Background Neuropsychiatric systemic lupus erythematosus (NPSLE) is a severe complication, which involves pathological damage to the brain and cognitive function. However, its exact mechanism of action still remains unclear. In this study, we explored the role of microglia in the cognitive dysfunction of NPSLE mice. We also analyzed and compared the metabolites in the hippocampal tissues of the lupus model and control mice. Methods MRL/MpJ-Faslpr (MRL/lpr) female mice were used as the NPSLE mouse model. Metabolomics was used to assess hippocampal glycolysis levels. Glucose, lactic acid, IL-6, and IL-1β of the hippocampus were detected by ELISA. Based on the glycolysis pathway, we found that pyruvate kinase isoform M2 (PKM2) in the hippocampus was significantly increased. Thus, the expression of PKM2 was detected by qRT-PCR and Western blotting, and the localization of PKM2 in microglia (IBA-1+) or neurons (NeuN+) was assessed by immunofluorescence staining. Flow cytometry was used to detect the number and phenotype of microglia; the changes in microglial phagocytosis and the β-catenin signaling pathway were detected in BV2 cells overexpressing PKM2. For in vivo experiments, MRL/lpr mice were treated with AAV9-shPKM2. After 2 months, Morris water maze and conditional fear tests were applied to investigate the cognitive ability of mice; H&E and immunofluorescence staining were used to evaluate brain damage; flow cytometry was used to detect the phenotype and function of microglia; neuronal synapse damage was monitored by qRT-PCR, Western blotting, and immunofluorescence staining. Results Glycolysis was elevated in the hippocampus of MRL/lpr lupus mice, accompanied by increased glucose consumption and lactate production. Furthermore, the activation of PKM2 in hippocampal microglia was observed in lupus mice. Cell experiments showed that PKM2 facilitated microglial activation and over-activated microglial phagocytosis via the β-catenin signaling pathway. In vivo, AAV9-shPKM2-treated mice showed decreased microglial activation and reduced neuronal synapses loss by blocking the β-catenin signaling pathway. Furthermore, the cognitive impairment and brain damage of MRL/lpr mice were significantly relieved after microglial PKM2 inhibition. Conclusion These data indicate that microglial PKM2 have potential to become a novel therapeutic target for treating lupus encephalopathy.
Background Systemic lupus erythematosus (SLE) is a chronic autoimmune disorder that results from widespread immune complex deposition and secondary tissue injury. Hydroxychloroquine (HCQ) has been used clinically to treat SLE, while its exact mechanism has still remained elusive. Some studies have shown that myeloid-derived suppressor cells (MDSCs) play a vital role in the regulation of SLE. In this study, we aimed to explore the effects of HCQ on the apoptosis of MDSCs in lupus mice and its possible molecular regulatory mechanism. Methods We constructed the imiquimod (IMQ)-induced lupus model in mice. The proportion and apoptosis of MDSCs were measured by flow cytometry. CD81-overexpressed adeno-associated virus was intraperitoneally injected into the lupus mice. We also transfected the CD81 siRNA into bone marrow-derived MDSCs, and employed qRT-PCR and Western blotting to quantify the level of CD81. Results The results showed that HCQ ameliorated IMQ-induced lupus symptoms, and simultaneously inhibited the expansion of MDSCs. In particular, HCQ induced the apoptosis of MDSCs, and also up-regulated the expression level of CD81 in MDSCs, which might indicate the relationship between the expression level of CD81 and the apoptosis of MDSCs. CD81 was further confirmed to participate in the apoptosis of MDSCs and lupus disease progression by overexpressing CD81 in vivo. Molecular docking experiment further proved the targeting effect of HCQ on CD81. And then we interfered CD81 in bone marrow derived MDSCs in vitro, and it was revealed that HCQ rescued the decreased expression level of CD81 and relieved the immune imbalance of Th17/Treg cells. Conclusion In summary, HCQ promoted the apoptosis of MDSCs by up-regulating the expression level of CD81 in MDSCs, and ultimately alleviated lupus symptoms. Our results may assist scholars to develop further effective therapies for SLE.
Background Neuropsychiatric systemic lupus erythematosus (NPSLE) is the severest complication of SLE, which often involves pathological damage to the brain and cognitive function. Glucose metabolic changes are observed in SLE patients with cognitive impairments by medical imaging. Pyruvate kinase isoform M2 (PKM2) is a vital catalyzer of glucose catabolic pathways and in neurological diseases. However, PKM2 regarding the progress of NPSLE remains poorly studied. Thus, this study aimed to analyze and compare the central carbon metabolites in the validated neuropsychiatric lupus model and control mice. Methods MRL/Mp-Faslpr (MRL/lpr) female mice were used as NPSLE mouse model, C57BL6 as control. Metabolomics to assess hippocampa glycolysis level. Glucose, lactic acid, IL-6 and IL-1β of hippocampal were detected by ELISA. The expression of PKM2 was detected by qRT-PCR and western blotting, and the localization of PKM2 in microglia and neurons was assessed with IBA-1, NeuN and PKM2 immunohistochemistry. Flow cytometry was used to detect the number and phenotype of microglia. In vitro, after transfected PKM2 overexpression plasmid on BV2, the effect on microglia and β-catenin signaling pathway were detected. Finally, PKM2 inhibitor Shikonin was injected into MRL/lpr mice, behavioral testing were performed to assess cognition, HE and FJB staining were used to evaluate brain damage.Results Glycolysis was elevated in the hippocampal tissues from MRL/lpr lupus mice, accompanied by an increase in glucose consumption and lactate production. Based on these metabolic variations, PKM2 activation was revealed in hippocampal microglia from lupus mice. Furthermore, PKM2 facilitated microglial phagocytic activity and engulfment of neurons via β-catenin signaling. In vivo, an inhibitor of PKM2, Shikonin, was shown to reduce microglial activation, loss of neuronal synapses, and block β-catenin signaling. Accordingly, the cognitive impairment and brain damage of MRL/lpr mice were relieved. Conclusion These results indicated that abnormal glycolytic metabolism in the brain tissue of NPSLE mice was induced by PKM2 overexpression, which increased the activation of microglia and the ability of phagocytizing neuronal synapses, leading to neuronal loss and cognitive dysfunction in lupus. These phenomena indicated that inhibition on PKM2 would be a novel therapeutic target for the treatment of lupus encephalopathy.
Background Out-of-bed mobilization is a significant component within the rehabilitation of people early after stroke. A top priority in acute stroke rehabilitation research is determining the optimal dose of out-of-bed mobilization exercises, such as frequency and timing. However, there is little evidence of the appropriate frequency out-of-bed mobilization for acute stroke patients. Aims The aim of the study described in this protocol is to investigate the effect of higher-frequency out-of-bed mobilization on the functional recovery of acute stroke patients. Methods This is a prospective randomized three-arm trial with the assessor blinded to the study intervention. 45 patients met the inclusion criteria will be randomly allocated to three groups, which are separately usual care (1 mobilization session/d), Frequency 1 group (2 mobilization sessions/d) and Frequency 2 group (4 mobilization sessions/d). Outcomes: The primary outcome is the modified Rankin Scale and the secondary outcomes are Rivermead Mobility Index, Fugl-Meyer (Lower Limb), Barthel Index and adverse events incidence. Outcomes will be measured at three time points. Summary: The proposed study will be beneficial for determining the frequency of out-of-bed mobilization, filling the gaps in the acute rehabilitation research field and optimising the recovery of people early after stroke. Trial registration: The proposed study has been registered at Chinese Clinical Trial on 11/03/2022, registry ID: ChiCTR2200057404.
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