Objective:To investigate the inhibitory effect and mechanism of puerarin(Pue)on hydrogen peroxide(H2 O2)induced ferroptosis in the rat Schwann cell derived cell line RSC96.Methods:The RSC96 cells were incuba-ted with H2 O2 to establish a cellular injury model,while a subset of cells were co-incubated with H2 O2 and Pue.The vi-ability of cells was examined using the CCK-8 assay.The intracellular levels of glutathione(GSH),total superoxide dismutase(T-SOD),reactive oxygen species(ROS),malondialdehyde(MDA),and ferrous ions(Fe2+)levels were quantified using commercial kits.Western blot was employed to detect the protein expression level of glutathione peroxi-dase 4(GPX4),cyclooxygenase-2(COX-2),solute carrier family 7 member 11(SLC7A11),nuclear factor erythroid 2-related factor 2(Nrf2),and heme oxygenase-1(HO-1).Immunofluorescence assay was performed to examine the expression and nuclear distribution of Nrf2.Results:Pue pretreatment significantly increased the survival rate of H2 O2-treated RSC96 cells,increased the intracellular content of GSH and T-SOD,and inhibited the concentration of ROS and MDA.It also activated the nuclear translocation of Nrf2 and upregulated GPX4,SLC7A11,HO-1,and Nrf2 proteins in RSC96 cells;This effect was abolished by the Nrf2 inhibitor ML385.Conclusion:Pue treatment alleviated the H2 O2-induced ferroptosis of RSC96 cells via the Nrf2/SLC7A11/GPX4 signaling pathway.

Objective:To investigate the inhibitory effect and mechanism of puerarin(Pue)on hydrogen peroxide(H2 O2)induced ferroptosis in the rat Schwann cell derived cell line RSC96.Methods:The RSC96 cells were incuba-ted with H2 O2 to establish a cellular injury model,while a subset of cells were co-incubated with H2 O2 and Pue.The vi-ability of cells was examined using the CCK-8 assay.The intracellular levels of glutathione(GSH),total superoxide dismutase(T-SOD),reactive oxygen species(ROS),malondialdehyde(MDA),and ferrous ions(Fe2+)levels were quantified using commercial kits.Western blot was employed to detect the protein expression level of glutathione peroxi-dase 4(GPX4),cyclooxygenase-2(COX-2),solute carrier family 7 member 11(SLC7A11),nuclear factor erythroid 2-related factor 2(Nrf2),and heme oxygenase-1(HO-1).Immunofluorescence assay was performed to examine the expression and nuclear distribution of Nrf2.Results:Pue pretreatment significantly increased the survival rate of H2 O2-treated RSC96 cells,increased the intracellular content of GSH and T-SOD,and inhibited the concentration of ROS and MDA.It also activated the nuclear translocation of Nrf2 and upregulated GPX4,SLC7A11,HO-1,and Nrf2 proteins in RSC96 cells;This effect was abolished by the Nrf2 inhibitor ML385.Conclusion:Pue treatment alleviated the H2 O2-induced ferroptosis of RSC96 cells via the Nrf2/SLC7A11/GPX4 signaling pathway.

ObjectiveTo explore the potential therapeutic targets and molecular mechanisms of menstrual blood-derived stem cells (MenSCs) transplantation combined with exercise training in promoting recovery in rats with spinal cord injury (SCI) through transcriptome sequencing analysis. MethodsFemale SD rats aged two months were selected and a SCI model was established by a hemisection at the tenth thoracic vertebra (T10). The rats were then divided into two groups: the Cell and Treadmill Training (CTMT) group, which received MenSCs transplantation and treadmill training after SCI, and the SCI group (control), with 12 rats in each group. One week after modeling, the CTMT group received a microinjection of 1×10⁵ MenSCs at the injury site, followed by two weeks of weight-supported aerobic exercise training. Spinal cord tissue from the injury site was selected for transcriptome sequencing, and mRNA expression data from both the SCI and CTMT groups were analyzed. Differential gene expression, GO (Gene Ontology) functional enrichment, KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment, and protein-protein interaction (PPI) network analyses were performed. Motor function recovery was assessed using the Basso, Beattie, and Bresnahan (BBB) score, while histopathological changes at the injury site were evaluated through hematoxylin-eosin (HE) staining. Real-time fluorescent quantitative PCR and Western blotting were used to verify the expression of differentially expressed genes. ResultsTranscriptome sequencing analysis showed 247 upregulated genes and 174 downregulated genes in the CTMT group compared to the SCI group. Notably, genes such as Bdnf, Hmox1, Sd4, Mmp3, and Cd163 were significantly upregulated [|log2(FoldChange)|≥0.66, P<0.05]. KEGG pathway enrichment analysis and GO functional enrichment analysis indicated that these differentially expressed genes were mainly involved in growth and development, metabolic reactions, and immune-inflammatory processes, such as axon growth and the electron transport chain. The Bdnf gene was notably enriched in the PI3K-Akt signaling pathway. The BBB score showed that MenSCs transplantation combined with exercise training significantly improved the motor function of SCI rats. HE staining revealed that pathological changes at the injury site were significantly reduced in the treatment group. Furthermore, real-time quantitative PCR and Western blotting confirmed that brain-derived neurotrophic factor (BDNF) mRNA and protein expression levels in the CTMT group were significantly higher than those in the SCI group (P<0.001). ConclusionThe combined exercise training with MenSCs effectively promotes the recovery of motor function in SCI rats by upregulating BDNF expression, providing a novel strategy for SCI treatment.

AIM:The study aimed to explore the effect of curcumin(Cur)on lipopolysaccharide(LPS)-in-duced RAW264.7 cell-derived osteoclasts,together with its underlying mechanism.METHODS:An osteoclast model was established by treating RAW264.7 cells with LPS.The viability of the cells was assessed by CCK-8 assays and osteo-clast formation was evaluated by tartrate-resistant acid phosphatase(TRAP)activity.The levels of reactive oxygen species(ROS),Fe2+,glutathione(GSH),and malondialdehyde(MDA)were examined by biochemical assays.Mitochondrial morphology was assessed by transmission electron microscopy.The mRNA and protein levels of p53,glutathione peroxi-dase 4(GPX4),and solute carrier family 7 member 11(SLC7A11)were determined by RT-qPCR and Western blot,re-spectively.RESULTS:Treatment with LPS successfully induced osteoclasts formation in RAW264.7 cells.The TRAP results showed that compared with the LPS-treated group,the number of osteoclasts and TRAP activity in the curcumin-treated group decreased dose-dependently(P<0.01).Compared with the control group,the LPS+Erastin group showed significantly increased TRAP activity(P<0.01),while after curcumin treatment,the TRAP activity declined in a dose-de-pendent manner(P<0.01).The results of the biochemical tests showed that compared with the control group,the LPS+Erastin group had significantly elevated levels of ROS,Fe2+,and MDA,while the GSH level was significantly reduced(P<0.01),and compared with the LPS+Erastin group,the ROS,Fe2+,and MDA levels in the curcumin group decreased(P<0.01)and GSH levels increased(P<0.01).These effects were all dose-dependent.Transmission electron microscopy showed that compared with the LPS group,the LPS+Erastin group had reduced mitochondrial cristae and increased mem-brane density,while after treatment with curcumin,both these effects were reversed.The RT-qPCR and Western blot re-sults showed that compared with the control group,the mRNA and protein levels of p53 in the LPS+Erastin group were sig-nificantly increased,while those of of SLC7A11 and GPX4 were significantly reduced(P<0.01).After curcumin treat-ment,the p53 mRNA and protein levels were reduced while the levels of SLC7A11 and GPX4 were increased(P<0.01).CONCLUSION:Curcumin can inhibit lipopolysaccharide-induced differentiation of RAW264.7 cells into osteoclasts,and its mechanism may be related to the inhibition of the p53/SLC7A11/GPX4 signaling pathway.

AIM:To explore the promotion of ferroptosis by artesunate(Art)and its underlying mechanism in osteosarcoma.METHODS:Human osteosarcoma U-2 OS cells were divided into 4 groups:control,ferrostatin-1(Fer-1),Art,and Art+Fer-1 groups.Cell viability was measured by CCK-8 assay,while reactive oxygen species(ROS),Fe2+,and glutathione(GSH)levels were measured using biochemical assays.Mitochondrial morphology was evaluated by trans-mission electron microscopy.The mRNA and protein levels of p53,solute carrier family 7 member 11(SLC7A11)and glutathione peroxidase 4(GPX4)were determined by RT-qPCR and Western blot,respectively.RESULTS:It was found that Art significantly reduced the growth of U-2 osteosarcoma cells,as well as inducing ferroptosis by promoting the accu-mulation of Fe2+and ROS and reducing GSH levels,while the ferroptosis inhibitor ferrostatin-1 inhibited ferroptosis.It was also observed that Art affected mitochondrial morphology,resulting in smaller mitochondria,higher mitochondrial mem-brane density,and reduced numbers of cristae.Treatment with Art also downregulated both the mRNA and protein expres-sion of the ferroptosis-associated genes SLC7A11 and GPX4,while upregulating expression of p53,an upstream regulator of ferroptosis,thus inducing ferroptosis.CONCLUSION:Artesunate induces ferroptosis in osteosarcoma cells through the p53/SLC7A11/GPX4 signaling pathway.

Objective:To investigate the inhibitory effect and mechanism of curcumin on the rat Schwann cell derived cell line RSC96 cells pyroptosis induced by hydrogen peroxide.Methods:The rat Schwann cell derived cell line RSC96 cells were selected and treated with hydrogen peroxide to establish the peripheral nerve oxidative injury mod-el,curcumin intervention was also given.Cell viability was detected by CCK-8 kit.Lactate dehydrogenase(LDH)kit was used to detect the LDH leakage rate.The level of IL-1 β was detected by enzyme linked immunosorbent assay(ELISA).The protein expression levels of NLRP3,caspase-1,cleaved caspase-1,GSDMD,GSDMD-N and IL-1 βwere detected by Western Blot.Results:Cur significantly increased the viability of RSC96 cells treated with H2O2,decreased LDH leakage rate,down-regulated IL-1 β secretion,and inhibited the protein expression of NLRP3,caspase-1,cleaved caspase-1,GSDMD,GSDMD-N,and IL-1 β.Conclusion:Curcumin can inhibit hydrogen peroxide-induced pyroptosis of RSC96 cells through the NLRP3/caspase-1/GSDMD signaling pathway,thus exerting neuroprotective effect.

Objective:To investigate the inhibitory effect and mechanism of curcumin on the rat Schwann cell derived cell line RSC96 cells pyroptosis induced by hydrogen peroxide.Methods:The rat Schwann cell derived cell line RSC96 cells were selected and treated with hydrogen peroxide to establish the peripheral nerve oxidative injury mod-el,curcumin intervention was also given.Cell viability was detected by CCK-8 kit.Lactate dehydrogenase(LDH)kit was used to detect the LDH leakage rate.The level of IL-1 β was detected by enzyme linked immunosorbent assay(ELISA).The protein expression levels of NLRP3,caspase-1,cleaved caspase-1,GSDMD,GSDMD-N and IL-1 βwere detected by Western Blot.Results:Cur significantly increased the viability of RSC96 cells treated with H2O2,decreased LDH leakage rate,down-regulated IL-1 β secretion,and inhibited the protein expression of NLRP3,caspase-1,cleaved caspase-1,GSDMD,GSDMD-N,and IL-1 β.Conclusion:Curcumin can inhibit hydrogen peroxide-induced pyroptosis of RSC96 cells through the NLRP3/caspase-1/GSDMD signaling pathway,thus exerting neuroprotective effect.

Diabetic foot with diabetic peripheral neuropathy(DPN)can lead to peripheral nerve dysfunction. Ex-ploring appropriate animal models of the disease can make a better understanding of the occurrence, development, patho-genesis,prevention and treatment of DPN. Therefore,a reasonable construction of animal models is related to the success of experimental research. Up to date, experimental animals used in the research of diabetic foot neuropathy include rats, nude mice,transgenic mice,and so on. Nerve injury models can be generated by different means,such as ischemia reper-fusion,resection or ligation of nerves and scalds. This article will review the recent research progress of diabetic foot neu-ropathy animal models.

Objective To study the effects of Puerarin(Pue)on survival rate,expression of GAP-43 and NGF in spinal motoneurons following brachial roots avulsion. Methods From March, 2014 to December, 2015, 192 adult Sprague Dawley rats were divided into four groups: Avulsion, Pue 50 treatment, Pue 100 and Pue 200 groups. The right C5-C7nerve roots were avulsed through the methods of cervical dorsal approach. Puerarin or normal saline was given immediatedly once daily to the rats respectively by the intraperitoneal injection. The rats were killed at 1, 2, 4 and 6 weeks after injury. The paraffin sections of C7 segment were stained with neutral red. Expression of GAP-43 and NGF were detected by Western blot. Results Compared to the avuled group, the motoneuron survival rates of Pue 100 and Pue 200 dose treatment groups increased on the second week and the sixth week(P < 0.05 or P < 0.01), and the Pue 200 dose treatment group increased on the fourth week(P < 0.01).In the anterior horn of all three groups, expression of GAP-43 increased from the 1st week to the 6 week after the operation, and reached the peak at the 4th week, and decreased at the 6th week. Compared to the avuled group, expression of GAP-43 protein increased in the Pue 100 and Pue 200 dose treatment groups at the 1st week and 2nd week,the expression of NGF protein increased in the three Pue treatment groups at four time points(P < 0.05 or P < 0.01). Conclusion Puerarin can improve the survival rates of spinal motoneurons after the brachial plexus root avulsion, and the expression of GAP-43 and NGF increased, suggesting that Puerarin may play an role in the repair of brachial plexus avulsions by promoting the ex-pression of nerve growth factors.

Breast cancer is one of the most common malignant tumors in women, which affects female physical and mental health seriously, and even has a life-threatening effect. TCM plays a more and more important role in the treatment of breast cancer today. Many veteran TCM doctors hold unique opinions about it, especially for postoperative complications. Doctor TAN Qing-gang is one of the most famous and experienced TCM doctors in the field of TCM in Enshi, Hubei Province. He is expert in the diagnosis and treatment on miscellaneous diseases of TCM. This article summarized the experience of Doctor TAN in treatment for postoperative breast cancer.