VSP-2 attenuates secretion of inflammatory cytokines induced by LPS in BV2 cells by mediating the PPARγ/NF-κB signaling pathway.

Neuroinflammation, characterized by microglial activation and the subsequent secretion of inflammatory cytokines, plays a pivotal role in neurodegenerative diseases and brain injuries, often leading to neuronal damage and death. Alleviating neuroinflammation has thus emerged as a promising strategy to protect neurons and ameliorate neurodegenerative disorders. While peroxisome proliferator-activated receptor gamma (PPARγ) agonists have demonstrated potential therapeutic actions on neuroinflammation, their prolonged use, such as with rosiglitazone, can lead to cardiac risks and lipid differentiation disorders. In this study, we investigated the effects of a newly synthesized PPARγ agonist, VSP-2, on secretion of inflammatory cytokines in BV2 cells. Treatment with VSP-2 significantly reduced the mRNA and protein levels of proinflammatory cytokines such as interleukin-1ß (IL-1ß), IL-6, and tumor necrosis factor-α (TNF-α). Furthermore, VSP-2 attenuated the phosphorylation of nuclear factor kappa B (NF-κB) (65 kD) and IκBα, as well as the nuclear translocation of NF-κB (65 kD). Additionally, the use of PPARγ small interfering RNA was able to attenuate the effects of VSP-2 on proinflammatory cytokines and the NF-κB pathway. In conclusion, our findings suggest that VSP-2 effectively suppressed the expressions of IL-1ß, IL-6, and TNF-α via the PPARγ/NF-κB signaling pathway. Given its potential therapeutic benefits, VSP-2 may emerge as a promising candidate for the treatment of neurodegenerative diseases or brain injuries associated with neuroinflammation.

Ethanolic extract of Pereskia aculeata induces Anti-Inflammatory Responses through P38/MK2/TTP-mediated signaling pathway.

Pereskia aculeata Miller, a member of the Cactaceae family, is a plant with pharmacological potential due to its containing compounds with various biological activities, which include anti-inflammatory, anti-cancer and analgesic activities. In this study, we evaluated the anti-inflammatory effects of an ethanolic extract of P. aculeata Miller (EEPA) and the signalling pathways by which it exerts these effects. In vitro, EEPA inhibited the secretion of inflammatory factors NO, IL-6 and PGE2 in ipopolysaccharide-stimulated RAW264.7 macrophages (P<0.05). Treatment of RAW264.7 cells with EEPA also significantly decreased the levels of P-P38 and P-MK2, while upregulating the expression of TTP (P<0.05). In vivo anti-inflammatory activity assays revealed that EEPA reduced the degree of foot and joint swelling, the splenic index and the serum concentrations of TNF-α and IL-6 in in adjuvant-induced arthritis rats (P<0.05). Similarly, EEPA treatment of mice inhibited the acetic acid-induced exudation of Evans blue dye from peritoneal capillaries and significantly prolonged heat-stimulated pain response time (P<0.05). Taken together, these results suggest that EEPA exerts anti-inflammatory effects in vitro and in vivo. Thus, this study provides experimental and technical support for the development of a novel anti-inflammatory treatment based on P. aculeata Miller.

EGCG attenuated acute myocardial infarction by inhibiting ferroptosis via miR-450b-5p/ACSL4 axis.

BACKGROUND: Epigallocatechin gallate (EGCG) has multiple biological effects such as anti-tumor multiple drug resistance, antioxidation and anti-inflammatory properties. Ferroptosis is the main driving factor of ischemic heart injury, thus inhibiting ferroptosis may prove to be an effective treatment strategy for cardiovascular diseases. However, the role of EGCG on ferroptosis in ischemic myocardium and underlying mechanisms remain uncertain. PURPOSE: This study was aimed to investigate the effects and potential mechanisms of EGCG on myocardial ischemic-induced ferroptosis both in vitro and in vivo. METHODS: Cardiomyocyte hypoxia model and mouse acute myocardial infarction (AMI) model were established in vitro and in vivo. MiR-450b-5p and ACSL4 silencing or overexpression plasmids were transfected, with or without EGCG pretreatment. Cell viability was determined by the CCK-8 assay. Hematoxylin and eosin (HE) staining and transmission electron microscopy (TEM) were used to evaluate the morphologic alterations. TTC staining was used to observe the infarction area, and echocardiography was adopted to appraise the heart function. Using flow cytometry, the presence of reactive oxygen species (ROS) was assessed. The content of cardiac troponin I (cTn I), glutathione (GSH), malondialdehyde (MDA), divalent iron ions (Fe2+) and superoxide dismutase (SOD) were detected using reagent kits. A luciferase activity assay was performed to assess the binding ability of miR-450b-5p to ACSL4. Expressions of related genes and proteins were measured by RT-qPCR and western blotting respectively. RESULTS: EGCG attenuated AMI-induced ferroptosis and improved myocardial ischemia injury, which was associated with reducing iron deposition and cTn I, inhibition of lipid peroxidation, decreasing TFR1 and ACSL4, and upregulating SLC7A11, FTH1 and GPX4. Meanwhile, EGCG pretreatment increased miR-450b-5p expression in ischemic myocardium. Further researches discovered that knockdown of miR-450b-5p partially compromised EGCG-generated protective effect in hypoxia HL-1 cells, while combination with miR-450b-5p mimic could strengthen the potency of EGCG on ischemic myocardium. The dual-luciferase test demonstrated that miR-450b-5p has binding to ACSL4. Furthermore, silencing of ACSL4 synergistically increased the cardioprotective effect of EGCG. More significantly, EGCG treatment regulated the ferroptosis-related proteins expression via miR-450b-5p/ACSL4 axis. CONCLUSION: In summary, the present study evidently demonstrated that EGCG attenuates myocardial ischemia injury by targeting ferroptosis. Our work revealed the role of miR-450b-5p/ACSL4 axis in AMI for the first time. Further, it also elucidated the molecular mechanisms of EGCG on inhibiting ferroptosis greatly depend on the miR-450b-5p/ACSL4 axis, suggesting that EGCG may act as a novel anti-ferroptosis agent and exert a therapeutic role in AMI.

Dual-energy spectral detector computed tomography differential diagnosis of adrenal adenoma and pheochromocytoma: Changes in the energy level curve, a phenomenon caused by lipid components?

Background and objectives: Pheochromocytoma and adrenal adenoma are common space-occupying lesions of the adrenal gland, and incorrect surgery may lead to adrenal crisis. We used a new method, dual-energy spectral detector computed tomography (SDCT), to differentiate between the two. Materials and methods: We analysed the imaging images of patients with SDCT scans and pathologically confirmed adrenal adenomas (n=70) and pheochromocytomas (n=15). The 40, 70, and 100 KeV virtual monoenergetic images (VMIs) were reconstructed based on the SCDT arterial phase, and the correlation between the arterial/venous phase iodine concentration (AP-IC/VP-IC), the effective atomic number (Z-effect), the slope of the Hounsfield unit attenuation plot (VMI slope) and the pathological results was tested. The Shapiro-Wilk test was used to determine whether the above data conformed to a normal distribution. For parameters with P greater than 0.05, Student's t test was used, and the Mann-Whitney test was used for the remaining parameters. A ROC curve was drawn based on the results. Results: Student's t test showed that the 40 KeV VMI and the VMI slope were both statistically significant (P<0.01). The Mann-Whitney U test showed that ID-A was statistically significant (P=0.004). ROC curve analysis showed that 40 keV VMI (AUC=0.818), AP-IC (AUC=0.736), difference (AUC=0.817) and VMI-Slope (0.817) could be used to differentiate adrenal adenoma from pheochromocytoma. Conclusion: The effect of lipid components on SDCT parameters can be used to differentiate adrenal adenoma from pheochromocytoma.

One-stage total hip arthroplasty for advanced hip tuberculosis combined with developmental dysplasia of the hip: A case report.

BACKGROUND: A patient with advanced tuberculosis of the hip joint combined with Crowe type IV developmental dysplasia of the hip (DDH) and a drainage sinus is a rare condition. There are no previous reports of this condition, and it is a complex challenge for surgeons to develop a treatment scheme. CASE SUMMARY: We report a 73-year-old male patient with severe hip pain and drainage sinus of the left hip for one month. Approximately 40 years ago, a drainage sinus occurred at the lateral left hip was healed at the local hospital with anti-infectious therapy. After the sinus healed, gradual pain occurred in the left hip for 40 years. Approximately one month prior, hip pain was sharply aggravated, and a drainage sinus reoccurred in the left hip. The X-ray and computed tomography examinations showed destruction of the head and neck of the left femur, as well as an acetabular deformity. The results of Mycobacterium tuberculosis antibody and Xpert were positive. Therefore, the patient was diagnosed with advanced TH combined with Crowe type IV DDH. After 22 d of treatment with anti-tuberculosis chemotherapy, the sinus healed, and the patient underwent one-stage total hip arthroplasty (THA) surgery consisting of debridement, osteotomy, and joint replacement. After surgery, the patient received anti-tuberculosis chemotherapy drugs for nine months, with no recurrent infection. After one year of follow-up, the Harris score of the patient increased from 21 pre-THA to 86. CONCLUSION: Although drainage sinuses are a contraindication to one-stage THA, one-stage THA is still an effective and safe surgical method after the sinus heals.

Arthroscopy-Assisted Treatment of Wear Debris Pseudotumor After Total Knee Arthroplasty: A Case Report.

BACKGROUND: Wear debris pseudotumors are a rare complication after total knee arthroplasty (TKA) and have seldom been reported in the recent literature. There is no consensus on the best therapeutic method, but the high quality curative treatment, safe, low invasive treatments are required for the patients. CASE PRESENTATION: In this paper, we present the case of a 74-year-old man with a wear debris pseudotumor after TKA with symptoms of severe pain and functional disability of his right knee. X-ray examination showed that the medial compartment of the right knee was narrowing. Magnetic resonance imaging (MRI) and Doppler ultrasound both revealed a polycystic mass at the posteromedial side of the patient's right knee. Considering the bad health condition and the minimally invasive surgery requirement of this senior patient, arthroscopic knee debridement and percutaneous cystic mass suction were carried out simultaneously. Video arthroscopy of the right knee showed visible inflammatory soft tissue, obvious polyethylene fragments, wear of the polyethylene prothesis, and a broken polyethylene insert. The intraarticular polyethylene wear debris was removed as much as possible, and inflammatory soft tissue was debrided and sent for pathology. Postoperative pathology showed polyethylene debris in the soft tissue with an apparent multinucleated giant cell response, which was consistent with foreign body granuloma. All clinical manifestation was improved and Lysholm scores were significantly better at one year with this treatment, increasing from 32 points to 71 points. CONCLUSION: After two years of follow-up, the patient's knee joint was significantly relieved from soreness and pain, and walking was not significantly restricted. Our treatment could not address the root cause of the wear debris pseudotumor, which was due to prosthesis failure, but sometimes, such an approach is the safest, most economical, and most effective choice for patients who are intolerant to reoperation.

MicroRNA-384-5p/Beclin-1 As Potential Indicators For Epigallocatechin Gallate Against Cardiomyocytes Ischemia Reperfusion Injury By Inhibiting Autophagy Via PI3K/Akt Pathway.

BACKGROUND/AIMS: Epigallocatechin gallate (EGCG) has established protective actions against myocardial ischemia/reperfusion (I/R) injury by regulating autophagy. However, little is known about the mechanisms of EGCG in posttranscriptional regulation in the process of cardioprotection. Here we studied whether microRNAs play a role in EGCG-induced cardioprotection. METHODS: The myocardial I/R injury in vitro and in vivo model were made, with or without EGCG pretreatment. The upregulation and silencing of microRNA-384-5p (miR-384) and Beclin-1 in H9c2 cell lines were established. Rats were transfected with miR-384 specific shRNA. Dual-luciferase reporter gene assay was conducted to verify the relationship between miR-384 and Beclin-1. TTC staining was performed to analyze the area of myocardial infarct size. Cell viability was monitored by cell counting kit-8 (CCK-8). The release of cardiac troponin-I (cTnI) was examined by ELISA. The levels of autophagy-related genes or proteins expression were evaluated by qRT-PCR or Western blotting. Autophagosomes of myocardial cells were detected by transmission electron microscopy and laser scanning confocal microscope. RESULTS: I/R increased both autophagosomes and autolysosomes, thereby increasing autophagic flux both in vitro and in vivo. Pretreatment with EGCG attenuated I/R-induced autophagic flux expression, accompanied by an increase in cell viability and a decrease in the size of myocardial infarction. MiR-384 expression was down-regulated in H9c2 cell lines when subjected to I/R, while this suppression could be reversed by EGCG pretreatment. The dual-luciferase assay verified that Beclin-1 was a target of miR-384. Both overexpression of miR-384 and knocking down of Beclin-1 significantly inhibited I/R-induced autophagy, accompanied by the activation of PI3K/Akt pathway, thus enhanced the protective effect of EGCG. However, these functions were abrogated by the PI3K inhibitor, LY294002. CONCLUSION: We confirmed that EGCG has a protective role in microRNA-384-mediated autophagy by targeting Beclin-1 via activating the PI3K/Akt signaling pathway. Our results unveiled a novel role of EGCG in myocardial protection, involving posttranscriptional regulation with miRNA-384.

Akt Inhibitor Perifosine Prevents Epileptogenesis in a Rat Model of Temporal Lobe Epilepsy.

Accumulating data have revealed that abnormal activity of the mTOR (mammalian target of rapamycin) pathway plays an important role in epileptogenesis triggered by various factors. We previously reported that pretreatment with perifosine, an inhibitor of Akt (also called protein kinase B), abolishes the rapamycin-induced paradoxical increase of S6 phosphorylation in a rat model induced by kainic acid (KA). Since Akt is an upstream target in the mTOR signaling pathway, we set out to determine whether perifosine has a preventive effect on epileptogenesis. Here, we explored the effect of perifosine on the model of temporal epilepsy induced by KA in rats and found that pretreatment with perifosine had no effect on the severity or duration of the KA-induced status epilepticus. However, perifosine almost completely inhibited the activation of p-Akt and p-S6 both acutely and chronically following the KA-induced status epilepticus. Perifosine pretreatment suppressed the KA-induced neuronal death and mossy fiber sprouting. The frequency of spontaneous seizures was markedly decreased in rats pretreated with perifosine. Accordingly, rats pretreated with perifosine showed mild impairment in cognitive functions. Collectively, this study provides novel evidence in a KA seizure model that perifosine may be a potential drug for use in anti-epileptogenic therapy.

Neural Progenitor Cells Rptor Ablation Impairs Development but Benefits to Seizure-Induced Behavioral Abnormalities.

AIMS: Previous study suggests that mTOR signaling pathway may play an important role in epileptogenesis. The present work was designed to explore the contribution of raptor protein to the development of epilepsy and comorbidities. METHODS: Mice with conditional knockout of raptor protein were generated by cross-bred Rptorflox/flox mice with nestin-CRE mice. The expression of raptor protein was analyzed by Western blotting in brain tissue samples. Neuronal death and mossy fiber sprouting were detected by FJB staining and Timm staining, respectively. Spontaneous seizures were recorded by EEG-video system. Morris water maze, open field test, and excitability test were used to study the behaviors of Rptor CKO mice. RESULTS: As the consequence of deleting Rptor, downstream proteins of raptor in mTORC1 signaling were partly blocked. Rptor CKO mice exhibited decrease in body and brain weight under 7 weeks old and accordingly, cortical layer thickness. After kainic acid (KA)-induced status epilepticus, overactivation of mTORC1 signaling was markedly reversed in Rptor CKO mice. Although low frequency of spontaneous seizure and seldom neuronal cell death were observed in both Rptor CKO and control littermates, KA seizure-induced mossy fiber spouting were attenuated in Rptor CKO mice. Additionally, cognitive-deficit and anxiety-like behavior after KA-induced seizures were partly reversed in Rptor CKO mice. CONCLUSION: Loss of the Rptor gene in mice neural progenitor cells affects normal development in young age and may contribute to alleviate KA seizure-induced behavioral abnormalities, suggesting that raptor protein plays an important role in seizure comorbidities.

Rapamycin prevents acute dendritic injury following seizures.

OBJECTIVE: Seizures cause acute structural changes in dendrites, which may contribute to cognitive deficits that occur in epilepsy patients. Disruption of the actin cytoskeleton of dendrites likely mediates the structural changes following seizures, but the upstream signaling mechanisms activated by synchronized physiological activity to cause seizure-induced dendritic injury are not known. In this study, we test the hypothesis that the mechanistic target of rapamycin complex 1 (mTORC1) pathway triggers structural changes in dendrites in response to seizures. METHODS: In vivo multiphoton imaging was performed in transgenic mice expressing green fluorescent protein in cortical neurons. The effect of rapamycin pre- and posttreatment was tested on kainate-induced dendritic injury and cofilin-mediated actin depolymerization. RESULTS: Kainate-induced seizures caused acute activation of mTORC1 activity, which was prevented by the mTORC1 inhibitor, rapamycin. Rapamycin pretreatment, and to a lesser degree, posttreatment attenuated acute seizure-induced dendritic injury and correspondingly decreased LIM kinase and cofilin-mediated depolymerization of actin. INTERPRETATION: The mTORC1 pathway mediates seizure-induced dendritic injury via depolymerization of actin. These findings have important mechanistic and translational applications for management of seizure-induced brain injury.

Effect of Chronic Administration of Low Dose Rapamycin on Development and Immunity in Young Rats.

Mammalian target of rapamycin (mTOR) regulates cell growth, cell differentiation and protein synthesis. Rapamycin, an inhibitor of mTOR, has been widely used as an immunosuppressant and anti-cancer drug. Recently, mTOR inhibitors have also been reported to be a potential anti-epileptic drug, which may be effective when used in young patients with genetic epilepsy. Thus, a suitable dose of rapamycin which can maintain the normal function of mTOR and has fewer side effects ideally should be identified. In the present study, we first detected changes in marker proteins of mTOR signaling pathway during development. Then we determined the dose of rapamycin by treating rats of 2 weeks of age with different doses of rapamycin for 3 days and detected its effect on mTOR pathway. Young rats were then treated with a suitable dose of rapamycin for 4 weeks and the effect of rapamycin on mTOR, development and immunity were investigated. We found that the expression of the marker proteins of mTOR pathway was changed during development in brain hippocampus and neocortex. After 3 days of treanent, 0.03 mg/kg rapamycin had no effect on phospho-S6, whereas 0.1, 0.3, 1.0 and 3.0 mg/kg rapamycin inhibited phospho-S6 in a dose-dependent manner. However, only 1.0 mg/kg and 3.0 mg/kg rapamycin inhibited phospho-S6 after 4 weeks treatment of rapamycin. Parallel to this result, rats treated with 0.1 and 0.3 mg/kg rapamycin had no obvious adverse effects, whereas rats treated with 1.0 and 3.0 mg/kg rapamycin showed significant decreases in body, spleen and thymus weight. Additionally, rats treated with 1.0 and 3.0 mg/kg rapamycin exhibited cognitive impairment and anxiety as evident by maze and open field experiments. Furthermore, the content of IL-1ß, IL-2, IFN-γ, TNF-α in serum and cerebral cortex were significantly decreased in 1.0 and 3.0 mg/kg rapamycin-treated rats. The expression of DCX was also significantly decreased in 1.0 and 3.0 mg/kg rapamycin-treated rats. However, rats treated with 1.0 mg/kg rapamycin exhibited fewer and milder side effects than those treated with 3.0 mg/kg. In summary, all these data suggest that there is not a rapamycin dose that can inhibit mTOR for epilepsy without causing any side effects, but 1 mg/kg may be the optimal dose for young rats for suppressing mTOR with relatively few side effects.

One hour of pilocarpine-induced status epilepticus is sufficient to develop chronic epilepsy in mice, and is associated with mossy fiber sprouting but not neuronal death.

Determining the minimal duration of status epilepticus (SE) that leads to the development of subsequent spontaneous seizures (i.e., epilepsy) is important, because it provides a critical time-window for seizure intervention and epilepsy prevention. In the present study, male ICR (Imprinting Control Region) mice were injected with pilocarpine to induce acute seizures. SE was terminated by diazepam at 10 min, 30 min, 1 h, 2 h and 4 h after seizure onset. Spontaneous seizures occurred in the 1, 2 and 4 h SE groups, and the seizure frequency increased with the prolongation of SE. Similarly, the Morris water maze revealed that the escape latency was significantly increased and the number of target quadrant crossings was markedly decreased in the 1, 2 and 4 h SE groups. Robust mossy fiber sprouting was observed in these groups, but not in the 10 or 30 min group. In contrast, Fluoro-Jade B staining revealed significant cell death only in the 4 h SE group. The incidence and frequency of spontaneous seizures were correlated with Timm score (P = 0.004) and escape latency (P = 0.004). These data suggest that SE longer than one hour results in spontaneous motor seizures and memory deficits, and spontaneous seizures are likely associated with robust mossy fiber sprouting but not neuronal death.

Rapamycin has paradoxical effects on S6 phosphorylation in rats with and without seizures.

PURPOSE: Accumulating data have demonstrated that seizures induced by kainate (KA) or pilocarpine activate the mammalian target of rapamycin (mTOR) pathway and that mTOR inhibitor rapamycin can inhibit mTOR activation, which subsequently has potential antiepileptic effects. However, a preliminary study showed a paradoxical exacerbation of increased mTOR pathway activity reflected by S6 phosphorylation when rapamycin was administrated within a short period before KA injection. In the present study, we examined this paradoxical effect of rapamycin in more detail, both in normal rats and KA-injected animals. METHODS: Normal rats or KA-treated rats pretreated with rapamycin at different time intervals were sacrificed at various time points (1, 3, 6, 10, 15, and 24 h) after rapamycin administration or seizure onset for western blotting analysis. Phosphorylation of mTOR signaling target of Akt, mTOR, Rictor, Raptor, S6K, and S6 were analyzed. Seizure activity was monitored behaviorally and graded according to a modified Racine scale (n = 6 for each time point). Neuronal cell death was detected by Fluoro-Jade B staining. KEY FINDINGS: In normal rats, we found that rapamycin showed the expected dose-dependent inhibition of S6 phosphorylation 3-24 h after injection, whereas a paradoxical elevation of S6 phosphorylation was observed 1 h after rapamycin. Similarly, pretreatment with rapamycin over 10 h before KA inhibited the KA seizure-induced mTOR activation. In contrast, rapamycin administered 1-6 h before KA caused a paradoxical increase in the KA seizure-induced mTOR activation. Rats pretreated with rapamycin 1 h prior to KA exhibited an increase in severity and duration of seizures and more neuronal cell death as compared to vehicle-treated groups. In contrast, rapamycin pretreated 10 h prior to KA had no effect on the seizures and decreased neuronal cell death. The paradoxical effect of rapamycin on S6 phosphorylation was correlated with upstream mTOR signaling and was reversed by pretreatment of perifosine, an Akt inhibitor. SIGNIFICANCE: These data indicate the complexity of S6 regulation and its effect on epilepsy. Paradoxical effects of rapamycin need to be considered in clinical applications, such as for potential treatment for epilepsy and other neurologic disorders.