Objective:To preliminarily explore whether sirtuin1 (SIRT1) activation can inhibit neuronal ferroptosis in rats after traumatic brain injury (TBI) by regulating hypoxia-inducible factor-1α (HIF-1α)-mediated glycolysis.Methods:(1) Six SD rats were randomly divided into sham-operated group and TBI group, with 3 rats in each group; TBI model in the TBI group was established by hydraulic impact method, and rats in the sham-operated group underwent same surgery without impact. Cortical tissues of the two groups were sent for tandem mass tag (TMT) labeled quantitative proteomics detection to analyze the differential expression proteome; Kyoto encyclopedia of genes and genomes (KEGG) and gene set enrichment analysis (GSEA) were used to detect pathway enrichment of the screened differential proteins. (2) Twelve SD rats were randomly divided into sham-operated group and 1-day, 3-day and 7-day post-TBI groups, with 3 rats in each group. Treatment methods were the same as above; Western blotting was used to detect SIRT1 protein expression. (3) Forty-eight rats were randomly divided into sham-operated group, TBI group, TBI+vehicle group and TBI+SIRT1 agonist group, with 12 rats in each group; rats in the sham-operated group and TBI group accepted treatment as above; rats in the TBI+SIRT1 agonist group were intraperitoneally injected with SRT1720 (dissolved in ≤ 5% dimethyl sulfoxide, at a dose of 20 mg/kg) within 30 minutes after modeling, twice a day (with an interval of 12 hours); and rats in the TBI+vehicle group were injected with same dose of dimethyl sulfoxide at the same time. One d after modeling, neurological deficit was assessed using modified Neurological severity score (mNSS), brain water content was measured by dry-wet weight method, histopathological changes in the cortical lesions were observed by HE staining, mitochondrial ultrastructure was examined by transmission electron microscopy, malondialdehyde (MDA) content and superoxide dismutase (SOD) activity in the brain tissues were detected by colorimetry, and protein expressions of SIRT1, HIF-1α (key protein in the glycolytic pathway), glutathione peroxidase 4 (GPX4, key protein in the ferroptosis pathway), and acyl-CoA synthetase long-chain family member 4 (ACSL4, key protein in the ferroptosis pathway) were evaluated by Western blotting.Results:(1) KEGG analysis revealed that the glycolysis pathway and HIF-1 signaling pathway were obviously enriched in the cortical tissues of rats in the TBI group compared with the sham-operated group; GSEA showed that the HIF-1 signaling pathway (mmu04066) and ferroptosis pathway (mmu04216) gene sets in the cortical tissues of rats in the TBI group exhibited enrichment trends compared with those in the sham-operated group. (2) Compared with the sham-operated group, the 1-day, 3-day, and 7-day post-TBI groups had significantly decreased SIRT1 protein expression ( P<0.05), with the most prominent decline in 1-day post-TBI group. (3) Compared with the TBI+vehicle group, rats in the TBI+SIRT1 agonist group showed significantly reduced mNSS score and brain tissue water content (9.83±1.17 vs. 7.66±1.21; [83.62±0.91]% vs. [80.09±0.68]%, P<0.05). HE staining indicated clearer structure of the cortical area at the injury sites, and improved neuron morphology in the TBI+SIRT1 agonist group compared with those in the TBI+vehicle group; and transmission electron microscopy showed reduced mitochondrial shrinkage and partial restoration of cristae structures in the TBI+SIRT1 agonist group compared with those in the TBI+vehicle group. Compared with the TBI+vehicle group, the TBI+SIRT1 agonist group exhibited significantly decreased MDA content ([62.72±9.20] nmol/g vs. [39.34±3.48] nmol/g), increased SOD activity ([1.95±0.23] U/mg vs. [2.48±0.14] U/mg), elevated GPX4 protein expression (0.37±0.04 vs. 0.46±0.03), and decreased HIF-1α and ACSL4 protein expressions (1.16±0.15 vs. 0.81±0.12; 1.14±0.06 vs. 1.29±0.04), with significant differences ( P<0.05). Conclusion:SIRT1 activation can exert neuroprotective effect by inhibiting HIF-1α-mediated glycolysis and reducing neuronal ferroptosis after TBI.

Objective:Explore the protective effect and mechanism of methyl badosolone (CDDO-Me) on rats with traumatic brain injury (TBI).Methods:A total of 72 SPF-grade SD rats aged 8 weeks were randomly (random number) divided into 4 groups ( n=18) using the random number table method: Sham, TBI, TBI+Vehicle, and TBI+CDDO-Me. The rat TBI model was established using the hydraulic impact head injury method. The TBI+CDDO-Me group was administered CDDO-Me (dissolved in 1% DMSO, at a dose of 10 mg/kg) via intraperitoneal injection 30 minutes after modeling, twice a day for a total of 3 days. On the third day after modeling, brain tissue was collected for pathological and water content detection after mNSS scoring. Immunofluorescence double staining was used to detect the expression of nuclear factor erythroid2 related factor 2 (Nrf2); immunohistochemical staining was used to detect the expression of ionized calcium binding adapter molecule-1(Iba-1); ELISA was used to detect the levels of tumor necrosis factor-α(TNF-α), interleukin (IL)-1β, and IL-18 in serum; kits were used to detect the levels of malondialdehyde (MDA) and reactive oxygen species (ROS); Western blot was used to detect the expression of the Nrf2 pathway, B-cell lymphoma-2 (BCL-2), and BCL-2 associated X protein (BAX). Results:(1) Compared with the Sham group, the mNSS scores and water content in the injured cortex of the TBI group rats were significantly increased (both P<0.05), and both significantly decreased after CDDO-Me intervention (both P<0.05). (2) Compared with the Sham group, the proportion of Nissl-stained injured neurons and apoptotic positive cells in the TBI group rats were significantly increased (both P<0.05), and both significantly decreased after CDDO-Me intervention (both P<0.05), accompanied by a decrease in BAX protein expression and upregulation of BCL-2 protein expression (both P<0.05). (3) Immunofluorescence and Western blot results showed that compared with the Sham group, the expression of total Nrf2, nuclear Nrf2, HO-1, and NQO1 proteins in the TBI group were all increased (all P<0.05), and the increase was more significant after CDDO-Me intervention (all P<0.05). (4) Immunohistochemistry and ELISA results showed that compared with the Sham group, the levels of MDA, ROS, Iba-1 in brain tissue and the levels of TNF-α, IL-1β, and IL-18 in serum in the TBI group rats were all significantly increased (all P<0.05), and all significantly decreased after CDDO-Me intervention (all P<0.05). Conclusion:CDDO-Me helps to reduce oxidative stress and inflammatory responses in TBI rats, and the mechanism may be related to the activation of the Nrf2/HO-1 antioxidant stress pathway.

Objective:To investigate the effect of asparagine synthetase (ASNS) knockdown on the senescence of retinal pigment epithelial (RPE) cells and its potential molecular mechanism.Methods:Human ARPE-19 RPE cells were divided into four groups: control group, short hairpin RNA targeting ASNS (shASNS) group, control+ (Janus kinase) JAK inhibitor group, and shASNS+ JAK inhibitor group, which were treated with short hairpin RNA control+ dimethyl sulfoxide (DMSO), shASNS+ DMSO, control+ JAK inhibitor and shASNS+ JAK inhibitor for 12 hours, respectively.An RPE cell senescence model was established by cell treatment with 500 μmol/L H 2O 2 for 24 hours.The mRNA and protein levels of ASNS and JAK were detected by real-time fluorescent quantitative PCR and Western blot, respectively.Reactive oxygen species (ROS) level within cells was measured using a kit.Cell cycle phase distribution and apoptosis rates were analyzed by flow cytometry.Cell viability from day 1 to day 5 of culturing was assessed via MTT assay.Senescent cell ratio was determined by β-galactosidase staining.Cellular damage was evaluated via immunofluorescence staining.Senescence-associated proteins (p16, pRb), and RPE markers (KRT18, CTNNB1, TJP1, BEST1) were quantified by Western blot. Results:Compared with the control group, mRNA and protein expression levels of ASNS and JAK were significantly reduced in the shASNS group, control+ JAK inhibitor group, and shASNS+ JAK inhibitor groups (all P<0.05).DCFH-DA staining revealed significantly lower ROS level in the shASNS group, control+ JAK inhibitor group, and shASNS+ JAK inhibitor group than in the control group (all P<0.05).Flow cytometry showed that there were more G2-phase cells and significantly reduced apoptosis rate in the shASNS group, control+ JAK inhibitor group, and shASNS+ JAK inhibitor group compared with the control group (all P<0.01).MTT assay indicated higher cell viability at all time points in the shASNS group, control+ JAK inhibitor group, and shASNS+ JAK inhibitor group compared with the control group, with statistically significant differences (all P<0.01).β-galactosidase-positive cell ratios in the shASNS, control+ JAK inhibitor, and shASNS+ JAK inhibitor groups were (42.36±1.28)%, (43.20±1.89)%, (25.97±1.13)%, respectively, which were significantly lower than (52.25±0.64)% in the control group (all P<0.001).p16 and pRb protein expression were decreased and γ-H2AX fluorescence intensity was attenuated in the shASNS group, control+ JAK inhibitor group, and shASNS+ JAK inhibitor group compared with the control group (all P<0.01).KRT18 and CTNNB1 expressions were upregulated, whereas TJP1 and BEST1 were downregulated in the shASNS group, control+ JAK inhibitor group, and shASNS+ JAK inhibitor group compared with the control group (all P<0.05).The shASNS+ JAK inhibitor group exhibited higher KRT18 and CTNNB1 expressions and lower TJP1 and BEST1 expressions than the shASNS and control+ JAK inhibitor groups (all P<0.05). Conclusions:ASNS knockdown can promote RPE cell proliferation, mitigate cellular damage, and delay senescence by suppressing the JAK pathway.

Objective:To investigate the effect of asparagine synthetase (ASNS) knockdown on the senescence of retinal pigment epithelial (RPE) cells and its potential molecular mechanism.Methods:Human ARPE-19 RPE cells were divided into four groups: control group, short hairpin RNA targeting ASNS (shASNS) group, control+ (Janus kinase) JAK inhibitor group, and shASNS+ JAK inhibitor group, which were treated with short hairpin RNA control+ dimethyl sulfoxide (DMSO), shASNS+ DMSO, control+ JAK inhibitor and shASNS+ JAK inhibitor for 12 hours, respectively.An RPE cell senescence model was established by cell treatment with 500 μmol/L H 2O 2 for 24 hours.The mRNA and protein levels of ASNS and JAK were detected by real-time fluorescent quantitative PCR and Western blot, respectively.Reactive oxygen species (ROS) level within cells was measured using a kit.Cell cycle phase distribution and apoptosis rates were analyzed by flow cytometry.Cell viability from day 1 to day 5 of culturing was assessed via MTT assay.Senescent cell ratio was determined by β-galactosidase staining.Cellular damage was evaluated via immunofluorescence staining.Senescence-associated proteins (p16, pRb), and RPE markers (KRT18, CTNNB1, TJP1, BEST1) were quantified by Western blot. Results:Compared with the control group, mRNA and protein expression levels of ASNS and JAK were significantly reduced in the shASNS group, control+ JAK inhibitor group, and shASNS+ JAK inhibitor groups (all P<0.05).DCFH-DA staining revealed significantly lower ROS level in the shASNS group, control+ JAK inhibitor group, and shASNS+ JAK inhibitor group than in the control group (all P<0.05).Flow cytometry showed that there were more G2-phase cells and significantly reduced apoptosis rate in the shASNS group, control+ JAK inhibitor group, and shASNS+ JAK inhibitor group compared with the control group (all P<0.01).MTT assay indicated higher cell viability at all time points in the shASNS group, control+ JAK inhibitor group, and shASNS+ JAK inhibitor group compared with the control group, with statistically significant differences (all P<0.01).β-galactosidase-positive cell ratios in the shASNS, control+ JAK inhibitor, and shASNS+ JAK inhibitor groups were (42.36±1.28)%, (43.20±1.89)%, (25.97±1.13)%, respectively, which were significantly lower than (52.25±0.64)% in the control group (all P<0.001).p16 and pRb protein expression were decreased and γ-H2AX fluorescence intensity was attenuated in the shASNS group, control+ JAK inhibitor group, and shASNS+ JAK inhibitor group compared with the control group (all P<0.01).KRT18 and CTNNB1 expressions were upregulated, whereas TJP1 and BEST1 were downregulated in the shASNS group, control+ JAK inhibitor group, and shASNS+ JAK inhibitor group compared with the control group (all P<0.05).The shASNS+ JAK inhibitor group exhibited higher KRT18 and CTNNB1 expressions and lower TJP1 and BEST1 expressions than the shASNS and control+ JAK inhibitor groups (all P<0.05). Conclusions:ASNS knockdown can promote RPE cell proliferation, mitigate cellular damage, and delay senescence by suppressing the JAK pathway.

Objective:To preliminarily explore whether sirtuin1 (SIRT1) activation can inhibit neuronal ferroptosis in rats after traumatic brain injury (TBI) by regulating hypoxia-inducible factor-1α (HIF-1α)-mediated glycolysis.Methods:(1) Six SD rats were randomly divided into sham-operated group and TBI group, with 3 rats in each group; TBI model in the TBI group was established by hydraulic impact method, and rats in the sham-operated group underwent same surgery without impact. Cortical tissues of the two groups were sent for tandem mass tag (TMT) labeled quantitative proteomics detection to analyze the differential expression proteome; Kyoto encyclopedia of genes and genomes (KEGG) and gene set enrichment analysis (GSEA) were used to detect pathway enrichment of the screened differential proteins. (2) Twelve SD rats were randomly divided into sham-operated group and 1-day, 3-day and 7-day post-TBI groups, with 3 rats in each group. Treatment methods were the same as above; Western blotting was used to detect SIRT1 protein expression. (3) Forty-eight rats were randomly divided into sham-operated group, TBI group, TBI+vehicle group and TBI+SIRT1 agonist group, with 12 rats in each group; rats in the sham-operated group and TBI group accepted treatment as above; rats in the TBI+SIRT1 agonist group were intraperitoneally injected with SRT1720 (dissolved in ≤ 5% dimethyl sulfoxide, at a dose of 20 mg/kg) within 30 minutes after modeling, twice a day (with an interval of 12 hours); and rats in the TBI+vehicle group were injected with same dose of dimethyl sulfoxide at the same time. One d after modeling, neurological deficit was assessed using modified Neurological severity score (mNSS), brain water content was measured by dry-wet weight method, histopathological changes in the cortical lesions were observed by HE staining, mitochondrial ultrastructure was examined by transmission electron microscopy, malondialdehyde (MDA) content and superoxide dismutase (SOD) activity in the brain tissues were detected by colorimetry, and protein expressions of SIRT1, HIF-1α (key protein in the glycolytic pathway), glutathione peroxidase 4 (GPX4, key protein in the ferroptosis pathway), and acyl-CoA synthetase long-chain family member 4 (ACSL4, key protein in the ferroptosis pathway) were evaluated by Western blotting.Results:(1) KEGG analysis revealed that the glycolysis pathway and HIF-1 signaling pathway were obviously enriched in the cortical tissues of rats in the TBI group compared with the sham-operated group; GSEA showed that the HIF-1 signaling pathway (mmu04066) and ferroptosis pathway (mmu04216) gene sets in the cortical tissues of rats in the TBI group exhibited enrichment trends compared with those in the sham-operated group. (2) Compared with the sham-operated group, the 1-day, 3-day, and 7-day post-TBI groups had significantly decreased SIRT1 protein expression ( P<0.05), with the most prominent decline in 1-day post-TBI group. (3) Compared with the TBI+vehicle group, rats in the TBI+SIRT1 agonist group showed significantly reduced mNSS score and brain tissue water content (9.83±1.17 vs. 7.66±1.21; [83.62±0.91]% vs. [80.09±0.68]%, P<0.05). HE staining indicated clearer structure of the cortical area at the injury sites, and improved neuron morphology in the TBI+SIRT1 agonist group compared with those in the TBI+vehicle group; and transmission electron microscopy showed reduced mitochondrial shrinkage and partial restoration of cristae structures in the TBI+SIRT1 agonist group compared with those in the TBI+vehicle group. Compared with the TBI+vehicle group, the TBI+SIRT1 agonist group exhibited significantly decreased MDA content ([62.72±9.20] nmol/g vs. [39.34±3.48] nmol/g), increased SOD activity ([1.95±0.23] U/mg vs. [2.48±0.14] U/mg), elevated GPX4 protein expression (0.37±0.04 vs. 0.46±0.03), and decreased HIF-1α and ACSL4 protein expressions (1.16±0.15 vs. 0.81±0.12; 1.14±0.06 vs. 1.29±0.04), with significant differences ( P<0.05). Conclusion:SIRT1 activation can exert neuroprotective effect by inhibiting HIF-1α-mediated glycolysis and reducing neuronal ferroptosis after TBI.

ObjectiveTo investigate the effects of Linggui Zhugantang on mitochondrial fission and fusion and silencing information regulator 3（Sirt3）/adenosine monophosphate dependent protein kinase （AMPK） signaling pathway in chronic heart failure （CHF） rats after myocardial infarction （MI）. MethodSD rats randomly divide into sham operation group （normal saline ，thread only without ligature）， model group （normal saline， ligation of the left anterior descending coronary artery proximal to the heart）， Linggui Zhugantang group （4.8 g·kg^-1） and Captopril group （0.002 57 g·kg^-1）， with 10 rats in each group. Administere drug continuously for 28 days. Echocardiography detected cardiac function parameters. Hematoxylin eosin （HE） staining observed the pathological changes of the heart. Immunofluorescence detected the levels of reactive oxygen species （ROS）. JC-1 detect mitochondrial membrane potential. Colorimetry measure adenosine triphosphate （ATP）， superoxide dismutase （SOD）， malondialdehyde （MDA）， mitochondrial respiratory chain complex activity （Ⅰ-Ⅳ）. TdT-mediated dUTP nick end labeling （TUNEL） staining detected the apoptosis rate of myocardial tissue. Western blot detected protein expression levels of Sirt3， phosphorylated AMPK （p-AMPK）， phosphorylated dynamic-related protein 1（p-Drp1）， mitochondrial fission protein 1（Fis1）， mitochondrial fission factor （MFF）， optic atrophy protein 1（OPA1）. ResultCompared to the sham group， the left ventricular end diastolic diameter （LVIDd） and left ventricular end systolic diameter （LVIDs） were significantly increased in model group （P<0.01）， while the left ventricular short axis shortening rate （LVFS） and left ventricular ejection fraction （LVEF） were significantly decreased （P<0.01）. There were inflammatory cell infiltration and obvious pathological injury in myocardial tissue. ROS， MDA levels and myocardial cell apoptosis rate were significantly increased （P<0.01）， SOD level， ATP content， and membrane potential were significantly decreased （P<0.01）. The activity of mitochondrial respiratory chain complexes （Ⅰ-Ⅳ） was significantly decreased （P<0.01）. Levels of p-Drp1， Fis1， MFF proteins were significantly up-regulated （P<0.01）， while Sirt3， p-AMPK， OPA1 proteins level were significantly down-regulated （P<0.01）. Compared with model group， LVIDd and LVIDs were significantly decreased （P<0.01）， LVEF and LVFS were significantly increased （P<0.01）. Inflammatory cell infiltration and pathological damage of myocardial tissue were significantly relieved. ROS， MDA levels and myocardial cell apoptosis rate were significantly decreased in Linggui Zhugantang group and Captopril group （P<0.01）， SOD level， ATP content， and membrane potential significantly increased （P<0.01）. The activity of mitochondrial respiratory chain complexes （Ⅰ-Ⅳ） increased significantly （P<0.01），and p-Drp1， Fis1， MFF protein levels were significantly down-regulated （P<0.01）， Sirt3， p-AMPK， OPA1 protein were significantly up-regulated （P<0.01）. ConclusionLinggui Zhugantang can alleviate oxidative stress and apoptosis damage of myocardial cells， maintain mitochondrial function stability， and its effect may be related to mitochondrial mitosis fusion and Sirt3/AMPK signaling pathway.

AIM: To evaluate the effect of ab-externo circumferential suture trabeculotomy(CST)on the 24 h pattern of intraocular pressure(IOP)in primary open angle glaucoma(POAG).METHODS: This retrospective study included 18 POAG patients who had poor control of IOP from March 2021 to May 2022. The ab-externo CST was performed, and IOP was tested preoperatively and 1 a postoperatively(9:00 a.m., 12:00 a.m., 3:00 p.m., 6:00 p.m., 9:00 p.m., 12:00 p.m., 3 a.m., and 6:00 a.m.). The mean, peak, trough, and range of IOP, as well as the average diurnal-nocturnal IOP change were calculated and compared.RESULTS: The 24 h IOP curves exhibited a decreasing trend during the diurnal period and an increasing trend during the nocturnal period, reaching a trough in the afternoon and peaking at night; the time of trough and peak IOP occurred several hours earlier compared to preoperative eyes. Postoperatively, the mean, peak, and trough IOP values were significantly lower compared to preoperative levels. The range of fluctuation showed no significant difference, while the average diurnal-nocturnal IOP change increased significantly.CONCLUSION: CST could reduce IOP of patients with POAG, but could not change the range of IOP fluctuation. However, an increase in the average diurnal-nocturnal IOP change was observed, indicating that CST might not necessarily reduce diurnal-nocturnal IOP fluctuations.

Tyrosine kinase inhibitors (TKIs) have revolutionized the treatment landscape for chronic myeloid leukemia (CML). However, TKI resistance poses a significant challenge, leading to treatment failure and disease progression. Resistance mechanisms include both BCR::ABL1-dependent and BCR::ABL1-independent pathways. The mechanisms underlying BCR::ABL1 independence remain incompletely understood, with CML cells potentially activating alternative signaling pathways, including the AKT/mTOR and JAK2/STAT5 pathways, to compensate for the loss of BCR::ABL1 kinase activity. This study explored tumoral VISTA (encoded by VSIR) as a contributing factor to TKI resistance in CML patients and identified elevated tumoral VISTA levels as a marker of resistance and poor survival. Through in vitro and in vivo analyses, we demonstrated that VSIR knockdown and the application of NSC-622608, a novel VISTA inhibitor, significantly impeded CML cell proliferation and induced apoptosis by attenuating the AKT/ mTOR and JAK2/STAT5 pathways, which are crucial for CML cell survival independent of BCR::ABL1 kinase activity. Moreover, VSIR overexpression promoted TKI resistance in CML cells. Importantly, the synergistic effect of NSC-622608 with TKIs offers a potent therapeutic avenue against both imatinib-sensitive and imatinib-resistant CML cells, including those harboring the challenging T315I mutation. Our findings highlight the role of tumoral VISTA in mediating TKI resistance in CML, suggesting that inhibition of VISTA, particularly in combination with TKIs, is an innovative approach to enhancing treatment outcomes in CML patients, irrespective of BCR::ABL1 mutation status. This study not only identified a new pathway contributing to TKI resistance but also revealed the possibility of targeting tumoral VISTA as a means of overcoming this significant clinical challenge.

Heat shock proteins (HSPs) are a group of highly conserved and functionally diverse molecular chaperones that play a crucial role in maintaining cellular homeostasis and coping with a variety of stress responses. HSP70 is one of the important members of the HSPs family, which is involved in the correct folding of proteins, preventing aggregation, and plays a key role in the formation of germ cells, DNA damage repair, fertilization process and embryo implantation in early embryonic development. HSP70 maintains the healthy development of embryos and protects cells from injury under various stress conditions by promoting the normal development and cellular homeostasis of fertilized eggs after fertilization. Through in-depth study of the complex functions and potential mechanisms of HSP70, it is expected to reveal its role in unexplained infertility, recurrent implantation failure, recurrent abortion and other diseases.

Heat shock proteins (HSPs) are a group of highly conserved and functionally diverse molecular chaperones that play a crucial role in maintaining cellular homeostasis and coping with a variety of stress responses. HSP70 is one of the important members of the HSPs family, which is involved in the correct folding of proteins, preventing aggregation, and plays a key role in the formation of germ cells, DNA damage repair, fertilization process and embryo implantation in early embryonic development. HSP70 maintains the healthy development of embryos and protects cells from injury under various stress conditions by promoting the normal development and cellular homeostasis of fertilized eggs after fertilization. Through in-depth study of the complex functions and potential mechanisms of HSP70, it is expected to reveal its role in unexplained infertility, recurrent implantation failure, recurrent abortion and other diseases.