously declined (P ＜ 0.05; P ＜ 0.01), and the phosphorylation level of JAK2 and STAT3 showed a significantlydecrease in the cortex of Migraine rats with FFLSP treatment (P ＜ 0.01) . Conclusion: This study demonstrates that thepharmacological mechanism of FFLSP in improving chronic migraine may be achieved by inhibiting neuroinflammationthrough the blockage of JAK2/STAT3 pathwayin the cortex of rat with nitroglycerin induction.

OBJECTIVE:To establish the quality standard for Gentiana scabra dispensing granule. METHODS:TLC was ad-opted to identify G. scabra in the preparation;HPLC was adopted to determine the content of gentiopicroside in the preparation:the column was Dionex C18 with mobile phase of methanol- water(25∶75,V/V)at a flow rate of 1.0 ml/min,the detection wave-length was 270 nm,column temperature was 30 ℃,the injection volume was 10 μl. RESULTS:TLC showed clear spots and good separation. The linear range of gentiopicroside injection volume was 0.302 6-3.026 μg (r=0.999 9);RSDs of precision,stability and reproducibility tests were lower than 2%;recovery was 98.27%-99.56%(RSD=0.68%,n=6). CONCLUSIONS:The estab-lished standard can be used for the quality control of Gentiana scabra dispensing granule.

Objective To establish the molecular weight distribution of anti-HBV placenta transfer factor injection (PSTF) by electrophoresis, HPLC and MS.Methods Using the methods of SDS-PAGE, HPSEC, MALDI-TOF-MS to test the molecular of PSTF.Results The Molecular was 8000 Da by SDS-PAGE.There were 5026.67,6783.44,7496.42,8736.55 Da components in PSTF by HPSEC.The main component molecular was 2972 Da and the maximum molecular component was 8194 Da.Conclusion HPSEC is simple and rapid to determine the maximum component molecular of PSTF.

Objective To compare the analgesic effect ofHuamoyan Keli(HMYKL) between Kunming mice and BALB/c mice.Methods Eighty Kunming mice and eighty BALB/c mice were randomly divided into five groups, respectively: a control group, a ibuprofen group, a HMYKL high-dose group(13.98 g crude dru g/kg), HMYKL middle-dose group(6.99 g crude drug/kg)and a HMYKL low-dose group(3.50 g crude dru g/kg). There were 16 mice in each group with 8 male mice and 8 female mice. Drugs were administered intragastrically daily for 5 days. After 1 h of drug treatment on day 4, the latency of tail-flick response was evaluated using illuminated pain measurement instrument. After the last drug treatment, pain model was established by i.p. acetic acid, writhing latency and writhing times were recorded to evaluate the analgesic effect of HMYKL.Results In tail-flick test, there was no statistical difference among male and female Kunming mice in the HMYKL groups. Among male BALB/c mice, the latency in HMYKL middle-dose group was significantly longer than that in the control group(4.84±1.16 minvs. 3.93±0.76 min,P<0.05). In writhing test, compared with control group(19.06±6.34), the writhing times among BALB/c mice were decreased in HMYKL high-dose group(8.56±6.19), HMYKL middle-dose group(5.73±3.17), HMYKL low-dose group(6.88±4.59)(allP<0.01).Conclusion All dose groups of HMYKL showed good analgesic effect on the pain induced by chemical stimulation and there was no sex difference. Kunming mice were not suitable for the evaluation of the analgesic pharmacodynamics because of their large individual difference. On contrast, BALB/c mice which had less individual difference could be used to produce the model of pain.

The morphologc observation of immunoreactive structures and the quantitative analysis of VIP, SP, L-ENK and SOM in the small intestine of the control and the irradiated rats were studied by using the methods of immunocytochemistry (ICC) and radioimmunoassay (RIA). Meanwhile, histopathologic study was carried out by light and electron microscope (LM, EM). The results and findings are as follows.. (1) Viewed on LM, no obvious change of the enteric ganglia was found in the small intestine of the irradiated rats; while under EM, the axons and synaptic vesicles showing swelling and vacuolar degeneration was observed. These stuctures have close relation to the transport and release of the neurotransmitter. (2) The normal morphology and the distribution of the immunoreactive nerves and endocrine cells of VIP, SP, L-ENK and SOM were revealed in the intestine of rats by ICC method. Four kinds of immunoreactive peptidergic nerves were confirmed as an important part of the enteric nervous system. No obvious change was found on the structure of the peptidergrc nerves after 2000 rad r-irradiation. The radiosensitivity of the peptide-containing endocrine cells was similar to that of the intestinal epithelium. (3) We first confirmed with RIA that the level of the 4 regulatory peptides remarkably changed in the small intestine of the irradiated rats. Combining the physiologic founctions and the trends of the 4 regulatory peptides, as well as the pathologic characteristics and process after r-irradiation, it is proposed that the 4 kinds of peptide may be involved in the pathologic process of the mucosa damage and the disturbance of intestinal motility etc. in the intestinal radiation sickness of rats.

SOM in sequence.The results of RIA wereconsistent with the density of the peptide-containing structures by ICC.