Objective To investigate the changes in eosinophil counts and the activation of microglial cells in the brain tissues of mice at different stages of Angiostrongylus cantonensis infection, and to examine the role of microglia in regulating the progression of angiostrongyliasis and unravel the possible molecular mechanisms. Methods Fifty BALB/c mice were randomly divided into the control group and the 7-d, 14-d, 21-day and 25-d infection groups, of 10 mice in each group. All mice in infection groups were infected with 30 stage III A. cantonensis larvae by gavage, and animals in the control group was given an equal amount of physiological saline. Five mice were collected from each of infection groups on days 7, 14, 21 d and 25 d post-infection, and 5 mice were collected from the control group on the day of oral gavage. The general and focal functional impairment was scored using the Clark scoring method to assess the degree of mouse neurological impairment. Five mice from each of infection groups were sacrificed on days 7, 14, 21 d and 25 d post-infection, and 5 mice from the control group were sacrificed on the day of oral gavage. Mouse brain tissues were sampled, and the pathological changes of brain tissues were dynamically observed using hematoxylin and eosin (HE) staining. Immunofluorescence staining with eosinophilic cationic protein (ECP) and ionized calcium binding adaptor molecule 1 (Iba1) was used to assess the degree of eosinophil infiltration and the counts of microglial cells in mouse brain tissues in each group, and the morphological parameters of microglial cells (skeleton analysis and fractal analysis) were quantified by using Image J software to determine the morphological changes of microglial cells. In addition, the expression of M1 microglia markers Fcγ receptor III (Fcgr3), Fcγ receptor IIb (Fcgr2b) and CD86 antigen (Cd86), M2 microglia markers Arginase 1 (Arg1), macrophage mannose receptor C-type 1 (Mrc1), chitinase-like 3 (Chil3), and phagocytosis genes myeloid cell triggering receptor expressed on myeloid cells 2 (Trem2), CD68 antigen (Cd68), and apolipoprotein E (Apoe) was quantified using real-time quantitative reverse transcription PCR (RT-qPCR) assay in the mouse cerebral cortex of mice post-infection. Results A large number of A. cantonensis larvae were seen on the mouse meninges surface post-infection, and many neuronal nuclei were crumpled and deeply stained, with a large number of bleeding points in the meninges. The median Clark scores of mouse general functional impairment were 0 (interquartile range, 0), 0 (interquartile range, 0.5), 6 (interquartile range, 1.0), 14 (interquartile range, 8.5) points and 20 (interquartile range, 9.0) points in the control group and the 7-d, 14-d, 21-d and 25-d groups, respectively (H = 22.45, P < 0.01), and the median Clark scores of mouse focal functional impairment were 0 (interquartile range, 0), 2 (interquartile range, 2.5), 7 (interquartile range, 3.0), 18 (interquartile range, 5.0) points and 25 (interquartile range, 6.5) points in the control group and the 7-d, 14-d, 21-d and 25-d groups, respectively (H = 22.72, P < 0.01). The mean scores of mice general and focal functional impairment were all higher in the infection groups than in the control group (all P values < 0.05). Immunofluorescence staining showed a significant difference in the eosinophil counts in mouse brain tissues among the five groups (F = 40.05, P < 0.000 1), and the eosinophil counts were significantly higher in mouse brain tissues in the 14-d (3.08 ± 0.78) and 21-d infection groups (5.97 ± 1.37) than in the control group (1.00 ± 0.28) (both P values < 0.05). Semi-quantitative analysis of microglia immunofluorescence showed a significant difference in the counts of microglial cells among the five groups (F = 17.66, P < 0.000 1), and higher Iba1 levels were detected in mouse brain tissues in 14-d (5.75 ± 1.28), 21-d (6.23 ± 1.89) and 25-d infection groups (3.70 ± 1.30) than in the control group (1.00 ± 0.30) (all P values < 0.05). Skeleton and fractal analyses showed that the branch length [(162.04 ± 34.10) μm vs. (395.37 ± 64.11) μm; t = 5.566, P < 0.05] and fractal dimension of microglial cells (1.30 ± 0.01 vs. 1.41 ± 0.03; t = 5.266, P < 0.05) were reduced in mouse brain tissues in the 21-d infection group relative to the control group. In addition, there were significant differences among the 5 groups in terms of M1 and M2 microglia markers Fcgr3 (F = 48.34, P < 0.05), Fcgr2b (F = 55.46, P < 0.05), Cd86 (F = 24.44, P < 0.05), Arg1 (F = 31.18, P < 0.05), Mrc1 (F = 15.42, P < 0.05) and Chil3 (F = 24.41, P < 0.05), as well as phagocytosis markers Trem2 (F = 21.19, P < 0.05), Cd68 (F = 43.95, P < 0.05) and Apoe (F = 7.12, P < 0.05) in mice brain tissues. Conclusions A. cantonensis infections may induce severe pathological injuries in mouse brain tissues that are characterized by massive eosinophil infiltration and persistent activation of microglia cells, thereby resulting in progressive deterioration of neurological functions.

Objective To identify long non-coding RNA (lncRNA) associated with rheumatoid arthritis-associated interstitial lung disease (RA-ILD) and investigate their mechanisms. Methods Peripheral blood samples were collected from RA-ILD patients (n＝3), RA patients without lung involvement (n＝3), and healthy controls (n＝3). Next-generation sequencing was performed to screen differentially expressed lncRNA. A human fibrotic lung cell model was established by inducing the MRC-5 cell line with transforming growth factor-β (TGF-β). Following siRNA-mediated knockdown of target genes, changes in inflammatory and oxidative stress-related genes were analyzed via real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). Western blotting and dual-luciferase reporter (DLR) assays were used to validate protein expression, ubiquitination levels, and nuclear translocation of oxidative stress regulators, and antioxidant response element (ARE) transcriptional activity. Rescue experiments were conducted to confirm the role of target lncRNA in oxidative stress and inflammation in fibrotic lung cells. Results High-throughput sequencing revealed significant downregulation of LINC00638 in RA-ILD patients. Knockdown of LINC00638 markedly reduced transcriptional levels of interleukin (IL)-4, nuclear factor erythroid-2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), and superoxide dismutase 2 (SOD2), while increasing IL-6, IL-1β, interferon-γ (IFN-γ), and reactive oxygen species (ROS) levels. Furthermore, LINC00638 knockdown decreased Nrf2 protein expression, increased its ubiquitination, reduced nuclear translocation, and suppressed ARE transcriptional activity. In MRC-5 cells, LINC00638 knockdown combined with N-acetylcysteine treatment restored Nrf2 and HO-1 levels while reducing IL-6 expression. Conclusions LINC00638 suppresses inflammatory responses in RA-ILD by activating the Nrf2/ARE antioxidant signaling pathway, suggesting its potential as a therapeutic target for diagnosis and treatment.

OBJECTIVES: To explore the mechanism of cinnamic acid (CA) for improving doxorubicin-induced myocardial injury (DIC) in mice. METHODS: Network pharmacology analysis was used to obtain the key targets of CA and DIC. Male C57BL/6J mice were randomized into Sham, DOX, CA (25, 50 and 100 mg/kg)+DOX, and CA+Ferrostatin-1+DOX groups, and their myocardial function and pathology were examined by echocardiography and HE staining. Serum levels of CK-MB, LDH, MDA, IL-6, TNF‑α and myocardial ROS level were detected, and the expression levels of TLR4 and ferroptosis pathway proteins in myocardial tissue were detected by Western blotting. Cultured murine cardiomyocytes (HL-1 cells) with or without transfection with a small interfering RNA targeting TLR4 (si-TLR4) were treated with DOX or Erastin, and the cellular ROS content was measured by DCFH-DA staining; the expression level of GPX4 was detected using immunofluorescence staining. RESULTS: Network pharmacology analysis suggested that CA may improve DIC through TLR4 signaling. DOX treatment caused obvious myocardial injury in mice, which showed significantly increased serum levels of CK-MB, LDH, MDA, IL-6, TNF-α and myocardial ROS level with decreased myocardial levels of SLC7A11 and GPX4 proteins and increased levels of TLR4 and PTGS2 proteins. All these changes in the mouse models were significantly alleviated by treatment with CA, and the mice receiving CA or ferrostatin-1 treatment exhibited increased myocardial expressions of SLC7A11 and GPX4 proteins and lowered expressions of TLR4 and PTGS2 proteins. In cultured HL-1 cells, treatment with DOX and Erastin both obviously increased intracellular ROS level and decreased cellular GPX4 expression level, and these changes were strongly attenuated by TLR4 interference. CONCLUSIONS CA, as a potent herbal monomer, can effectively alleviate DIC in mice by inhibiting TLR4-mediated ferroptosis.
Animals ; Ferroptosis/drug effects* ; Toll-Like Receptor 4/metabolism* ; Myocytes, Cardiac/metabolism* ; Mice, Inbred C57BL ; Mice ; Male ; Doxorubicin/adverse effects* ; Cinnamates/pharmacology* ; Signal Transduction ; Reactive Oxygen Species/metabolism*

Metabolic dysfunction-associated fatty liver disease (MAFLD), characterized by fatty acid overload, secondary chronic inflammation, and fibrosis, has become the most prevalent chronic liver disease globally. While no effective pharmacotherapy exists for MAFLD, mitigating inflammatory responses represents a promising approach to preventing the progression from steatosis to severe steatohepatitis. The NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome, which detects endogenous danger and stress signals, has emerged as a significant target for inflammatory disease treatment, as transcriptional inactivation of its components demonstrates the therapeutic potential for MAFLD. Natural products targeting NLRP3 inflammasome activation have shown promising efficacy in MAFLD therapy. This review synthesizes the current understanding of NLRP3 inflammasome activation and therapeutic targets for NLRP3 homeostasis. Additionally, natural products reported to inhibit NLRP3 inflammasome for MAFLD improvement are categorized according to their mechanisms of action. The review also addresses limitations and future directions regarding natural products targeting NLRP3 inflammasome in MAFLD treatment. Enhanced understanding of NLRP3 inflammasome activation mechanisms in MAFLD and the identification of novel natural products supported by mechanistic research will significantly advance MAFLD treatment.
Humans ; NLR Family, Pyrin Domain-Containing 3 Protein/immunology* ; Inflammasomes/metabolism* ; Biological Products/therapeutic use* ; Animals ; Fatty Liver/immunology*

Objective To investigate whether curcumin alleviates septic lung injury by inhibiting ferroptosis through modulating the TXNIP/TRX-1/GPX4 pathway.Methods Male C57BL/6 mice were randomly divided into Sham group,cecal ligation puncture(CLP)-induced sepsis group,CLP with curcumin treatment(50,100,and 200 mg/kg)groups,and CLP with both curcumin(200 mg/kg)and TRX-1 inhibitor PX-12(25 mg/kg)treatment group.Inflammatory factors,MDA,MPO,and GSH levels in the lung tissue of the mice were detected.Beas-2B cells stimulated with lipopolysaccharide(LPS;1 μg/mL)were treated with 2.5,5,or 10 μmol/L curcumin or with 10 μmol/L curcumin combined with 5 μmol/L PX-12,and the changes in MDA,Fe2+and ROS levels were assessed.Western blotting was performed to detect the protein expressions of TXNIP,TRX-1,GPX4 and X-CT in both the mouse lung tissues and Beas-2B cells.Results The mice with CLP-induced sepsis showed severe lung injury with elevated expressions of IL-6,IL-1β,TNF-α,MDA and MPO and decreased GSH expression.In Beas-2B cells,LPS stimulation significantly increased MDA and Fe2+levels and ROS release,increased TXNIP protein expression,and lowered the protein expression levels of TRX-1,GPX4 and X-CT,and these changes were also observed in the septic mice.Curcumin treatments at different concentrations obviously alleviated lung injury in the septic mice and reduced LPS-induced injury in Beas-2B cells.Curcumin significantly decreased the release of inflammatory factors,MDA and MPO,increased GSH level,lowered Fe2+,MDA and ROS levels,increased TXNIP protein expression,and lowered the protein expressions of TRX-1,GPX4 and X-CT in both septic mouse lung tissues and LPS-stimulated Beas-2B cells.The protective effect of curcumin was effectively blocked by PX-12 treatment.Conclusion Curcumin inhibits ferroptosis and alleviates septic lung injury in mice by elevating TRX-1 and GPX4 and decreasing TXNIP in the lung tissue.

Objective To explore the mechanism of plumbagin for protecting against sepsis-induced myocardial injury in mice.Methods Network pharmacology analysis was used to obtain the key targets of plumbagin and diseases,which were subjected to GO and KEGG analysis,and the binding energy was verified using molecular docking.In a mouse model of cecal ligation and puncture(CLP),the protective effect of plumbagin treatment prior to CLP against sepsis-induced myocardial injury was evaluated by examination of myocardial function and pathology using echocardiography and HE staining.Serum levels of CK-MB,LDH,MDA,IL-1β and IL-18 and myocardial ROS level in the mice were detected,and Western blotting was used to determine the protein expression levels of STAT3,GSDMD,caspase-11,JAK2,P-STAT3,P-JAK2,GSDMD-N and HMGB1 in the myocardial tissues.Results Five core targets were screened from the 10 intersecting genes.Molecular docking showed strong binding affinity of plumbagin to STAT3,p-STAT3,and JAK2.Compared with the sham-operated mice,the mouse models of CLP-induced sepsis had significantly decreased CO,LVEF,LVFS and SV and increased serum levels of CK-MB,LDH,MDA and myocardial inflammatory factors and ROS.HE staining and Western blotting showed obvious myocardial injury in the septic mice with increased expressions of JAK2/STAT3 signaling pathway and pyroptosis-related proteins(P<0.05).Pretreatment with plumbagin significantly improved cardiac functions of CLP mice,lowered serum levels of CK-MB,LDH,MDA,inflammatory factors and myocardial ROS,and decreased the expression levels of JAK2/STAT3 signaling pathway and pyroptosis-related proteins.Conclusion Plumbagin pretreatment alleviates myocardial injury in septic mice possibly by inhibiting the STAT3 signaling pathway to reduce cardiomyocyte pyroptosis.

Objective To investigate whether curcumin alleviates septic lung injury by inhibiting ferroptosis through modulating the TXNIP/TRX-1/GPX4 pathway.Methods Male C57BL/6 mice were randomly divided into Sham group,cecal ligation puncture(CLP)-induced sepsis group,CLP with curcumin treatment(50,100,and 200 mg/kg)groups,and CLP with both curcumin(200 mg/kg)and TRX-1 inhibitor PX-12(25 mg/kg)treatment group.Inflammatory factors,MDA,MPO,and GSH levels in the lung tissue of the mice were detected.Beas-2B cells stimulated with lipopolysaccharide(LPS;1 μg/mL)were treated with 2.5,5,or 10 μmol/L curcumin or with 10 μmol/L curcumin combined with 5 μmol/L PX-12,and the changes in MDA,Fe2+and ROS levels were assessed.Western blotting was performed to detect the protein expressions of TXNIP,TRX-1,GPX4 and X-CT in both the mouse lung tissues and Beas-2B cells.Results The mice with CLP-induced sepsis showed severe lung injury with elevated expressions of IL-6,IL-1β,TNF-α,MDA and MPO and decreased GSH expression.In Beas-2B cells,LPS stimulation significantly increased MDA and Fe2+levels and ROS release,increased TXNIP protein expression,and lowered the protein expression levels of TRX-1,GPX4 and X-CT,and these changes were also observed in the septic mice.Curcumin treatments at different concentrations obviously alleviated lung injury in the septic mice and reduced LPS-induced injury in Beas-2B cells.Curcumin significantly decreased the release of inflammatory factors,MDA and MPO,increased GSH level,lowered Fe2+,MDA and ROS levels,increased TXNIP protein expression,and lowered the protein expressions of TRX-1,GPX4 and X-CT in both septic mouse lung tissues and LPS-stimulated Beas-2B cells.The protective effect of curcumin was effectively blocked by PX-12 treatment.Conclusion Curcumin inhibits ferroptosis and alleviates septic lung injury in mice by elevating TRX-1 and GPX4 and decreasing TXNIP in the lung tissue.

Objective To explore the mechanism of plumbagin for protecting against sepsis-induced myocardial injury in mice.Methods Network pharmacology analysis was used to obtain the key targets of plumbagin and diseases,which were subjected to GO and KEGG analysis,and the binding energy was verified using molecular docking.In a mouse model of cecal ligation and puncture(CLP),the protective effect of plumbagin treatment prior to CLP against sepsis-induced myocardial injury was evaluated by examination of myocardial function and pathology using echocardiography and HE staining.Serum levels of CK-MB,LDH,MDA,IL-1β and IL-18 and myocardial ROS level in the mice were detected,and Western blotting was used to determine the protein expression levels of STAT3,GSDMD,caspase-11,JAK2,P-STAT3,P-JAK2,GSDMD-N and HMGB1 in the myocardial tissues.Results Five core targets were screened from the 10 intersecting genes.Molecular docking showed strong binding affinity of plumbagin to STAT3,p-STAT3,and JAK2.Compared with the sham-operated mice,the mouse models of CLP-induced sepsis had significantly decreased CO,LVEF,LVFS and SV and increased serum levels of CK-MB,LDH,MDA and myocardial inflammatory factors and ROS.HE staining and Western blotting showed obvious myocardial injury in the septic mice with increased expressions of JAK2/STAT3 signaling pathway and pyroptosis-related proteins(P<0.05).Pretreatment with plumbagin significantly improved cardiac functions of CLP mice,lowered serum levels of CK-MB,LDH,MDA,inflammatory factors and myocardial ROS,and decreased the expression levels of JAK2/STAT3 signaling pathway and pyroptosis-related proteins.Conclusion Plumbagin pretreatment alleviates myocardial injury in septic mice possibly by inhibiting the STAT3 signaling pathway to reduce cardiomyocyte pyroptosis.

Objective:To investigate the predictive value of thrombus permeability for poor outcome and symptomatic intracranial hemorrhage (sICH) after endovascular therapy (EVT) in patients with middle cerebral artery occlusion stroke.Methods:Patients with middle cerebral artery occlusion stroke underwent EVT at the Cardiocerebrovascular Disease Hospital, the Affiliated Hospital of Yan'an University from January 2018 to January 2024 were included retrospectively. Thrombus attenuation increase (TAI) was used to evaluate thrombus permeability. The poor outcome was defined as a modified Rankin Scale score >2 at 90 days after procedure. sICH was defined as an increase of ≥4 of the National Institutes of Health Stroke Scale (NIHSS) score relative to baseline or lowest within 7 days after EVT, and CT scan showed cerebral hemorrhage. Multivariate logistic regression analysis was used to determine the independent influencing factors of poor outcome and sICH. Receiver operating characteristic (ROC) curves was used to evaluate the predictive value of TAI for poor outcome and sICH. Results:A total of 77 patients with middle cerebral artery occlusion stroke received EVT treatment were enrolled, including 44 males (57.1%), aged 62.1±12.4 years. Thirty-three patients (48.1%) had poor outcome, 35 (45.5%) experienced hemorrhagic transformation, of which 12 (15.6%) were sICH. Multivariate logistic regression analysis showed that TAI (odds ratio [ OR] 0.930, 95% confidence interval [ CI] 0.883-0.980; P=0.007) and sICH ( OR 0.868, 95% CI 0.784-0.961; P=0.006) were the independent influencing factors of poor outcome. ROC curve analysis showed that the area under the curve of TAI for predicting poor outcome at 90 days was 0.836 (95% CI 0.742-0.930). The cut-off value was 10.135 HU. The sensitivity and specificity were 73.0% and 92.5%, respectively. The area under the curve of TAI for predicting sICH was 0.750 (95% CI 0.637-0.902). The cut-off value was 18.200 HU. The sensitivity and specificity were 94.1% and 64.5%, respectively. Conclusions:TAI has certain predictive value for poor outcome and sICH after EVT in patients with middle cerebral artery occlusion stroke. Patients with higher thrombus permeability are less likely to develop sICH after EVT and have a higher likelihood of good outcome.

Membranous nephropathy(MN)is the predominant cause of primary nephrotic syn-drome(NS)among adults.The identification of PLA2R as target antigen has brought about a pro-found transformation in the management of MN,offering a basis for the utilization of B-cell deplet-ing agents such as rituximab(RTX).The question of whether early intervention targeting antibodies can effectively impede the progression of MN,contrib-uting to enhanced disease control and long-term renal outcomes for patients,remains further explo-ration.We analyzed demographic data,laboratory parameters,and renal involvement in 13 patients with PLA2R antibody-related MN who received at least one RTX treatment at our center from Octo-ber 2019 to March 2023.Early-stage medium-to-high-risk MN was defined as baseline or admission anti-PLA2R antibody levels exceeding 50 RU/mL,ex-cluding patients who already presented with ne-phrotic syndrome at baseline.The median duration of MN at the initiation of the first RTX treatment was 4.1 months(IQR 1-7.7),and the median follow-up time after RTX therapy was 27 months(IQR 23-45).All patients had commenced renin-angiotensin system inhibitors before receiving RTX.Following RTX therapy,none of the 13 patients progressed to NS during the follow-up period,and 12 patients achieved complete or partial remission at the 2-year follow-up or the last visit.No deaths,severe infections,or other serious adverse reactions oc-curred during the follow-up period.In conclusion,RTX demonstrates favorable efficacy and safety in early-stage,medium-to-high-risk MN patients.Initi-ating antibody clearance therapy in these patients may be beneficial for long-term disease control and distant renal outcomes.