Rehabilitation motivation is more important than any other factor in terms of treatment effects among stroke patients. The goal of this study is to explore the variables related to rehabilitation motivation that affect treatment effects and analyze their effect sizes, in order to manage the psychosocial interventions required by stroke patients.Thirteen electronic databases will be searched from November to December 2020. The search terms will be composed of the disease term part  and the intervention term part . Selected studies the for systematic review and meta-analysis will include randomized, quasi-randomized, and nonrandomized controlled trials, and research programs on rehabilitation motivation; qualitative research and case studies will be excluded. The participants will be stroke patients. Two authors will independently assess each study for eligibility and risk of bias, and to extract data.This study will comprehensively explore the psychosocial and physical behavioral variables related to the rehabilitation motivation of stroke patients and provide their priorities and effect sizes. In addition, we will report the magnitude of the correlation effect on the rehabilitation motivation of stroke patients according to each demographic variable.The conclusions of our study will provide effective evidence of psychosocial variables that influence the treatment outcomes of stroke patients.CRD42020207467 It is a sensitive work through interaction with the patient that results in the patient's will and motivation to engage in treatment. In particular, rehabilitation treatment is effective or discontinued not only according to the patient's rehabilitation motivation but also to the family's economic ability and psychological support to help the patient undergo long-term treatment.,5 However, in the past, rehabilitation treatment from a traditional point of view focused on simply recovering the impaired function rather than prioritizing the patient's will or goal of treatment, and viewed function recovery as the main treatment effect. However, around the world, the concept of disability has shifted from permanent damage to the body to the possibility of activity and participation in society. Rehabilitation treatment no longer regards recovery of physical function as the goal of treatment, but has started to pay attention to the patient's return to daily routine. This change in perspective has made it possible to understand the cases in which patients who received rehabilitation treatments were unable to return to their daily lives even though their functional impairment had resolved. In addition, it was predicted that the inability to return to their daily life may occur in patients who have a low probability of cure and require long-term rehabilitation. According to Choi et al,,10 such patients would include those with severe diseases that are caused by brain damage, such as stroke, and for which a cure is unlikely at 6 months after onset. In fact, most stroke patients have to completely or partially depend on others, and 12% to 18% of them also experience speech impairment.The ultimate goal of all treatment is to allow stroke patients to move by themselves and achieve an independent life. To achieve this, the doctor diagnoses the patient, develops a treatment plan, and implements it. and, in severe cases, lead to stopping rehabilitation. Stroke patients may experience anger, frustration, and depression as well as increased economic burden due to the rehabilitation treatment and family discord due to long-term treatment period.,15 This negative emotional experience and the persistence of a disability that is difficult to resolve lowers patients’ rehabilitation motivation and may cause them to stop rehabilitation. Accordingly, among severely ill patients, especially stroke patients who need long-term rehabilitation treatment, the lower the rehabilitation motivation, the more difficult the rehabilitation treatment becomes.,17These physical impairments and perceptions of continuous rehabilitation treatment reduce the adherence of stroke patientsIn addition, it is important to understand patients’ rehabilitation from a psychological point of view, and not only physical, as the patient has to leave the treatment facility and live life with a disability even when physical function is recovered. Since treatment is performed mainly focusing on functional recovery, stroke patients with a low probability of cure may face frustration in the rehabilitation process. In other words, the key to a successful rehabilitation may involve setting a realistic period of rehabilitation. However, there have been no studies investigating the factors related to the rehabilitation motivation, which is a key variable for rehabilitation outcomes and for the ultimate goal of rehabilitation, namely “return to daily life.” However, stroke studies have analyzed the relationship between self-efficacy, self-esteem, motivation-related rehabilitation, family support, economic status, and ability as an environmental factor influencing rehabilitation performance. However, it has become necessary to comprehensively analyze and organize these variables related to rehabilitation motivation. Therefore, this study aims to identify variables related to rehabilitation motivation in stroke patients by conducting a systematic literature review and a correlation meta-analysis, and to comprehensively organize the findings of previous studies.This is not a period of rehabilitation for the recovery of function as it was before the occurrence of the disease, but a goal to return to daily life in a state of recognizing the extent of realistic recovery and accepting the persisting disability.For this purpose, we aim to identify factors related to “rehabilitation motivation” among stroke patients and provide important information for future rehabilitation interventions. In addition, by classifying and organizing the psychosocial variables and physical behavior factors related to the rehabilitation motivation of stroke patients, the role of psychosocial intervention methods in rehabilitation treatment in the future will be provided as basic data in the fields of medical welfare and medical humanities.22.1 and the Cochrane Handbook for Systematic Reviews of Interventions. If the protocol represents an amendment of a previously completed or published protocol, identify as such and list changesThe protocol for this systematic review was registered in the International Prospective Register of Systematic Reviews (PROSPERO) (registration number: CRD42020207467) on November 10, 2020. This study will involve and update a systematic review according to this protocol. This protocol will be reported in accordance with the Preferred Reporting Items for Systematic Review and Meta-Analysis Protocols 2015 statement2.2The following databases will be searched comprehensively from their inception to November 2020 by 2 independent researchers (MJC and BHJ): 6 English-language databases , 5 Korean-language databases , and 2 Chinese-language databases . We will also search the reference lists of the relevant articles and perform a manual search on Google Scholar to identify additional articles. We will include not only the literature published in journals but also “gray literature” such as theses and conference proceedings. There will be no language restrictions.2.3The search terms will be composed of the disease term part  and the intervention term part . The search strategies for the MEDLINE and EMBASE databases are shown in Table 2.42.4.1Selected studies for systematic review and meta-analysis will include randomized controlled clinical trials, quasi-randomized controlled trials, controlled (nonrandomized) clinical trials, and research programs on rehabilitation motivation; qualitative research and case studies will be excluded.2.4.2We will include studies with stroke patients. There will be no restriction on the gender, age, or race of the participants.2.4.3Studies using psychosocial variables or factors related to rehabilitation motivation will be included. We will also include studies using social behavioral variables such as socioeconomic status and family support, and individual internal and external variables related with the rehabilitation adherence or treatment. There are no comparators.2.52.5.1 like other rehabilitation motivation assessment tools used as a measurement and evaluation tool in each study.The primary outcome measure is the rehabilitation motivaton, assessed with the Patient Questionnaire Rehabilitation Motivation, developed by Hafen, Jastrebow, Nubling, and Bengel (2001),2.5.2The secondary outcome measures will use tools that can be evaluated in terms of psychological factors and physical behaviors related to rehabilitation motivation.1) consists of a total of 29 questions: 5 on medications, 3 on rehabilitation exercises, 3 on bedsores prevention, 2 on aspiration prevention, and 2 on health behaviors.Rehabilitation adherence The rehabilitation adherence assessment tool for stroke patients developed by Park (2014):Adherence measures are of 3 types(1)Patient monitoring: Patient attendance to rehabilitation sessions is monitored. For each participant, the ratio of sessions attended to scheduled sessions is calculated. Attendance has been used as an adherence measure in previous sports injury research.(2)  at each physical therapy appointment, the practitioner  responsible for the rehabilitation of each participant on that day completes the sport injury rehabilitation adherence scale.Sport injury rehabilitation adherence scale(3)Patient self-reports of home exercise: At each rehabilitation session, patients report their degree of completion of prescribed home exercises on a scale ranging from 1 (none) to 10 .2)Modified Barthel indexThe modified Barthel index developed by Austrian occupational therapists will be used to evaluate the performance of daily activities.3)National Institutes of Health Stroke Scale, stroke impact scale, Scandinavian stroke scale, stroke specific quality of life scale2.6 . The extracted data will be recorded using Excel 2016  and will be shared among researchers using Dropbox  folders. We will contact the corresponding authors of the included studies via email to request additional information if the data are insufficient or ambiguous.2.8 We will assess random sequence generation, allocation concealment, blinding of participants and personnel, blinding of outcome assessments, incomplete outcome data, selective reporting, and other biases for each included study. Each domain will be categorized into 1 of 3 groups: “low risk,” “unclear,” or “high risk.” Each evaluation will be recorded in an Excel 2016 spreadsheet and will be shared among researchers using Dropbox  folders. The evaluated results will be presented in a full review using Review Manager version 5.3 . The results of the quality of evidence will be presented through a summary-of-findings table. The evaluation process will be shared and discussed by researchers.Two independent researchers, MJC and BHJ, will assess the methodological quality of the included studies and the quality of the evidence for each main finding. Discrepancies will be resolved through discussion with other researchers. The methodological quality of the included studies will be assessed using the Cochrane Collaboration risk-of-bias tool.2.9Data synthesis and analysis will be performed using Review Manager Version 5.3 (Cochrane) and Excel 2016, and files will be shared among researchers using Dropbox  folders. Descriptive analyses of the details of participants, interventions, and outcomes will be conducted for all included studies. A quantitative synthesis will be performed if there are studies using the same types of intervention, comparison, and outcome measures. The collected data will be analyzed in 2 stages by first synthesizing and analyzing the data according to the systematic review process, and then classifying the studies with figures that can be meta-analyzed. In the first stage, a systematic review aims to comprehensively organize and analyze psychosocial variables related to the rehabilitation motivation of stroke patients. A Study on the effect of psychological intervention in the recovery of stroke patients and exploring individual psychological and environmental variables, such as support for rehabilitation motivation of stroke patients. Therefore, this study will be classified and coded to “author (year of publication),” “subjects (patients),” psychosocial factors and sub-factors that affect the rehabilitation motivation of stroke patients, measurement tools of rehabilitation motivation and research methods, research procedures, and research results. We will synthesize and analyze each paper in this way. In the second step, the psychosocial factors related to the rehabilitation motivation of stroke patients used in the meta-analysis will be systematized through discussions and reviews among researchers.z  by checking the correlation coefficient in the 95% confidence interval.The framework of the analysis category will be nominated and coded based on the following items in order to calculate the size of the correlation for each study. The data coding for the meta-analysis will be as follows. First, the psychosocial variables related to the rehibiliatation of stroke patients will be classified as psychological or social variables. Second, psychological and social variables will be divided into risk factors having a negative correlation and protective factors having a positive correlation with rehabilitation motivation. Third, the sub-variables of risk factors and protection factors will be synthesized by identifying the correlation code in studies on psychosocial variables in stroke patients, reviewing the theoretical background, and classifying each variable into an easy frame for analysis. After that, we will analyze the overall publishing bias, homogeneity verification, overall correlation effect size analysis, and correlation effect size between all factors related rehabilitation motivation. The correlation effect size will be analyzed using Fisher I-squared statistic. We will consider I-squared values greater than 50% and 75% indicative of substantial and high heterogeneity, respectively. In the meta-analyses, a random effects model will be used when the heterogeneity is significant , while a fixed effects model will be used when the heterogeneity is non-significant. A fixed effects model will be also used when the number of studies included in the meta-analysis is very small, where inter-study variance estimates have poor accuracy. When it is considered that the heterogeneity is too high for the results to be synthesized , a subgroup analysis will be conducted as follows to determine the cause of heterogeneity.Heterogeneity between the studies in terms of effect measures will be assessed using both the chi-squared test and the 2.10 which was rated according to the following 5 categories: risk of bias, imprecision, inconsistency, indirectness, and other factors such as publication bias.The quality of the evidence was assessed using the Grading of Recommendations, Assessment, Development, and Evaluation,2.11I-squared value > 75%) and the necessary data are available, we will conduct a subgroup analysis to account for the heterogeneity. A subgroup analysis will be conducted according to the following criteria:If heterogeneity is evaluated as significant ((1)the stroke rehabilitation period,(2)the hospital stay period,(3)demographic variables, and(4)socioeconomic status.2.12To identify the robustness of the meta-analysis result, we will perform sensitivity analyses by determining the effects of excluding(1)studies with high risks of bias,(2)studies with missing data, and(3)outliers.2.13If there are more than 10 trials included in the analysis, reporting biases such as publication bias will be assessed using funnel plots. When reporting bias is implied by funnel plot asymmetry, we will attempt to explain possible reasons.3Ethical approval will not be needed because the data used in this systematic review will not include individual patient data and there will be no concerns regarding privacy. The results will be disseminated by the publication of a manuscript in a peer-reviewed journal and/or presentation at a relevant conference.4 As such, rehabilitation treatment requires a long period of time, and it is difficult to expect a satisfactory rehabilitation effect without the patient's active participation, a clear goal setting for the rehabilitation period, and the economic support of the family. Even if being an economically rich or with competent therapist, rehabilitation is likely to be stopped if the patient has no or low rehabilitation motivation. In particular, stroke patients show decreased willingness to rehabilitate as well as feelings of frustration and anger when they are not in the shape or situation they expect at a particular stage of recovery through rehabilitation. Therefore, it will be useful to design effective therapeutic interventions to identifying the variables that affect the rehabilitation motivation of stroke patients. However, until now, systematic searches for such variables have not been conducted. Therefore, in this study, we aim to explore the variables related to the rehabilitation motivation, one of the major factors in the treatment of stroke patients. We believe the results of this systematic review will help clinicians optimize treatment protocols for stroke patients. It is also expected that social welfare and health policy makers will be able to identify areas in the public health setting that require intervention to improve treatment for stroke patients.Rehabilitation treatment for stroke patients is not performed over a short period of time, and the concept of cure does not apply; thus, it is considered that patients are in rehabilitation for a lifetime .Conceptualization: Moon Joo Cheong, Byeonghyeon Jeon.Data curation: Moon Joo Cheong.Formal analysis: Moon Joo Cheong.Funding acquisition: Se-Eung Noh.Investigation: Moon Joo Cheong.Methodology: Moon Joo Cheong.Project administration: Moon Joo Cheong.Resources: Se-Eung Noh.Supervision: Moon Joo Cheong, Se-Eung Noh.Writing – original draft: Moon Joo Cheong.Writing – review & editing: Moon Joo Cheong, Byeonghyeon Jeon.


In familial migraine, dysfunction of nonneuronal cells alters frontal cortex physiology and promotes cranial pain. +,K+-ATPases. We used whole-cell electrophysiology, two-photon microscopy, and astrocyte gene rescue to demonstrate that an impairment in astrocytic glutamate uptake promotes NMDA spike generation in dendrites of cingulate cortex pyramidal neurons and enhances output firing of these neurons. Astrocyte compensation of the defective ATPase in the cingulate cortex rescued glutamate uptake, prevented abnormal NMDA spikes, and reduced sensitivity to cranial pain triggers. Together, our results demonstrate that impaired astrocyte function alters neuronal activity in the cingulate cortex and facilitates migraine-like cranial pain states in a mouse model of migraine.Astrocytes are essential contributors to neuronal function. As a consequence, disturbed astrocyte-neuron interactions are involved in the pathophysiology of several neurological disorders, with a strong impact on brain circuits and behavior. Here, we describe altered cortical physiology in a genetic mouse model of familial hemiplegic migraine type 2 (FHM2), with reduced expression of astrocytic Na Atp1a2 gene, which encodes the α2 subunit of the Na+, K+-dependent adenosine triphosphatase (ATPase) (α2 NKA) (2 NKA is reduced in the heterozygous +/R887Atp1a2 FHM2 knock-in (KI) mice, which carry an Atp1a2 missense mutation, causing a complete loss of function of recombinant α2 NKA , the neuronal correlate of the aura symptoms that precede migraine headache  spikes in layer 5 (L5) pyramidal neurons, and enhances their output firing. Moreover, we reveal that FHM2 mice display increased sensitivity to head pain triggers. Last, we show that rescuing the disease-causing mutation in astrocytes of the Cg recovers neuronal function and reduces the pain phenotype. Our results provide a clear example of how astrocyte dysfunction produced by a genetic defect affects neuronal activity in the Cg and affects sensitivity to head pain triggers.In this study, we took advantage of the FHM2 KI mouse model to understand whether and how a mutation in an astrocyte-specific protein affects neuronal functions in the cingulate cortex (Cg). This cortical region is crucial for pain processing and displays altered functionality in patients with migraine  expressed on perisynaptic astrocyte processes (2 NKA in heterozygous +/R887Atp1a2 mice (FHM2 mice) results in a reduction in glutamate and K+ buffering capacity of astrocytes in the primary somatosensory cortex  and FHM2 τdecay = 3.69 ± 0.15 ms ; 50 Hz: WT τdecay = 2.81 ± 0.11 ms (n = 10) and FHM2 τdecay = 3.34 ± 0.18 ms ; 100 Hz: WT τdecay = 2.54 ± 0.13 ms (n = 10) and FHM2 τdecay = 3.22 ± 0.18 ms ]. The decay kinetics of the K+ currents following a train of pulses at 100 Hz were also significantly slower in the FHM2 mice [decay = 1.72 ± 0.08 s  and FHM2 τdecay = 2.25 ± 0.18 s ]. We observed no difference in astrocytic resting membrane potential nor input resistance between the two groups (fig. S1). These data indicate that in the Cg of adult FHM2 mice, astrocytic uptake of neuron-derived glutamate and K+ was impaired.The αl slices . We recoSlower glutamate clearance by astrocytes may lead to prolonged and increased glutamate levels in the extracellular space. To directly test this prediction, we took advantage of the intensity-based glutamate fluorescent sensor iGluSnFr . To estidecay = 75.56 ± 3.62 ms  and FHM2 τdecay = 100.9 ± 5.33 ms ; 100 Hz: WT τdecay = 65.6 ± 3.32 ms (n = 14) and FHM2 τdecay = 84.57 ± 4.77 ms ]. Note that we and others have previously demonstrated that the size and location of ROIs, stimulation intensity, and sulforhodamine 101 dye (SR-101) do not influence the iGluSnFr decay kinetics   .n = 12 cells, N = 8 mice) and FHM2, 9.13 ± 2.48 mV ; second pulse AUC: WT, 40.64 ± 13.9 mV ms (n = 11) and FHM2, 411.5 ± 131 mV ms ; stimulation intensity, 2 mA]. Partial blockade of GluTs with subsaturating concentrations of the GluT blocker dl-TBOA (3 μM) significantly enhanced NMDA spike generation in L5 pyramidal neurons in WT mice (fig. S3), mimicking the effect of the FHM2 genetic mutation.As predicted from the reduced glutamate clearance in FHM2 mice, these mice displayed facilitated NMDA spike generation reflected by higher amplitude and AUC of the voltage responses evoked by L1 synaptic stimulations . We additionally recorded AMPA-mediated EPSCs evoked with single-pulse and paired-pulse extracellular synaptic stimulations (n = 7 cells) and FHM2 KI, −118.6 ± 12.03 pA ; decay: WT, 17.7 ± 2.1 ms (n = 7 cells) and FHM2 KI, 18.29 ± 1.4 ms ]. Similarly, upon paired-pulse stimulation at 20 Hz, the second-over-first EPSC amplitude ratio was comparable in WT and FHM2 mice . These datasets strongly argue against alterations in glutamate release or in AMPA-mediated synaptic transmission in the Cg of FHM2 mice.We then investigated whether synaptic activity is altered in FHM2 mice. We first recorded miniature excitatory postsynaptic currents (mEPSCs) from L5 pyramidal cells of WT and FHM2 mice  and founulations . Upon si2 NKA (Atp1a2) in astrocytes. To deliver Atp1a2, we used an adeno-associated virus (AAV) of the 5/2 serotype that preferentially targets astrocytes and took advantage of the astrocyte promoter hGFAP (AAV.hGFAP.ATP1A2)  and rescue τdecay = 2.14 ± 0.13 s ] and active glutamate clearance by astrocytes became significantly faster in FHM2 mice in which WT Atp1a2 was expressed compared to FHM2 mice that were injected with the control virus . The decay kinetics of the uptake currents in the rescued FHM2 mice became comparable to those observed in WT mice (fig. S7A). The astrocytic resting membrane potential was slightly but significantly hyperpolarized in rescued FHM2 mice compared to those injected with control virus, with no difference in input resistance (fig. S8).We then performed electrophysiological recordings in the same manner as in Atp1a2 compared to FHM2 mice injected with the control virus . The values in the rescued FHM2 mice were comparable to those in WT mice (fig. S7B). This rescue was also accompanied by a significantly lower probability of AP firing following NMDA spikes in the rescued FHM2 mice ] triggered facial mechanical hypersensitivity in both WT and FHM2 mice at 30, 60, and 120 min post NTG injection compared to mice injected with saline . FHM2 mice developed orofacial hypersensitivity upon NTG doses that were ineffective in WT littermates . These results suggest that the FHM2 mutation promotes the development of NTG-induced facial mechanical hypersensitivity. The same phenotype was observed with a lighter von Frey filament of 0.025 g (fig. S9). Activity tests showed no difference between saline- and NTG-treated mice or between FHM2 and WT littermates, suggesting that the different responses to mechanical stimulation were not accompanied by alterations in motor function .The Cg is a critical cortical region in encoding cephalic pain. Altered neuronal activity in this brain area has been reported to influence the activation and sensitization of pain pathways in pathological pain conditions (Atp1a2 (AAV.hGFAP.ATP1A2) in Cg astrocytes of FHM2 mice. As a control, FHM2 mice were injected with AAV.hGFAP.eGFP. Three weeks after viral injections, we triggered facial mechanical sensitivity by NTG injections (5 mg kg−1) and performed facial von Frey tests as described above (N = 14 mice) and rescue, 91.40 ± 13.98 ].Since the Cg is implicated in pain signaling and development of mechanical hypersensitivity in several pathological pain syndromes, we wondered whether rescuing local astrocyte dysfunction in this brain area could ameliorate the increased nocifensive responses detected in FHM2 mice upon NTG treatment  in WT mice. The probability of AP firing following NMDA spikes was also increased in FHM2 mice. In contrast, no changes were observed in spontaneous and evoked synaptic transmission in FHM2 mice.The slowdown in glutamate clearance by astrocytes prolonged the presence of elevated glutamate levels in the extracellular space. Increase in glutamate spillover enhances cortical excitatory neurotransmission, particularly NMDA receptor–mediated transmission  and midcingulate cortex (MCC) play a key role in pain processing  heterozygous KI mice harboring the W887R FHM2 mutation [f]quinoxaline-2,3-dione (NBQX), and picrotoxin were obtained from Sigma-Aldrich. 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), AP-V, dl-TBOA, and d-serine were obtained from Tocris. NBQX, CNQX, and dl-TBOA were dissolved in dimethyl sulfoxide. Tetrodotoxin (TTX) was obtained from Abcam. NTG was obtained from Sigma-Aldrich. Picrotoxin was dissolved in ethanol (EtOH). AP-V, d-serine, and TTX were dissolved in ddH2O.Reagents for artificial cerebrospinal fluid (ACSF) and internal solutions, biocytin, 6-nitro-7-sulfamoylbenzo for a day each (www.mesospim.org) with an Olympus MVX10 macroscope in the detection path and an MVPLAPO 1× objective , the samples were incubated with Laemmli sample buffer (Bio-Rad) for 30 min at 37°C. Aliquots containing 10 μg (GLAST) or 15 μg of protein (ATPase α2 and GLT-1) were subjected to SDS–polyacrylamide gel electrophoresis using 10% (GLAST and GLT-1) or 7.5% (ATPase α2) mini gels . Proteins were transferred onto nitrocellulose membranes in a Trans-Blot semi-dry transfer cell (Bio-Rad) at 15 V for 60 min using 39 mM glycine, 48 mM tris, 1.3 mM SDS, and 20% methanol as transfer buffer. After blotting, the transferred proteins were stained with REVERT Total Protein Stain (LI-COR) and immediately imaged using the Odyssey CLx imager (LI-COR). For immunodetection, the blots were blocked for 1 hour in PBS containing 5% nonfat dry milk at room temperature, followed by incubation at 4°C overnight with anti–α2 NKA antibodies , anti–GLT-1 antibodies , or anti-GLAST antibodies , diluted in PBST [PBS (pH 7.4) and 0.05% Tween 20] containing 5% nonfat dry milk. The blots were then washed five times for 5 min with Tris Buffered Saline + Tween 20 (TBST)  and incubated with secondary antibodies  for 1 hour at room temperature. Following extensive washing (see above), immunoreactivity was detected using the Odyssey CLx imager (LI-COR). Immunoreactivity was quantified with the Image Studio software (LI-COR) and normalized to total protein in the corresponding lanes. Each antibody was tested for its linear detection range using different protein and antibody concentrations.Two weeks following stereotaxic viral injections in the Cg, tissue of the Cg was rapidly dissected and immediately frozen on dry ice and stored at −80°C until used. On the day of the experiment, the tissue (10 to 20 mg) was thawed and homogenized by sonication in 20 volumes of 20 mM tris (pH 7.4) containing the protease inhibitor cocktail cOmplete Mini (Roche Diagnostics). The homogenate was centrifuged for 10 min at 1000−1, i.p.) before transcardiac perfusion with 20 ml of phosphate buffer followed by 100 ml of 4% ice-cold PFA [in 0.1 M sodium phosphate buffer (pH 7.4)]. Brain tissue was postfixed for 4 hours with 4% PFA on ice and cryoprotected in 30% sucrose solution (in 0.1 M sodium phosphate buffer) overnight at 4°C. Brains were embedded in NEG50 frozen section medium (Richard-Allan Scientific) and cut into 40-μm free-floating sections . Brain sections were left in antifreezing solution at −20°C until use. Following incubation in blocking solution  for 1 hour, brain sections were incubated at 4°C overnight in a primary antibody solution  containing combinations of the following antibodies: chicken anti-GFP , guinea pig anti-NeuN , and rabbit anti-S100B . Three washing steps of 10 min each in PBS were performed before incubating brain sections with secondary antibodies (1:500): Cyanine Cy3 donkey anti-rabbit , Alexa Fluor 647 donkey anti-guinea pig , and Alexa Fluor 488 donkey anti-chicken  for 90 min at room temperature in PBS supplemented with 0.3% Triton X-100. Images were taken with an LSM 800 with Airyscan confocal microscopes (Carl Zeiss) controlled with ZEN 2.3 (blue edition) software and using a Plan-Apochromat ×40/1.4 Oil DIC M27 oil-immersion objective. Z stack images of 10 optical sections and 1.5-μm step size were used for the analysis of fluorescence colocalization and to create maximum intensity projections images. Images were processed using ImageJ software.Three weeks after stereotaxic viral injections in the Cg, FHM2 KI mice were anaesthetized with pentobarbital  with Bonferroni post hoc test, one-way ANOVA with Tukey post hoc test, or two-way repeated-measures ANOVA test. All graphs and statistical tests were performed using GraphPad Prism, and figures were prepared using Adobe Illustrator CS5. P values less than 0.05 were considered statistically significant.Data are displayed as means ± SEM in all experiments except for the Western blot data that are displayed as means ± SD. Statistical details can be found in Results, figures, and figure legends. Statistical comparisons were made with two-tailed paired or unpaired


Purpose: Polycystic ovarian morphology (PCOM) is one of the key features of polycystic ovary syndrome (PCOS). The diagnosis of PCOM according to the Rotterdam criteria  is debated because of the high prevalence of PCOM in the general population. Androgen receptor (AR) is associated with the PCOS phenotype and might as well play a role during folliculogenesis. This study is aimed to investigate the expression of the AR in PCOS granulosa cells (GCs) and its relationship with the PCOM phenotype. Methods: 106 PCOS cases and 63 controls were included from the Center for Reproductive Medicine, Shandong University. The diagnosis of PCOS was following the Rotterdam criteria (2003). Total RNA was extracted from GCs retrieved from ovarian stimulation. The expression of AR was amplified by means of quantitative real-time polymerase chain reaction. Results: The AR expression was significantly decreased in PCOS cases, especially in the tPCOM subgroup . Correlation analyses showed that AR expression was significantly correlated with serum FSH levels in controls and non-tPCOM. In the tPCOM subgroup, the AR expression was significantly correlated with serum LH levels. Interestingly, the significance of these correlations gradually disappeared as the threshold of antral follicles increased above 24 for PCOM. Conclusions:AR was differently expressed in PCOS and especially in the tPCOM subtype. The correlation of AR expression with serum FSH and LH might be associated with the number of follicles in PCOM. Polycystic ovarian morphology (PCOM) is one of the key features of PCOS, characterized by increased recruitment of pre-antral and antral follicles but fail to progress to ovulation. Due to the observer’s variability and the improvements in imaging technology, the threshold of ≥12 antral follicles per ovary defining the diagnosis of PCOM might lead to over-diagnosis. Moreover, several studies show a high prevalence of PCOM in the population leading to a continuous debate considering the best threshold for PCOM4. The recently published international PCOS guideline recommends based on the existing evidence ≥20 follicles per ovary5. Up until now, molecular evidence has not been included in the discussion considering PCOM threshold6.Polycystic ovary syndrome (PCOS) is a worldwide female endocrine disorder, with a prevalence up to 10–15% of reproductive-aged women leading to infertility because of anovulation, hyperandrogenism, and metabolic abnormalitiesAR mutations are found in complete androgen insensitivity patients7. The AR signalling pathway has been recognised as a potential factor influencing ovarian function leading to anovulation in PCOS. Studies found that the AR (CAG)n polymorphic trinucleotide repeats in the N-terminal domain8 and rs6152 gene polymorphisms9 are associated with PCOS. The AR exhibits distinct expression patterns at different stages of follicle growth. The AR expression is high in GCs of pre- and early antral follicles and decreases as the follicles maturate. This might indicate that AR-mediated androgens might play a role during folliculogenesis11.The majority of women with PCOS suffer from hyperandrogenism. Androgens act via the androgen receptor (AR) in a variety of tissues. AR knockout (ARKO) mice exhibit subfertility13. The lack of androgen activity in the GCs leads to prolonged oestrous cycle, increased number of pre-antral and atretic follicles with decreased corpora lutea and ovulation rates15. In addition, the theca cell and the oocyte-specific ARKO mice show normal fertility and follicle populations16, implying the important role of AR signalling in GCs. Studies of the expression levels of AR in PCOS GCs are few and the results of these studies are not consistent. The AR expression of GCs from small and large antral follicles from PCOS women is controversial. It is inconsistent that the AR expression is higher or lower in different GCs from PCOS women18. We supposed that the controversial results might be related to the different phenotypes of PCOS, due to its heterogeneity. Therefore, we studied the expression of AR in luteinized GCs in a large group of PCOS patients and further analyzed its relationship with PCOM phenotype.There is some evidence that global 19. PCOS was diagnosed when at least two of the following criteria were present: oligo- or anovulation, clinical and/or biochemical signs of hyperandrogenism, polycystic ovaries with exclusion of other etiologies . Control women had a regular menstrual cycle (26–35 days) and steroid hormone levels within normal range. They had a normal ovarian morphology. Control women visited the IVF center because of oviduct and/or male factors related infertility.A total of 169 Chinese women were recruited in the Center for Reproductive Medicine, Shandong University from October 2015 to June 2016. The participants consisted of 106 PCOS cases and 63 controls. The diagnosis of PCOS was defined according to the Rotterdam criteriaAll participants’ anthropometric variables, including age, height, body weight, and menstrual cycle were recorded. The levels of day 3 serum hormones including follicle stimulating hormone (FSH), luteinizing hormone (LH), oestradiol (E2), progesterone (P), total testosterone (TT) and anti-Müllerian hormone (AMH) were measured in the clinical laboratory of Center for Reproductive Medicine, Shandong University by chemiluminescence immunoassay (CLIA) and Enzyme-Linked Immuno-sorbent Assay (ELISA). Antral follicle count (AFC) was assessed by transvaginal ultrasound.20.For ovarian stimulation, the long gonadotropin-releasing hormone agonist protocol was used. All participants were injected with a gonadotropin-releasing hormone (GnRH) agonist at the beginning of the mid-luteal phase, and the ultrasound scan for follicle development and serum oestradiol assays were performed every 1 to 3 days. When more than 3 follicles measured ≥18 mm in diameter, moderate human chorionic gonadotropin (hCG) was administrated. Ultrasound-guided oocyte retrieval was performed 36 hours after hCG injection. The GCs were collected in sterile tubes from the follicular fluid and isolated with Ficoll-Percoll  as previously described18sRNA was used for normalization and the relative expression of AR mRNA was calculated based on the 2−ΔCt method21.Total RNA was extracted from GCs using TRIzol Reagent  following the manufacturer’s instructions, and was reversely transcribed to cDNA using Prime Script RT reagent Kit with gDNA Eraser . qRT-PCR was performed using SYBR Premix Ex Taq  on a LightCycler 480 system according to the manufacturer’s instructions. The primers were shown as Supplementary Table 5. Based on the preliminary correlation analyses, the threshold of AFC gradually increased, and these cases were divided into PCOM subgroup. The remaining PCOS cases were defined as non-PCOM. New grouped non-PCOM and PCOM subgroups were only performed association analyses between the AR expression and endocrine parameters. The study group design was shown as Fig. The PCOS cases were grouped into non-true-PCOM  and true-PCOM  for preliminary data analysis based on the threshold of PCOM suggested by the recently published international PCOS guidelineAR expression with endocrine parameters were performed using Spearman test. p < 0.05 was statistically significant.Data were analyzed using SPSS 20.0. Data distribution was assessed using the Kolmogorov-Smirnov test to determine whether continuous variables were normally distributed. Abnormal distribution data were transformed into normal distribution data. Student’s t-test was used to determine statistical significance for baseline characteristics between PCOS cases and the controls. Two-way ANOVA followed by Bonferroni and Dunnett-T3 test was performed for multiple comparisons amongst controls, non-tPCOM and tPCOM groups. The association analyses of the All experimental protocols performed in studies involving human participants were in accordance with the ethical standards of the Ethics Committee of Shandong University and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Written informed consent was obtained from each patient. All experimental protocols were performed in accordance with relevant guidelines and regulations approved by the Institutional Review Board of Shandong University.p < 0.05). No significant differences were found in age and serum oestradiol levels (p > 0.05).We collected GCs from 63 controls and 106 PCOS cases. All participants were 20 to 35 years old. The comparison of the anthropometric, biochemical and endocrine parameters between PCOS patients and controls was shown in Table AR mRNA expression in PCOS patients was significantly lower compared to the controls  and non-tPCOM group . The significant correlation between the AR expression and serum FSH levels was lost at the threshold of 24 follicles per ovary in non-PCOM subgroup, and with the threshold increased the correlation coefficient gradually reduced. While in PCOM group, there was no significant correlation between the AR expression and serum FSH levels. In the meanwhile, the relationship between the AR expression and serum LH levels showed non-significant in PCOM group at this threshold  in human pre-antral follicles. A positive correlation has been described previously between AR and FSHR mRNA levels in the GCs from normal cyclic, androgen or FSH-treated primates26. Moreover, this is described in GCs of antral follicles in PCOS27. Although exogenous androgens stimulate mRNA expression of the FSHR in follicles at all development stages, FSH only increases AR mRNA levels in primary follicles26. It suggests some interaction between AR and FSH in early follicular development. It has been suggested28 that transgenic FSH can partially rescue the subfertility phenotype and ovarian function in ARKO mice. This principle is also used in ovulation induction treatment in anovulatory women with PCOS. The positive correlation of the AR expression in GCs with FSH levels in controls and non-PCOM cases found in our study was in line with the previously published studies. It also indicated that there might be a larger number of antral follicles as well as atretic follicles in true PCOM cases.We then analyzed the correlations of the 30. We found the significant negative correlation between AR expression and serum LH levels in true PCOM cases, indicating that the interrelationship of androgen and LH might have an impact on the medium to large-sized antral follicles. Together with our previous reports31, which showed LHCGR is increased in PCOS GCs, the abnormal expression of these receptors might contribute to the abnormal antral follicles in PCOS.In normal ovaries, only the GCs from a large (13 mm in diameter) and dominant follicle respond to LH. Cells derived from women with PCOS have inappropriate responsiveness to LH in some follicles as small as 2–4 mm. Also, high basal levels of LH show an exaggerated response in a proportion of medium-sized antral folliclesAR was significantly positively correlated with FSH in control and non-tPCOM groups while had a negative correlation tendency with FSH in tPCOM group when defined by 20 follicles per ovary. And the AR expression was significantly negatively correlated with LH in tPCOM but did not show a similar correlation in the other two groups. It made us wonder why those correlations of AR and endocrine levels were different in tPCOM and non-tPCOM. We suspected that these correlations might represent special features of PCOM except the ultrasound change. Hence, we increased the threshold of follicles per ovary and analyzed the correlation changes. To our surprise, we found that the significance of correlation disappeared above a follicle threshold of 24 follicles per ovary. It indicated that different phenotypes of PCOS arisen over 24 follicles per ovary, which made us reconsider the definition of PCOM.In this study, we accidentally found that the expression of 5. While some researches supported that ≥25 follicles per ovary might be more suitable for PCOM definition4. However, it is hard to define an appropriate strict threshold for PCOM. Though AR expression in GCs could not be considered as a diagnostic marker of PCOM, our findings of the interesting correlation pattern below and over 24 follicles might provide evidence from the molecular level that a stricter threshold might be more suitable for true PCOM.The recently published international PCOS guideline recommends based on the existing evidence ≥20 follicles per ovaryAR expression in GCs of PCOS patients was significantly reduced, and the reduction was much more significant in the tPCOM subgroup, indicating that the AR-mediated action might play important roles for the folliculogenesis of PCOS. The significant correlations of the AR expression in GCs with FSH and LH were lost above a follicle threshold of 24 follicles per ovary.To summarize, we have investigated that the Supplementary Figure S1.Supplementary Figure S2.Supplementary information.


Acute hyperglycemia is closely related to kidney injury. Oxidative stress activation and notable mitochondria damages were found under acute hyperglycemia treatment in our previous work. In the present study, we explored the dose-effect relationship and the pivotal role of mitophagy in acute hyperglycemia induced tubular injuries.Forty non-diabetic SD rats were randomly divided and treated with different concentrations of hyperglycemia respectively during the 6-h clamp experiment. Renal morphological and functional alterations were detected. Rat renal tubular epithelial cells were treated with different concentrations of glucose for 6 h. Markers and the regulation pathway of mitophagy were analyzed.P < 0.05). ROS was activated, mitochondrial membrane potential and LC3-II/LC3-I ratio were decreased but P62 and BNIP3L/Nix were increased in hyperglycemia groups (P < 0.05), which were reversed by AMPK activation or mTOR inhibition.Significant tubular injuries but not glomeruli were observed under both light and electron microscope after acute hyperglycemia treatment, which manifested as enlargement of tubular epithelial cells, disarrangement of epithelial cell labyrinths and swelling of mitochondria. Urinary microalbumin, β2-MG, CysC, NAG, GAL, and NGAL were increased significantly with the increase of blood glucose (Acute hyperglycemia causes obvious tubular morphological and functional injuries in a dose-dependent manner. Acute hyperglycemia could inhibit mitophagy through AMPK/mTOR pathway, which would aggravate mitochondria damage and renal tubular impairment. Diabetes mellitus (DM) is becoming a more threatening public health problem not only because of its high prevalence, but also owing to its high incidence and poor outcomes of vascular complications such as diabetic kidney disease (DKD). People have used to concentrate on the bad effects of chronic, longstanding hyperglycemia for a long period, but neglect the injuries resulting from acute hyperglycemia. In peoples’ traditional view, acute hyperglycemia occurred in diabetic ketosis and hypertonic hyperglycemia state could induce a series of metabolic disorders; in addition, acute hyperglycemia could suppress the insulin secretion function of β-cells transiently which is called “acute hyperglycemic toxicity”. We often encounter patients with diabetic ketosis accompanied by transient albuminuria, who are usually misdiagnosed as “diabetic kidney disease”. Does it mean rapidly elevated blood glucose could also lead to “acute hyperglycemic renal toxicity”? Patients admitted in intensive care unit (ICU) with acute hyperglycemia usually have a high prevalence of acute kidney injury (AKI), and strict control of blood glucose could obviously improve this outcome . In ordeThe abnormal increase of reactive oxygen species (ROS) in renal tubular epithelial cells is the pivotal mechanism of renal tubular injury , 4. In oForty male Sprague-Dawley rats (body weight 250–280 g) supplied by Beijing HuaFuKang Bioscience Co., LTD were included in this study. All rats were maintained at 20–25°C and 50%–60% humidity on a 12-h–12-h light-dark cycle with free access to food and water. This study was permitted by the Tianjin Medical University Animal Committee, and all the animals were maintained according to the guidelines for the care and use of laboratory animals.All animals received surgical placement of catheters into the left internal jugular vein which were then externalized to the back of the neck under anesthesia  after 1 week of adaptation. All the rats regained their presurgical body weight and kept in a good health condition before the clamp, which were preformed 5 days after surgery.Rats were randomly assigned to four groups, control group, hyperglycemia group A , hyperglycemia group B , and hyperglycemia group C , 10 in each group. Rats were kept in a postabsorptive state before the clamp, and stayed awake in the fixator during the clamp. A 6-h hyperglycemia-clamp procedure was performed as described in our previous study  stain. After staining, tissue sections were dehydrated in increasing concentrations of ethanol and xylene and sealed with gum. The changes of glomerular and tubular morphology were observed using light microscope (OLYMPUS IX5O/BX5O).3) were fixed in 2.5% glutaraldehyde and 1% osmium tetraoxide, dehydrated with gradient alcohol  and epoxypropane. Samples were then oriented longitudinally and embedded in Epon 812. Ultrathin sections were cut into 50 μm ± using ultramicrotome and then dyed with uranyl acetate and lead citrate. Transmission electron microscope (HITACHI-7500) was used to observe glomerular and tubular morphology and autophagosome.Kidney tissue specimens  for 6 h. 3- -2, 5-diphenyltetrazolium bromide (MTT) assay was used to assess cell viability. NRK-52E cells were treated with hyperglycemia (16.7 mmol/L) in the presence or absence of 5’-AMP- activated protein kinase (AMPK) activator 5-Aminoimidazole-4-carboxamide1-β-D-ribofuranoside (AICAR)  or mammalian target of rapamycin complex (mTOR) inhibitor rapamycin  for 6 h in order to detect the role of AMPK or mTOR in acute hyperglycemia induced kidney injury. Total RNA and protein were extracted for further analysis. Studies were replicated three times.Rat renal tubular epithelial cells (NRK-52E cells) purchased from Chinese Academy of Sciences Cell Library were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum  and 1% penicillin/streptomycin (Gibco) at 37°C and 5% COCells were incubated with H2-DCFDA  for ROS detection. Media was aspirated from the cells grown in 24-well plates and the cells were washed twice with 500 μl PBS. 10μM H2-DCFDA was added to the monolayer of cells. The plate was incubated in the dark at 37°C for 30 min. After incubation, cells were washed with 500-μl PBS and then observed using fluorescence microscope .Mitochondrial Membrane Potential Assay Kit   was used for quantifying changes in mitochondrial membrane potential (MMP) in NRK-52E cells. JC-1 exhibits potential-driven accumulation in mitochondria, decreased red fluorescence and corresponding increased green fluorescence suggest depolarized mitochondria. Cells grown in 24-well plates were incubated with JC-1 dyeing working solution in the dark at 37°C for 20 min. After incubation, cells were washed with JC-1 dyeing buffer and examined using fluorescence microscope .Renal tissues and cells were homogenized in RIPA buffer containing Protease/Phosphatase Inhibitor Cocktail  after washing with PBS, and total protein concentration was estimated using BCA Protein Assay Kit . Protein samples (40-80μg) were submitted to SDS-PAGE and then transferred to nitrocellulose membranes. Membranes were blocked for 2 h in 5% non-fat milk and then incubated with primary antibodies against LC3 , SQSTM1/p62 , BNIP3L/Nix , mTOR , Phospho-mTOR , AMPKα , Phospho-AMPKα , SGLT2 , and β-actin  at 4°C overnight. After being washed with TBST, the membranes were incubated with peroxidase-conjugated secondary antibodies . The reactive bands were detected using the ECL system (Advansta). Signal intensity was then assessed using automatic gel imaging system (SYNGENE). Studies were replicated 3 times.−ΔΔCT method . The cDNA was synthesized using Reverse Transcription Kit  according to the following protocol: 25°C for 5min, 42°C for 60min, 70°C for 5 min. All primers were designed and synthesized by Beijing oak Biotech Corp. Primers are listed in T method . The β-aP < 0.05 were considered statistically significant.SPSS 20.0 was used to analyze the data. All values were tested for normality and homogeneity of variance. Normally distributed values were expressed as means ± SD. Independent t-test was used to compare differences between two groups. One-way analysis of variance was used to analyze the differences among multiple groups. Parameters that were not normally distributed were expressed as  and compared using Rank sum test. Values of P < 0.05].During the hyperglycemic clamp, as shown in We detected the renal glomerular and tubular morphology alterations in rats using both optical microscope and transmission electron microscope under different concentrations of acute hyperglycemia treatment.Under optical microscope, swelling of tubular epithelial cells and tubular stenosis in each hyperglycemic group were noticed, which became more severe with the increase of blood glucose. However, no obvious differences in glomeruli were detected compared with that in control group . However, 24-h UMA increased significantly in hyperglycemia groups compared with that in control group, which increased gradually with blood glucose increase  . As shown in P < 0.05), and urinary NAG, β2-MG and CysC began to elevate significantly since blood glucose reached 16.7 mmol/L .Urinary β2-MG and CysC were used to assess the reabsorption function of renal tubules, while NAG, GAL and NGAL were used to evaluate injuries of tubular epithelial cells. All these indicators increased significantly in hyperglycemia groups compared with that in control group   with different concentrations of glucose to study the effects of acute hyperglycemia on tubular epithelial cells.P > 0.05). Consistent with the results in vivo, we also detected the gradually down-regulated LC3-II/LC3-I ratio accompanied with the gradually up-regulated P62 and BNIP3L/Nix in hyperglycemia groups (P < 0.05) (As shown in in vivo and in vitro in this study (P < 0.05 for HG-C vs. Control). The ratio of p-AMPK/AMPK were decreased and p-mTOR/mTOR were increased by high glucose treatment (P < 0.05 for HG-B and C vs. Control) (P < 0.05)  Figure 6As the prevalence of diabetes is increasing rapidly, people are used to concentrate more on chronic hyperglycemia, the main clinical sign of diabetes, and its poor outcomes. But acute hyperglycemia, often occurs in diabetic patients with ketoacidosis and hyperosmolar states and non-diabetic patients with severe injuries, is usually neglected. Studies have revealed a strong relationship between acute hyperglycemia and kidney injuries. Albuminuria occurs commonly in new diagnosed diabetic patients especially those with diabetic ketosis, who are usually misdiagnosed as “diabetic kidney disease”. For critically ill patients with or without pre-diagnosed diabetes, acute elevated blood glucose could increase the incidence of acute kidney injury, and strict control of blood glucose could improve this outcome , 16–18. Renal tubules manifested to be more seriously damaged under acute hyperglycemic treatment in the present study. Tubular morphological changes could be detected since blood glucose reached 11.1 mmol/L under both light and electron microscope, and became more obvious with blood glucose concentration increase. Urinary microalbumin (UMA), a traditional marker representing damages of glomerular filtration barrier and glomerular hyperfiltration, increased gradually in a dose-dependent manner since blood glucose reached 11.1 mmol/L. In recent years, UMA is believed to be primarily controlled by renal tubular epithelial cells, which represents tubular reabsorption dysfunction defined as “diabetic tubulopathy” –24. A goP < 0.05). Urinary GAL and NGAL even began to increase significantly since blood glucose reached 11.1 mmol/L (P < 0.05). In this study, we could conclude that acute hyperglycemia could induce obvious morphological and functional damages of tubular epithelial cells in a dose-dependent manner.Plasma low molecular weight proteins (LMWP) β2-MG and CysC are freely filtered through glomerular filtration membrane and almost reabsorbed by proximal tubular epithelial cells. The raise of urinary β2-MG and CysC reflect the impaired proximal tubular reabsorption . In the Renal tubule is responsible for the reabsorption, secretion, and excretion of many important molecules and is susceptible to various kinds of harmful factors such as ischemia, hypoxia, and oxidative stress because of its structural and functional characteristics. Proximal tubular epithelial cells are very vulnerable to high glucose damage, as they cannot decrease glucose transport to prevent excessive changes of intracellular glucose concentration when exposed to hyperglycemia . Blood sTubular epithelial cells contain large amounts of mitochondria for their energy requirement, and an impairment of mitochondrial bioenergetics can result in renal functional decline. In our previous study, mitochondria in tubular epithelial cells were seriously damaged, and oxidative stress was obviously activated after 6-h hyperglycemia treatment . Oxidatiin vivo and in vitro, which suggested the inhibited autophagy under acute hyperglycemic treatment.Autophagy is a lysosome degradation pathway that plays an important role in maintaining intracellular homeostasis and cell integrity through removing protein aggregates and damaged or excess organelles. Autophagy was up-regulated and provided a protected effect in acute kidney injuries induced by drugs or toxins –9. WhileExcess oxidative stress activation could aggravate mitochondria damages and even cell apoptosis, and clearing away damaged mitochondria through mitophagy in time is crucial for cellular homeostasis. Mitophagy, firstly proposed by Lemasters in 2005 , was defin vivo and in vitro. Mitophagy was inhibited in a dose-dependent manner in tubular epithelial cells both in vivo and in vitro after acute hyperglycemia treatment, which was in consistence with long term hyperglycemia treatment . In the present study, SGLT2 increased gradually in hyperglycemia groups which aggravated glucose reabsorption in renal tubular epithelial cells. P62 accumulation decreased in type 1 diabetic mice after sorption .AMPK and mTOR play a central role in the regulation of autophagy initiation. AMPK is a metabolic master-switch that regulates and maintains cellular energy homeostasis. Loss of sensitivity of AMPK activation to cellular stress impairs metabolic regulation, increases oxidative stress and apoptosis, and reduces autophagic clearance. AMPK activation will then turn off mTOR signaling and resulting in autophagy induction .Activating mTOR signaling pathway could aggravate podocytes damage and glomerular filtration rate decline in patients with DKD . Acute gOur results also showed that acute hyperglycemia could improve the phosphorylation level of mTOR, but decrease phosphorylation level of AMPK both in renal tissues of healthy rats and NRK-52E cells. Restoration through AMPK activation or mTOR inhibition could improve mitophagy activity, decrease ROS production and improve MMP in NRK-52E cells, suggesting AMPK/mTOR pathway was directly involved in the regulation of mitophagy inhibited by acute hyperglycemia induced tubular injuries.Our study still has some limitations. First, gene knockout or overexpression model should be used to further verify the possible regulation pathway of acute hyperglycemic tubular injury; second, the reversibility of acute hyperglycemic tubular injury should be discussed in the future study.In conclusion, acute hyperglycemia could lead to obvious tubular morphological and functional injuries when the blood glucose level was greater than 11.1 mmol/L, and behaved in a dose-dependent manner. Acute hyperglycemia could inhibit mitophagy through AMPK/mTOR pathway, which would aggravate damaged mitochondria accumulation and renal tubular injuries. The protective role of mitophagy improvement under acute hyperglycemic stress is in urgent need to be verified in the future.All datasets generated for this study are included in the article/supplementary material.JW, XY, BaiC, and BaoC conceptualized the study. XY, ZW, and XJ contributed to the methodology. XC and ZG were in charge of the data curation. JY and CS contributed to the project administration. YY and JL were in charge of the validation. JW, and BaoC acquired the funding. JW and XY wrote the original draft. BaiC and BaoC wrote, reviewed, and edited the manuscript. CM contributed to the methology and validation. All authors contributed to the article and approved the submitted version.This work was supported by the National Key R&D Program of China (No. 2018YFC1314000), National Natural Science Foundation of China , Tianjin Natural Science Foundation , and the fund of the State Key Laboratory of Kidney Diseases in PLA General Hospital (KF-01-133).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Curcuma longa. Ar-turmerone exhibited anti-inflammatory properties. So far, antiplasmodial data for ar-turmerone is still not reported. The data showed the in vitro antiplasmodial effect of ar-turmerone against Plasmodium falciparum 3D7 (chloroquine-sensitive) via Plasmodium lactate dehydrogenase assay (pLDH) and cytotoxic effect against Vero mammalian kidney cells using 3--2, 5-diphenyltetrazolium bromide (MTT) colourimetric assay. Selectivity indexes of ar-turmerone were calculated based on inhibition concentration at 50% of parasite growth (IC50) from MTT and pLDH assays and the effects of ar-turmerone were compared to the antimalarial reference drug chloroquine diphosphate. The inhibitory effect of ar-turmerone at the intraerythrocytic stages of plasmodial lifecycles was evaluated via a stage-dependant susceptibility test. The antiplasmodial and cytotoxic activities of ar-turmerone revealed IC50 values of 46.8 ± 2.4 μM and 820.4 ± 1.5 μM respectively. The selectivity index of ar-turmerone was 17.5. Ar-turmerone suppressed the ring-trophozoite transition stage of the intraerythrocytic life cycle of P. falciparum 3D7.Aromatic (ar)-turmerone is one of the aromatic constituents abundant in turmeric essential oil from  Specifications Table•in vitro antimalarial and cytotoxic properties of Ar-turmerone which is useful for further animal (in vivo) and clinical studies.The research data is important to understand the •Curcuma longa as an adjunctive therapy remedy for malaria treatment.The data obtained from this work will add to the knowledge about the medicinal value of •Parasitologists, pharmacologists, pharmacists, and nutritionists can give thought to the data obtained as it may be a new antimalarial drug template and as natural supplements for immunity against malarial infection.•The research data is useful as a reference for further drug development of experiments for malarial drug discovery involving drug pharmacology, toxicity and pharmacokinetic fields.1in vitro antiplasmodial and cytotoxic activities of ar-turmerone were reported in this dataset. The antiplasmodial effect of ar-turmerone against P. falciparum 3D7 (chloroquine-sensitive) using plasmodium lactate dehydrogenase (pLDH) assay showed an IC50 of 46.8 ± 2.4 µM as demonstrated in 50 = 820.4 ± 1.5 µM) was also shown in 50=0.0058±0.0012 µM; MTT IC50= 1020.9±10.7 µM) was used as an antimalarial reference drug. The selectivity index (SI) of ar-turmerone showed a value of 17.5. This data suggested that ar-turmerone is a promising antiplasmodial compound and non-toxic. Chloroquine diphosphate exhibited high SI value (SI>2000). High SI value shown by the antimalarial drug, chloroquine diphosphate indicated that the drug is potent and non-toxic. The stage-inhibitory effect of ar-turmerone revealed 85% suppression at the ring stage, 65% suppression at the trophozoite stage, and 50% suppression at the schizont stage .The data on nt stage . The raw22.1P. falciparum was collected from the Malaria Research and Reference Reagent Resource Centre (MR4), Virginia, USA. The parasite was revived from cryopreservation and cultured in growth medium at 2% haematocrit in purified O+ human erythrocytes at 37 °C, 5% CO2. Human erythrocytes were provided by the University Health Centre. This study protocol received ethical approval from the Research Ethics Committee of Universiti Kebangsaan Malaysia (JEP-2020-334). In this study, all chemicals and reagents were purchased from Sigma-Aldrich, St. Louis, MO, USA unless stated otherwise. The growth medium contained RPMI 1640  supplemented with 0.5% Albumax I , 25 mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES) , 100 μM hypoxanthine, 12.5 μg/mL gentamicin and 1.77 mM sodium bicarbonate P. falciparum 3D7 to ar-turmerone  (CAS Number:532-65-0) was evaluated using parasite lactate dehydrogenase (pLDH) assay. PLDH assay was conducted in flat-bottomed 96-well microtiter plates 50) of the tested compound. Experiments were performed in triplicate and repeated three times.Chloroquine-sensitive strain (3D7) of 2.24 cells/mL in DMEM complete medium  2). MTT reagent was used as a colorimetric reagent in this assay and then the reagent was added to each well before 3 h incubation. The medium in the mixture was then removed and MTT formazan product was then dissolved in dimethyl sulphoxide (DMSO). Absorbance was measured at 540 nm (Fluorostar OPTIMA). The concentration of test compounds at 50% growth inhibition (IC50) was determined from a sigmoidal curve generated from a statistical analysis software, GraphPad Prism 5. Selectivity indexes were measured as the ratio of cytotoxic over antiplasmodial activity, each one expressed with IC50 (ratio IC50 MTT/IC50 pLDH assay) Vero (ATCC: CCL-81), normal epithelial monkey kidney cells were obtained from the American Type Culture Collection (ATCC), USA. Cytotoxicity of ar-turmerone was evaluated using the 3--2, 5-diphenyltetrazolium bromide (MTT) assay in Vero cells seeded at 2 × 102.3P. falciparum 3D7 was exposed to ar-turmerone at the concentration of IC90 (463.5 µM) for 5 h at each stage; ring stage (at 0 to 5 h), trophozoite stage (at 28 to 33 h), and schizont stage (at 42 to 47 h). Chloroquine diphosphate was used as a reference drug at the IC90 value (0.052 µM). The IC90 value used in the study was to evaluate the maximum parasite inhibitory effect of ar-turmerone and to compare the compound potency with the reference antimalarial drug, chloroquine diphosphate. After incubation, parasites were washed with incomplete medium (medium without Albumax I) to remove the test compound and were then further cultured using normal culture conditions (growth medium supplemented with Albumax I). Untreated synchronous cultures served as a control and were processed in the same way as the treated cultures. After the next ring cycle, blood smears were stained with Field A (methylene blue) and B (eosin) staining. Parasitaemia level in treated cultures was measured by counting the number of new rings formed in a total of 2 000 cells during the second intraerythrocytic cycle. The parasitaemia counting was carried out to observe the effect of the treatments given at the different stages . Chemo-suppression percentage was calculated from the parasitaemia level of treated cultures as compared to a control. The stage counting was done blinded to treatment. Morphological changes were observed at each stage by microscopic examination of thin and thick blood smear of the treated and untreated cultures at 1000 × magnification using immersion oil.The stage-specific developmental inhibition of ar-turmerone was studied according to an established protocol Amatul Hamizah Ali: Methodology, Writing–Original draft preparation. Hani Kartini Agustar: Writing, Reviewing, Editing. Nurul Izzaty Hassan: Revising. Jalifah Latip: Revising. Noor Embi: Supervision. Hasidah Mohd Sidek: Supervision.The authors declare that they have no known competing for financial interests or personal relationships exist.


NRP1 mRNA expression and its correlation with patient survival and immune cell infiltration using various databases. NRP1 mRNA expression was significantly higher in STAD than normal tissues, and Kaplan-Meier survival analysis showed that NRP1 expression was significantly associated with poor prognosis in patients with STAD. To elucidate the related mechanism, we analyzed the correlation between NRP1 expression and immune cell infiltration level. In particular, the infiltration of immune-suppressive cells, such as regulatory T (Treg) cells and M2 macrophage, was significantly increased by NRP1 expression. In addition, the expression of interleukin (IL)-35, IL-10, and TGF-β1 was also positively correlated with NRP1 expression, resulting in the immune suppression. Collectively in this study, our integrated analysis using various clinical databases shows that the significant correlation between NRP1 expression and the infiltration of Treg cells and M2 macrophage explains poor prognosis mechanism in STAD, suggesting the clinical relevance of NRP1 expression as a prognostic biomarker for STAD patients.Neuropilin1 (NRP1) plays a critical role in tumor progression and immune responses. Although the roles of NRP1 in various tumors have been investigated, the clinical relevance of NRP1 expression in stomach adenocarcinoma (STAD) has not been studied. To investigate the use of NRP1 as a prognostic biomarker of STAD, we analyzed  Stomach adenocarcinoma (STAD) is one of the most common malignancies and is third in cancer-related deaths worldwide . In addiNeuropilin1 (NRP1) is a cell surface glycoprotein involved in various cellular processes, including angiogenesis, cell migration, and axon growth . EspeciaIn the tumor microenvironment (TME), tumor cells interact with various cells, including immune cells, fibroblasts, and stromal cells . The intNRP1 in tumor infiltrating macrophages exerts an anti-cancer function through suppression of an immune suppression mechanism, and is associated with a better prognosis [NRP1 mRNA expression and its correlation with prognosis of cancer patients using various databases. As shown in the results, NRP1 mRNA expression was significantly higher in STAD, compared with normal tissues. The higher expression of NRP1 was associated with poor patient survival in STAD. Furthermore, NRP1 expression showed positive correlation with tumor infiltration of Treg cells and M2 macrophages. Collectively, our study suggests that NRP1 expression could act as an effective prognostic marker by predicting the infiltration of Treg cells and M2 macrophages, indicating the role of NRP1 as a prognosis biomarker in patients with STAD.Interestingly, it has been reported that NRP1 expression increases infiltration of Treg cells in the TME. In vivo study also shows that gene deletion of rognosis ,26. TherNRP1 expression in various cancers and normal tissues was analyzed using the Oncomine, Gene Expression Profiling Analysis (GEPIA2) and Tumor Immune Estimation Resource (TIMER) databases. In the Oncomine database, a tumor microarray database, was used to compare the transcription levels of NRP1 between tumor and corresponding normal tissues in different types of cancer [−4, fold-change > 2, and gene ranking top 5%. GEPIA2 can assess the effect of 9736 tumors and 8587 normal samples from The Cancer Genome Atlas (TCGA) and the GTEx projects [NRP1 across 33 TCGA tumors was compared to normal TCGA and GTEx data using GEPIA2. TIMER database supplies an analysis of relative expression of the gene across tumor and normal tissues [NRP1 expression was analyzed in cancers to compare with normal tissues.f cancer ,28. The projects ,30. Expr tissues ,32. NRP1NRP1 and clinicopathological features was analyzed to determine the prognostic value of NRP1 in patients with stomach adenocarcinoma (STAD). mRNA levels and promoter methylation of NRP1 were separately analyzed with STAD patient characteristics, including individual cancer stage, age, histological subtype, race, gender, and tumor grade, compared to the normal tissues.UALCAN database, using TCGA transcriptome and clinical patient data, provides the expression level of genes and patient characteristics ,34. The NRP1 expression and survival in various cancers was assessed by the GEPIA2 and Kaplan-Meier survival plotter [NRP1 expression levels in STAD and lung adenocarcinoma (LUAD) of the TCGA database. NRP1 high and low patient groups were split by median NRP1 expression. We assessed cancer prognosis, including overall survival (OS), first progression (FS), and post progression survival (PPS) using gene chip datasets of Kaplan-Meier survival plotter with best cut off option, which split patient groups at the NRP1 expression level to minimize log rank P-value [P-values [The correlation between  plotter . We used P-value . These dP-values .NRP1 expression in STAD using the TIMER database. The correlation between NRP1 expression and genetic markers of tumor-infiltrating immune cells was explored through the correlation module [NRP1 was used for the x-axis with gene symbols, and related marker genes of tumor-associated macrophages (TAM), M1 macrophages, M2 macrophages, and Treg cells were represented on the y-axis as gene symbols. Gene expression was displayed with log2 RSEM. Correlation of immune signatures and NRP1 expression was also confirmed in Tumor Gastric- Tan-192-fRMA-u133p2 dataset in R2: Genomics Analysis and Visualization platform [We analyzed the correlation between immune cell and n module . The corplatform .NRP1 mRNA expression between tumors and normal tissues, we identified NRP1 mRNA levels using three independent bioinformatics databases. In the Oncomine database, NRP1 mRNA expression demonstrated upregulation of NRP1 in lymphoma, brain and central nervous system (CNS), kidney, leukemia, sarcoma, and gastric cancer tissues compared to normal tissues  , KICH (kidney chromophobe), LUSC (lung squamous cell carcinoma), OV (ovarian serous cystadenocarcinoma), UCEC , and UCS (uterine carcinosarcoma). However, it was significantly increased in STAD (stomach adenocarcinoma), DLBC (lymphoid neoplasm diffuse large B-cell lymphoma), ESCA , GBM (glioblastoma multiforme), HNSC (head and neck squamous cell carcinoma), KIRC , PAAD (pancreatic adenocarcinoma), and THYM (thymoma). In addition, we further analyzed the NRP1 expression among 36 types of cancer and normal tissues from TCGA data. NRP1 transcript levels in CHOL (cholangio carcinoma), ESCA , HNSC (head and neck squamous cell carcinoma), KIRC, and LIHC (liver hepatocellular carcinoma), including STAD. However, downregulation of NRP1 transcription level was confirmed in BRCA (breast invasive carcinoma), COAD (colon adenocarcinoma), KICH, LUSC, READ (rectum adenocarcinoma), and UCEC. Collectively, data from all three databases shows that NRP1 mRNA expression in STAD was significantly higher compared to in normal tissues.To analyze ange: 2) a. As shoNRP1 mRNA expression and the clinicopathological characteristics of STAD, we analyzed TCGA data using the UALCAN database. Compared to normal tissues, NRP1 mRNA expression was significantly increased in most clinicopathological characteristics of STAD; tumor stage , race (Caucasian and Asian), gender , age , and tumor grade (Grade 2) as shown in NRP1 gene has a positive correlation with poor prognosis in gastric cancer [NRP1 mRNA expression and downregulation of promoter methylation in STAD.To investigate the association between c cancer . Here, FNRP1 expression in STAD, we analyzed the correlation between NRP1 expression and patient survival in STAD using the Kaplan-Meier survival curve. As shown in NRP1 expression was significantly correlated with poor prognosis in STAD. To further confirm the correlation between NRP1 expression and patient survival, the Kaplan-Meier plotter database was used. Survival curves of overall survival (OS), first progression (FP), and post progression survival (PPS) showed that patient survival rates were considerably decreased by NRP1 expression  with no difference in expression of NRP1 between tumor tissues and normal tissues as shown in NRP1 expression was increased in patients with STAD and had a negative correlation with patient survival in STAD, suggesting that poor prognosis could be predicted through the levels of NRP1 expression.To elucidate the prognostic value of pression c. Intere+ T cells, and γδ T cells [NRP1 expression, association of NRP1 expression and infiltration of immune cells was analyzed in STAD. As shown in NRP1 expression and various gene expressions of immune cell markers was determined using TIMER web tool. Most of the immune cell marker genes showed a positive correlation with NRP1 expression, but among them the marker gene expression of Treg cells and M2 macrophages showed the most strong positive correlations with NRP1 expression in STAD. Strong positve correlation was confirmed in different dataset using R2 platform as shown in Tumor microenvironment (TME) consists of immune cells, stromal cells, and soluble factors, as well as cancer cells. It is known that TME plays an important role in the progression of tumors by interacting with other cells ,42. Espe T cells ,43,44. T+ T cells, and B cells, NRP1 expression also had a weak or moderate positive correlation with their marker gene expression. Additionally, despite the positive correlation between NRP1 expression and the infiltration of effector cells, higher NRP1 expression was associated with lower patient survival in STAD, indicating that the infilrated Treg cells and M2 macrophages might effectively inhibit the anti-cancer functions of effector cells. The anti-cancer effects of M1 macrophage and the pro-cancer effects of M2 macrophages and tumor-associated macrophages (TAM) [NRP1 expression and the infiltration level of TAM and M2 macrophages  was much greater than the correlation between NRP1 expression and the infiltration of M1 macrophages . In addition, high correlation between NRP1 expression and both Treg cells and M2 macrophages was confirmed using the GEPIA database  ,46 are adatabase . These r+ T cells [NRP1 expression and gene expression of Treg cell, M2 macrophage, and TAM markers, indicating the increased infiltration of the immune-suppressive cells  using the TIMER database . However, NRP1 expression was less correlated with the gene expression of immune-suppressive cytokine in LUAD compared to STAD . Collectively, our data indicate that NRP1 expression had a positive correlation with immune-suppressive functions by up-regulating inhibitory cytokines, resulting in the poor prognosis in STAD.Immunosuppression is a well-known mechanism for tumor progression, leading to tumor growth and metastasis. Many studies have reported that Treg cells, M2 macrophages, and TAM contribute to suppress effector cells by various mechanisms, and one of these mechanisms is a secretion of inhibitory cytokines, such as transforming growth factor (TGF)-β1, interleukin (IL)-10, and IL-35 ,47. Thes T cells ,49. In tve cells . Therefodatabase . Colony-NRP1 correlates with poor prognosis of stomach adenocarcinoma (STAD). In addition, the expression of NRP1 was highly correlated to the invasion of various immune cells. Particularly there was a clear difference in correlation of NRP1 expression with infiltrated Treg and M2 macrophages between STAD and lung adenocarcinoma (LUAD). Therefore, our study suggests differential TME between STAD and LUAD that affects prognostic value of NRP1 expression. Increased NRP1 mRNA and protein expression in stomach cancer tissue was already suggested in a previous report [NRP1 mRNA in stomach cancer at multiple datasets using the various databases   acts as a co-receptor of various growth factors and plays a major role in tumor progression. Overexpression of NRP1 is associated with poor prognosis in various cancers including prostate cancer, lung cancer, and melanoma. However, it has been reported to be associated with favorable prognosis in colon cancer ,52,53. Ts report . We conf UALCAN) . In addi dataset .NRP1 exhibited a significant decrease across almost all clinical characteristics. DNA methylation mainly regulates the level of gene expression. Especially, abnormal methylation is frequently observed in tumors by affecting the cell division cycle, therefore it is associated with tumor malignant biological properties [NRP1 methylation, among the tumor-related genes, is found to be associated with survival of colon and liver cancers [NRP1 is a hypomethylated and upregulated gene in tumor tissues and is co-expressed with platelet-derived growth factor receptor beta (PDGFRB), which is associated with the malignant phenotype for patients with gastric cancer, thereby potentially serving as a prognostic biomarker of gastric cancer [NRP1 and lower DNA methylation of NRP1 gene in STAD than in normal control. Taken together, it suggests that the hypomethylation of NRP1 could be associated with poor prognosis of STAD.Interestingly, the promoter methylation of operties . The cytoperties . In part cancers ,56. In ac cancer . Here, oNRP1 as a marker to predict the prognosis of STAD, we analyzed the correlation between NRP1 expression and patient survival using the Kaplan-Meire curve. NRP1 expression, as based on the Kaplan-Meier survival curves, was significantly correlated with overall survival (OS), first progression (FP), and post progression survival (PPS) in STAD, showing that NRP1 expression is negatively correlated with patient survival. , which present properties of the M2 macrophages in the tumor microenvironment (TEM), are known to induce immune suppression and stimulate tumor progression [+ and CD8+ T cells [NRP1 expression induces the production of the inhibitory cytokines IL-10, IL-35, and TGFβ1 of the Treg cells and M2 macrophages, and the expressed cytokines serve as major signals in the immune suppression mechanism of STAD.This study also demonstrates that rophages . Despitegression ,66. The gression ,68. Treggression ,70,71. A T cells . In addi T cells . NRP1 en T cells ,74. TherNRP1 expression correlated with cytokine gene markers  that were secreted from Treg cells and M2 macrophages , interleukin-10 (IL-10), and colony stimulating factor 1 (CSF1) . CSF1, wrophages . TherefoNRP1 expression is positively correlated in Treg cells and M2 macrophages in STAD. Further, we found a significant correlation between NRP1 expression and cytokines in Treg cells and M2 macrophages. High correlation of NRP1 expression with immunosuppressive cells may be one of the causes of the prognostic value of NRP1 in STAD. Thus, this systematic analysis provides evidence suggesting the potential use of NRP1 as an effective biomarker for patient survival in STAD and a therapeutic target modulating the tumor immune microenvironment.The main finding of this study is that