RESUMO
This study aims to investigate the mechanism of the anti-atherosclerosis effect of Huayu Qutan Recipe (HYQT) on the inhibition of foam cell formation. In vivo, the mice were randomly divided into three groups: CTRL group, MOD group and HYQT group. The HYQT group received HYQT oral administration twice a day (20.54 g/kg/d), and the plaque formation in ApoE-/- mice was observed using haematoxylin-eosin (HE) staining and oil red O (ORO) staining. The co-localization of aortic macrophages and lipid droplets (LDs) was examined using fluorescent labelling of CD11b and BODIPY fluorescence probe. In vitro, RAW 264.7 cells were exposed to 50 µg/mL ox-LDL for 48 h and then treated with HYQT for 24 h. The accumulation of LDs was evaluated using ORO and BODIPY. Cell viability was assessed using the CCK-8 assay. The co-localization of LC3b and BODIPY was detected via immunofluorescence and fluorescence probe. LysoTracker Red and BODIPY 493/503 were used as markers for lysosomes and LDs, respectively. Autophagosome formation were observed via transmission electron microscopy. The levels of LC3A/B II/LC3A/B I, p-mTOR/mTOR, p-4EBP1/4EBP1, p-P70S6K/P70S6K and TFEB protein level were examined via western blotting, while SQSTM1/p62, Beclin1, ABCA1, ABCG1 and SCARB1 were examined via qRT-PCR and western blotting. The nuclear translocation of TFEB was detected using immunofluorescence. The components of HYQT medicated serum were determined using Q-Orbitrap high-resolution MS analysis. Molecular docking was employed to identify the components of HYQT medicated serum responsible for the mTOR signalling pathway. The mechanism of taurine was illustrated. HYQT has a remarkable effect on atherosclerotic plaque formation and blood lipid level in ApoE-/- mice. HYQT decreased the co-localization of CD11b and BODIPY. HYQT (10% medicated serum) reduced the LDs accumulation in RAW 264.7 cells. HYQT and RAPA (rapamycin, a mTOR inhibitor) could promote cholesterol efflux, while chloroquine (CQ, an autophagy inhibitor) weakened the effect of HYQT. Moreover, MHY1485 (a mTOR agonist) also mitigated the effects of HYQT by reduced cholesterol efflux. qRT-PCR and WB results suggested that HYQT improved the expression of the proteins ABCA1, ABCG1 and SCARB1.HYQT regulates ABCA1 and SCARB1 protein depending on the mTORC1/TFEB signalling pathway. However, the activation of ABCG1 does not depend on this pathway. Q-Orbitrap high-resolution MS analysis results demonstrated that seven core compounds have good binding ability to the mTOR protein. Taurine may play an important role in the mechanism regulation. HYQT may reduce cardiovascular risk by promoting cholesterol efflux and degrading macrophage-derived foam cell formation. It has been observed that HYQT and ox-LDL regulate lipophagy through the mTOR/TFEB signalling pathway, rather than the mTOR/4EBP1/P70S6K pathway. Additionally, HYQT is found to regulate cholesterol efflux through the mTORC1/TFEB/ABCA1-SCARB1 signal axis, while taurine plays a significant role in lipophagy.
Assuntos
Aterosclerose , Compostos de Boro , Proteínas Quinases S6 Ribossômicas 70-kDa , Animais , Camundongos , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Colesterol/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Simulação de Acoplamento Molecular , Células Espumosas/metabolismo , Aterosclerose/tratamento farmacológico , Aterosclerose/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Autofagia , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Taurina/metabolismoRESUMO
Obesity, which has unknown pathogenesis, can increase the frequency and seriousness of acute myocardial infarction (AMI). This study evaluated effect of Huayu Qutan Recipe (HQR) pretreatment on myocardial apoptosis induced by AMI by regulating mitochondrial function via PI3K/Akt/Bad pathway in rats with obesity. For in vivo experiments, 60 male rats were randomly divided into 6 groups: sham group, AMI group, AMI (obese) group, 4.5, 9.0 and 18.0 g/kg/d HQR groups. The models fed on HQR with different concentrations for 2 weeks before AMI. For in vitro experiments, the cardiomyocytes line (H9c2) was used. Cells were pretreated with palmitic acid (PA) for 24 h, then to build hypoxia model followed by HQR-containing serum for 24 h. Related indicators were also detected. In vivo, HQR can lessen pathohistological damage and apoptosis after AMI. In addition, HQR improves blood fat levels, cardiac function, inflammatory factor, the balance of oxidation and antioxidation, as well as lessen infarction in rats with obesity after AMI. Meanwhile, HQR can diminish myocardial cell death by improving mitochondrial function via PI3K/Akt/Bad pathway activation. In vitro, HQR inhibited H9c2 cells apoptosis, improved mitochondrial function and activated the PI3K/Akt/Bad pathway, but effects can be peripeteiad by LY294002. Myocardial mitochondrial dysfunction occurs following AMI and can lead to myocardial apoptosis, which can be aggravated by obesity. HQR can relieve myocardial apoptosis by improving mitochondrial function via the PI3K/Akt/Bad pathway in rats with obesity.
Assuntos
Infarto do Miocárdio , Fosfatidilinositol 3-Quinases , Animais , Apoptose , Masculino , Mitocôndrias/metabolismo , Infarto do Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Obesidade/complicações , Obesidade/tratamento farmacológico , Obesidade/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Adult neurogenesis plays a vital role in maintaining cognitive functions in mammals and human beings. Mobilization of hippocampal neurogenesis has been regarded as a promising therapeutic approach to restore injured neurons in neurodegenerative diseases including Alzheimer's disease (AD). Icarisid II (ICS II), an active ingredient derived from Epimedii Folium, has been reported to exhibit multiple neuroprotective effects. In the present study, we investigated the effects of ICS II on the proliferation and differentiation of neural stem cells (NSCs) and amyloid precusor protein (APP)-overexpressing NSCs (APP-NSCs) in vitro. Our results demonstrated that ICS II dose-dependently suppressed apoptosis and elevated viability of APP-NSCs. ICS II (1 µM) potently promoted proliferation and neuronal differentiation of NSCs and APP-NSCs. ICS II (1 µM) significantly upregulated Wnt-3a expression, increased the phosphorylation of glycogen synthase kinase-3ß and enhanced the nuclear transfer of ß-catenin. Moreover, ICS II also promoted astrocytes to secrete Wnt-3a, which positively modulates Wnt/ß-catenin signaling pathway. These findings demonstrate that ICS II promotes NSCs proliferation and neuronal differentiation partly by activating the Wnt/ß-catenin signaling pathway.
RESUMO
BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common digestive system cancers with high mortality rates worldwide. The main ingredients in Mu Ji Fang Granules (MJF) are alkaloids, flavonoids, and polysaccharides. MJF has been used in the clinical treatment of hepatitis, cirrhosis and HCC for more than 30 years. Few previous studies have focused on the mechanism of MJF on tumor immu-nology in the treatment of HCC. AIM: To explore the mechanism of action of MJF on tumor immunology in the treatment of HCC. METHODS: The absorbable ingredients of MJF were identified using Molecule Network related to High Performance Liquid Chromatography-Electron Spray Ionization-Time of Flight- Mass Spectrometry, and hub potential anti-HCC targets were screened using network pharmacology and pathway enrichment analysis. Forty male mice were randomly divided into the Blank, Model, and MJF groups (1.8, 5.4, and 10.8 g/kg/d) following 7 d of oral administration. Average body weight gain, spleen and thymus indices were calculated, tumor tissues were stained with hematoxylin and eosin, and Interferon gamma (IFN-γ), Tumor necrosis factor α (TNF-α), Interleukin-2, aspartate aminotransferase, alanine aminotransferase, alpha-fetoprotein (AFP), Fas, and FasL were measured by Enzyme-linked Immunosorbent Assay. Relevant mRNA expression of Bax and Bcl2 was evaluated by Real Time Quantitative PCR (RT-qPCR) and protein expression of Transforming growth factor ß1 (TGF-ß1) and Mothers against decapentaplegic homolog (SMAD) 4 was assessed by Western blotting. The HepG2 cell line was treated with 10 mg/mL, 20 mg/mL, 30 mg/mL, 40 mg/mL of MJF, and another 3 groups were treated with TGF-ß1 inhibitor (LY364947) and different doses of MJF. Relevant mRNA expression of TNF-α, IFN-γ, Bax and Bcl2 was evaluated by RT-qPCR and protein expression of TGF-ß1, SMAD2, p-SMAD2, SMAD4, and SMAD7 was assessed by Western blotting. RESULTS: It was shown that MJF improved body weight gain and tumor inhibition rate in H22 tumor-bearing mice, protected immune organs and liver function, reduced the HCC indicator AFP, affected immunity and apoptosis, and up-regulated the TGF-ß1/SMAD signaling pathway, by increasing the relative expression of TGF-ß1, SMAD2, p-SMAD2 and SMAD4 and decreasing SMAD7, reducing immune factors TNF-α and IFN-γ, decreasing apoptosis cytokines Fas, FasL and Bcl2/Bax, and inhibiting the effect of LY364947 in HepG2 cells. CONCLUSION: MJF inhibits HCC by activating the TGF-ß1/SMAD signaling pathway, and affecting immune and apoptotic cytokines, which may be due to MJF adjusting immune escape and apoptosis.
RESUMO
Tobacco mosaic virus (TMV) causes significant losses in many economically important crops. Contaminated soils may play roles as reservoirs and sources of transmission for TMV. In this study we report the development of an immunocapture real-time RT-PCR (IC-real-time RT-PCR) assay for direct detection of TMV in soils without RNA isolation. A series of TMV infected leaf sap dilutions of 1:101, 1:102, 1:103, 1:104, 1:105 and 1:106 (w/v, g/mL) were added to one gram of soil. The reactivity of DAS-ELISA and conventional RT-PCR was in the range of 1:102 and 1:103 dilution in TMV-infested soils, respectively. Meanwhile, the detection limit of IC-real-time RT-PCR sensitivity was up to 1:106 dilution. However, in plant sap infected by TMV, both IC-real-time RT-PCR and real-time RT-PCR were up to 1:106 dilution, DAS-ELISA could detect at least 1:103 dilution. IC-real-time RT-PCR method can use either plant sample extracts or cultivated soils, and show higher sensitivity than RT-PCR and DAS-ELISA for detection of TMV in soils. Therefore, the proposed IC-real-time RT-PCR assay provides an alternative for quick and very sensitive detection of TMV in soils, with the advantage of not requiring a concentration or RNA purification steps while still allowing detection of TMV for disease control.
Assuntos
Reação em Cadeia da Polimerase em Tempo Real/métodos , Microbiologia do Solo , Vírus do Mosaico do Tabaco/isolamento & purificação , Ensaio de Imunoadsorção Enzimática , Folhas de Planta/virologia , RNA Viral , Nicotiana/virologiaRESUMO
Polycystic ovary syndrome (PCOS) is a universal endocrine and metabolic disorder prevalent in reproductive aged women. PCOS is often accompanied with insulin resistance (IR) which is an essential pathological factor. Although there is no known cure for PCOS, cangfudaotan (CFDT) decoction is widely used for the treatment of PCOS; nevertheless, the underlying mechanism is not clear. In this study, 40 Sprague-Dawley (SD) rats (female) were randomized to 4 groups, namely the control group, PCOS group, PCOS+CFDT group, and PCOS+metformin group. The rats in the control group were fed a normal-fat diet, intraperitoneally injected with 0.5% carboxymethyl cellulose (CMC, 1 mL/kg/d) for 21 days and orally given saline (1 mL/kg/d) for the next 4 weeks. The rats in the PCOS group, PCOS+CFDT group, and PCOS+Metformin group were fed a high-fat diet (HFD) and intraperitoneally injected with letrozole (1.0 mg/kg) for 21 days. During this period, we recorded the body weight, estrous cycles, and rate of pregnancy in all rats. We also observed the ovarian ultrastructure. Blood glucose indices, serum hormones, and inflammatory factors were also recorded. Then, we detected apoptotic and mitochondrial function, and observed mitochondria in ovarian granular cells by transmission electron microscopy. We also detected genes of ASK1/JNK pathway at mRNA and protein levels. The results showed that CFDT alleviated pathohistological damnification and apoptosis in PCOS rat model. In addition, CFDT improved ovarian function, reduced inflammatory response, inhibited apoptosis of granular cells, and inhibited the operation of ASK1/JNK pathway. These findings demonstrate the occurrence of ovary mitochondrial dysfunction and granular cell apoptosis in PCOS. CFDT can relieve mitochondria-dependent apoptosis by inhibiting the ASK1/JNK pathway in PCOS rats.
Assuntos
Metformina , Síndrome do Ovário Policístico , Feminino , Gravidez , Humanos , Ratos , Animais , Síndrome do Ovário Policístico/tratamento farmacológico , Ratos Sprague-Dawley , Células da Granulosa , Mitocôndrias , Apoptose , Metformina/farmacologiaRESUMO
As a widely applied traditional Chinese medicine (TCM), Jian-Pi-Yi-Shen (JPYS) decoction maybe applied in curing premature ovarian failure (POF) besides chronic kidney disease (CKD). In vivo experiments, 40 female SD (8-week-old) rats were randomized into four groups, namely, control group (negative control), POF model group, JPYS treatment group, and triptorelin treatment group (positive control). JPYS group was treated with JPYS decoction (oral, 11 g/kg) for 60 days, and the triptorelin group was treated with triptorelin (injection, 1.5 mg/kg) for 10 days before the administration of cyclophosphamide (CTX) (50 mg/kg body weight) to establish POF model. We examined apoptosis, mitochondrial function, and target gene (ASK1/JNK pathway and mitochondrial fusion/fission) expression. In vitro experiments, the KGN human granulosa cell line was used. Cells were pretreated with CTX (20, 40, and 60 µg/mL) for 24 h, followed by JPYS-containing serum (2, 4, and 8 %) for 24 h. Thereafter, these cells were employed to assess apoptosis, mitochondrial function, and target gene levels of protein and mRNA. In vivo, JPYS alleviated injury and suppressed apoptosis in POF rats. In addition, JPYS improved ovarian function. JPYS inhibit apoptosis of granulosa cells through improving mitochondrial function by activating ASK1/JNK pathway. In vitro, JPYS inhibited KGN cell apoptosis through inhibited ASK1/JNK pathway and improved mitochondrial function. The effects of GS-49977 were similar to those of JPYS. During POF, mitochondrial dysfunction occurs in the ovary and leads to granulosa cell apoptosis. JPYS decoction improves mitochondrial function and alleviates apoptosis through ASK1/JNK pathway.
Assuntos
Medicamentos de Ervas Chinesas , Insuficiência Ovariana Primária , Ratos , Feminino , Humanos , Animais , Insuficiência Ovariana Primária/metabolismo , Pamoato de Triptorrelina/metabolismo , Pamoato de Triptorrelina/farmacologia , Medicamentos de Ervas Chinesas/farmacologia , Medicamentos de Ervas Chinesas/uso terapêutico , Apoptose , Células da Granulosa/metabolismo , Mitocôndrias/metabolismoRESUMO
OBJECTIVE: Obesity enhances the frequency and severity of acute kidney injury (AKI) induced by renal ischemia-reperfusion (IR). Tanshinone IIA (TIIA) pre-treatment was used to alleviate renal injury induced by renal IR, and whether TIIA can attenuate renal cell apoptosis via modulating mitochondrial function through PI3K/Akt/Bad pathway in obese rats was examined. METHODS: Male rates were fed a high-fat diet for 8 weeks to generate obesity, followed by 30 min of kidney ischemia and 24 h reperfusion induced AKI. The male obese rates were given TIIA (5 mg/kg.d, 10 mg/kg.d, and 20 mg/kg.d) for 2 weeks before renal IR. RESULTS: TIIA alleviated the pathohistological injury and apoptosis induced by IR. In addition, TIIA improved renal function, inflammatory factor, and balance of oxidation and antioxidation in obese rats after renal IR. At the same time, TIIA can inhibit cell apoptosis by improving mitochondrial function through the PI3K/Akt/Bad pathway. Mitochondrial dysfunction was supported by decreasing intracellular ATP, respiration controlling rate (RCR), mitochondrial membrane potential (MMP), and mitochondrial respiratory chain complex enzymes, and by increasing ROS, the opening of mitochondrial permeability transition pore (mPTP), and the mtDNA damage. The injury to mitochondrial dynamic function was assessed by decreasing Drp1, and increasing Mfn1/2; and the injury of mitochondrial biogenesis was assessed by decreasing PGC-1, Nrf1, and TFam. CONCLUSIONS: Renal mitochondrial dysfunction occurs along with renal IR and can induce renal cell apoptosis. Obesity can aggravate apoptosis. TIIA can attenuate renal cell apoptosis via modulating mitochondrial function through PI3K/Akt/Bad pathway in obese rats.
Assuntos
Injúria Renal Aguda , Traumatismo por Reperfusão Miocárdica , Ratos , Masculino , Animais , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Rim/patologia , Injúria Renal Aguda/tratamento farmacológico , Injúria Renal Aguda/etiologia , Injúria Renal Aguda/prevenção & controle , Apoptose , Traumatismo por Reperfusão Miocárdica/metabolismo , Reperfusão , Isquemia/patologia , Obesidade/complicações , Obesidade/patologiaRESUMO
To assess the variations in pulmonary function and vascular endothelial function in their early stages (without related complications). A total of 162 type 2 diabetes mellitus (T2DM) patients without diabetes complications and 55 healthy people were selected, comprising the T2DM group and the control group, respectively, to evaluate changes in vascular endothelial function and lung function and determine the correlation between them. In this study, the T2DM group exhibited significantly lower pulmonary function than that of the control group (P < 0.05). The T2DM group also showed significantly lower flow-mediated dilation (FMD) and nitric oxide (NO) (P < 0.05) than those of the control group. Pulmonary functional indexes correlated positively with FMD and NO (P < 0.05) and correlated negatively with endothelin-1 (ET-1) (P < 0.05). FMD and NO correlated negatively with diabetes duration/HbA1c (P < 0.05), whereas ET-1 correlated positively with glycosylated hemoglobinA1c (HbA1c)/diabetes duration (P < 0.05). Pulmonary functional indexes negatively correlated with HbA1c/diabetes duration (P < 0.05). Multiple linear regression was used to analyze the relationship between vascular endothelial function indexes (FMD, ET-1, and NO) and pulmonary functional indexes. The results indicated that each vascular endothelial function index (FMD, ET-1, and NO) was significantly correlated with the pulmonary functional index (P < 0.05). The patients with T2DM presented changes in the subclinical vascular endothelial and pulmonary function. They also had impaired vascular endothelial functions, which were characterized by reduced vascular endothelial function relative to those of healthy people. Regulating glycemia may improve vascular endothelial and pulmonary functions. Moreover, microvascular lesions in preclinical stages, vascular endothelial function indexes (FMD, ET-1, and NO) were valid predictors of alterations in pulmonary function in T2DM patients without related complications. Clinical Trial Registration: ClinicalTrials.gov, identifier NCT03575988.
Assuntos
Glicemia , Diabetes Mellitus Tipo 2/fisiopatologia , Endotélio Vascular/fisiopatologia , Pulmão/fisiopatologia , Adulto , Diabetes Mellitus Tipo 2/sangue , Endotelina-1/sangue , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Óxido Nítrico/sangueRESUMO
Acute lung injury (ALI), which is induced by renal ischemia-reperfusion (IR), is one of the leading causes of acute renal IR-related death. Obesity raises the frequency and severity of acute kidney injury (AKI) and ALI. Tanshinone IIA (TIIA) combined with cyclosporine A (CsA) was employed to lessen the lung apoptosis led by renal IR and to evaluate whether TIIA combined with CsA could alleviate lung apoptosis by regulating mitochondrial function through the PI3K/Akt/Bad pathway in obese rats. Hematoxylin-eosin (HE) staining was used to assess the histology of the lung injury. Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) was used to assess apoptosis of the lung. Electron microscopy was used to assess mitochondrial morphology in lung cells. Arterial blood gas and pulmonary function were used to assess the external respiratory function. Mitochondrial function was used to assess the internal respiratory function and mitochondrial dynamics and biogenesis. Western blot (WB) was used to examine the PI3K/Akt/Bad pathway-related proteins. TIIA combined with CsA can alleviate lung apoptosis by regulating mitochondrial function through the PI3K/Akt/Bad pathway in obese rats.
RESUMO
BACKGROUND: Acute myocardial injury (AMI), which is induced by renal ischemia-reperfusion (IR), is a significant cause of acute kidney injury (AKI)-related associated death. Obesity increases the severity and frequency of AMI and AKI. Tanshinone IIA (TIIA) combined with cyclosporine A (CsA) pretreatment was used to alleviate myocardial cell apoptosis induced by renal IR, and to determine whether TIIA combined with CsA would attenuate myocardial cell apoptosis by modulating mitochondrial function through the PI3K/Akt/Bad pathway in obese rats. METHODS: Male rates were fed a high fat diet for 8 weeks to generate obesity. AKI was induced by 30 min of kidney ischemia followed 24 h of reperfusion. Obese rats were given TIIA (10 mg/kg·d) for 2 weeks and CsA (5 mg/kg) 30 min before renal IR. After 24 h of reperfusion, the rats were anaesthetized, the blood were fetched from the abdominal aorta and kidney were fetched from abdominal cavity, then related indicators were examined. RESULTS: TIIA combined with CsA can alleviate the pathohistological injury and apoptosis induced by renal IR in myocardial cells. TIIA combined with CsA improved cardiac function after renal ischemia (30 min)-reperfusion (24 h) in obese rats. At the same time, TIIA combined with CsA improved mitochondrial function. Abnormal function of mitochondria was supported by decreases in respiration controlling rate (RCR), intracellular adenosine triphosphate (ATP), oxygen consumption rate, and mitochondrial membrane potential (MMP), and increases in mitochondrial reactive oxygen species (ROS), opening of the mitochondrial permeability transition pore (mPTP), mitochondrial DNA damage, and mitochondrial respiratory chain complex enzymes. The injury of mitochondrial dynamic function was assessed by decrease in dynamin-related protein 1 (Drp1), and increases in mitofusin1/2 (Mfn1/2), and mitochondrial biogenesis injury was assessed by decreases in PPARγ coactivator-1-α (PGC-1), nucleo respiratory factor1 (Nrf1), and transcription factor A of mitochondrial (TFam). CONCLUSION: We used isolated mitochondria from rat myocardial tissues to demonstrate that myocardial mitochondrial dysfunction occurred along with renal IR to induce myocardial cell apoptosis; obesity aggravated apoptosis. TIIA combined with CsA attenuated myocardial cell apoptosis by modulating mitochondrial function through the PI3K/Akt/Bad pathway in obese rats.
Assuntos
Abietanos/farmacologia , Apoptose/efeitos dos fármacos , Ciclosporina/farmacologia , Mitocôndrias Cardíacas/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Traumatismo por Reperfusão/tratamento farmacológico , Animais , DNA Mitocondrial , Coração/efeitos dos fármacos , Rim/fisiopatologia , Masculino , Potencial da Membrana Mitocondrial , Mitocôndrias Cardíacas/patologia , Poro de Transição de Permeabilidade Mitocondrial , Obesidade , Ratos , Espécies Reativas de Oxigênio/metabolismo , Traumatismo por Reperfusão/patologia , Transdução de SinaisRESUMO
OBJECTIVE: To determine whether microRNAs (miRs) are elevated in the plasma of individuals with the inherited peripheral neuropathy Charcot-Marie-Tooth disease type 1A (CMT1A), miR profiling was employed to compare control and CMT1A plasma. METHODS: We performed a screen of CMT1A and control plasma samples to identify miRs that are elevated in CMT1A using next-generation sequencing, followed by validation of selected miRs by quantitative PCR, and correlation with protein biomarkers and clinical data: Rasch-modified CMT Examination and Neuropathy Scores, ulnar compound muscle action potentials, and motor nerve conduction velocities. RESULTS: After an initial pilot screen, a broader screen confirmed elevated levels of several muscle-associated miRNAs (miR1, -133a, -133b, and -206, known as myomiRs) along with a set of miRs that are highly expressed in Schwann cells of peripheral nerve. Comparison to other candidate biomarkers for CMT1A (e.g., neurofilament light) measured on the same sample set shows a comparable elevation of several miRs (e.g., miR133a, -206, -223) and ability to discriminate cases from controls. Neurofilament light levels were most highly correlated with miR133a. In addition, the putative Schwann cell miRs (e.g., miR223, -199a, -328, -409, -431) correlate with the recently described transmembrane protease serine 5 (TMPRSS5) protein biomarker that is most highly expressed in Schwann cells and also elevated in CMT1A plasma. CONCLUSIONS: These studies identify a set of miRs that are candidate biomarkers for clinical trials in CMT1A. Some of the miRs may reflect Schwann cell processes that underlie the pathogenesis of the disease. CLASSIFICATION OF EVIDENCE: This study provides Class III evidence that a set of plasma miRs are elevated in patients with CMT1A.
Assuntos
Doença de Charcot-Marie-Tooth/diagnóstico , Doença de Charcot-Marie-Tooth/genética , MicroRNAs/análise , MicroRNAs/genética , Potenciais de Ação , Adulto , Envelhecimento , Biomarcadores/análise , Biologia Computacional , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Pessoa de Meia-Idade , Neurônios Motores , Músculo Esquelético/fisiopatologia , Condução Nervosa , Proteínas de Neurofilamentos/química , Nervos Periféricos/metabolismo , Reprodutibilidade dos Testes , Células de Schwann/metabolismo , Nervo Ulnar/fisiopatologiaRESUMO
Receptor interacting protein kinase 1 (RIPK1) mediates cell death and inflammatory signaling and is increased in multiple sclerosis (MS) brain samples. Here, we investigate the role of glial RIPK1 kinase activity in mediating MS pathogenesis. We demonstrate RIPK1 levels correlate with MS disease progression. We find microglia are susceptible to RIPK1-mediated cell death and identify an inflammatory gene signature that may contribute to the neuroinflammatory milieu in MS patients. We uncover a distinct role for RIPK1 in astrocytes in regulating inflammatory signaling in the absence of cell death and confirm RIPK1-kinase-dependent regulation in human glia. Using a murine MS model, we show RIPK1 inhibition attenuates disease progression and suppresses deleterious signaling in astrocytes and microglia. Our results suggest RIPK1 kinase activation in microglia and astrocytes induces a detrimental neuroinflammatory program that contributes to the neurodegenerative environment in progressive MS.
Assuntos
Microglia/metabolismo , Esclerose Múltipla/genética , Doenças Neuroinflamatórias/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Animais , Modelos Animais de Doenças , Progressão da Doença , Humanos , Camundongos , Esclerose Múltipla/patologia , Transdução de SinaisRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: According to the theory of traditional Chinese medicine (TCM), Alzheimer's disease (AD) is identified as "forgetfulness" or "dementia", and is mainly caused by "kidney essence deficiency" which ultimately induces "encephala reduction". Therefore, herbal formulas possessing the efficacy of nourishing kidney essence or replenishing brain marrow are commonly served as effective strategies for AD treatment. Shenzao jiannao oral liquid (SZJN), a traditional Chinese preparation approved by the China Food and Drug Administration (CFDA), is used for the treatment of insomnia and mind fatigue at present for its efficacy of nourishing kidneys. In present study, we found that SZJN could improve cognitive function of AD-like mice. AIMS OF STUDY: This study aims to investigate the effects of SJZN on ameliorating cognitive deficits of AD-like mouse model, and to illuminate the underlying mechanisms from the perspective of neuroprotection and neurogenesis. MATERIALS AND METHODS: Kunming mice (28 ± 2 g) were randomly allocated into seven groups: control, sham, model, donepezil and SZJN groups (low, middle and high). The AD mouse model was established by Aß42 combined with scopolamine. SZJN were intragastrically administrated at doses of 0.3, 1.5 and 7.5 g/kg for 28 days. Morris water maze (MWM) test was applied to determine the cognitive function. Hematoxylin eosin (HE) and Nissl staining were carried out to evaluate pathological damages in the cortex and hippocampal tissues. To explore the protective effects of SZJN on multiple pathogenic factors of AD, protein levels of Aß42, glial fibrillary acidic protein (GFAP), Bax, Bcl-2, Caspase-3, synaptophysin (SYP), brain-derived neurotrophic factor (BDNF), and neurogenesis related proteins were assessed using Immunofluorescence (IF) and western blot analysis. In vitro, the AD cell model was established by transduction of APP695swe genes into Neural stem cells (NSCs) isolated from the hippocampal tissues of neonatal C57BL/6 mice. Cell viability assay and neurosphere formation assay were carried out to verify the efficacy of SZJN on proliferation of NSCs. RESULTS: Our results demonstrated that SZJN (1.5 g/kg and 7.5 g/kg) treatment significantly ameliorated cognitive deficits of AD-like mice. SZJN (7.5 g/kg) treatment significantly retarded the pathological damages including neuronal degeneration, neuronal apoptosis, Aß peptides aggregation and reaction of astrocytes in AD-like mice. In addition, SZJN (7.5 g/kg) increased the expression of BDNF and SYP, and restored the abnormal level of MDA and SOD in the brain of AD-like mice. Furthermore, SZJN treatment for 28 days remarkably increased the proliferation of NSCs evidenced by more Nestin+ and BrdU+ cells in the hippocampal DG regions, and increased the amount of mature neurons marked by NeuN both in the cortex and hippocampal DG regions. In vitro, SZJN treatement (16, 32, 64 mg/ml) promoted the proliferation of NSCs evidenced by the increased amount and enlarged size of the neurospheres (p < 0.05). CONCLUSIONS: Our findings indicated that SZJN could ameliorate cognitive deficits by protecting neurons from death and triggering endogenous neurogenesis. Therefore, SZJN may be considered as a promising agent to restore neuronal loss and deter the deterioration in AD patients.
Assuntos
Doença de Alzheimer/prevenção & controle , Comportamento Animal/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Cognição/efeitos dos fármacos , Medicamentos de Ervas Chinesas/farmacologia , Neurogênese/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Nootrópicos/farmacologia , Administração Oral , Doença de Alzheimer/induzido quimicamente , Doença de Alzheimer/patologia , Doença de Alzheimer/psicologia , Peptídeos beta-Amiloides , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Morte Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Medicamentos de Ervas Chinesas/administração & dosagem , Reação de Fuga/efeitos dos fármacos , Feminino , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Atividade Motora/efeitos dos fármacos , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/patologia , Neurônios/metabolismo , Neurônios/patologia , Fármacos Neuroprotetores/administração & dosagem , Nootrópicos/administração & dosagem , Fragmentos de Peptídeos , Escopolamina , Transdução de SinaisRESUMO
OBJECTIVE: Development of biomarkers for Charcot-Marie-Tooth (CMT) disease is critical for implementing effective clinical trials. The most common form of CMT, type 1A, is caused by a genomic duplication surrounding the PMP22 gene. A recent report (Neurology 2018;90:e518-3524) showed elevation of neurofilament light (NfL) in plasma of CMT1A disease patients, which correlated with disease severity. However, no plasma/serum biomarker has been identified that is specific to Schwann cells, the most directly affected cells in CMT1A. METHODS: We used the Olink immuno PCR platform to profile CMT1A patient (n = 47, 2 cohorts) and normal control plasma (n = 41, two cohorts) on five different Olink panels to screen 398 unique proteins. RESULTS: The TMPRSS5 protein (Transmembrane protease serine 5) was elevated 2.07-fold (P = <0.0001) in two independent cohorts of CMT1A samples relative to controls. TMPRSS5 is most highly expressed in Schwann cells of peripheral nerve. Consistent with early myelination deficits in CMT1A, TMPRSS5 was not significantly correlated with disease score (CMTES-R, CMTNS-R), nerve conduction velocities (Ulnar CMAP, Ulnar MNCV), or with age. TMPRSS5 was not significantly elevated in smaller sample sets from patients with CMT2A, CMT2E, CMT1B, or CMT1X. The Olink immuno PCR assays confirmed elevated levels of NfL (average 1.58-fold, P < 0.0001), which correlated with CMT1A patient disease score. INTERPRETATION: These data identify the first Schwann cell-specific protein that is elevated in plasma of CMT1A patients, and may provide a disease marker and a potentially treatment-responsive biomarker with good disease specificity for clinical trials.
Assuntos
Doença de Charcot-Marie-Tooth/sangue , Doença de Charcot-Marie-Tooth/diagnóstico , Proteínas de Membrana/sangue , Proteínas Mitocondriais/sangue , Células de Schwann , Serina Endopeptidases/sangue , Adulto , Animais , Biomarcadores/sangue , Células Cultivadas , Doença de Charcot-Marie-Tooth/fisiopatologia , Estudos de Coortes , Diagnóstico Diferencial , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Condução Nervosa/fisiologia , Reação em Cadeia da Polimerase , RatosRESUMO
PURPOSE: The main objectives of this study were to assess the early changes in pulmonary function and intrarenal haemodynamics and to determine the correlation between pulmonary function and intrarenal haemodynamics in patients with type 2 diabetes mellitus (T2DM). METHODS: 96 patients with T2DM (diabetes group) without diabetes kidney disease (DKD) and 33 healthy subjects (control group) were enrolled in studies intended to assess the early changes in pulmonary function and intrarenal haemodynamics associated with diabetes, as well as to determine the correlation between pulmonary function and intrarenal haemodynamics. RESULTS: Pulmonary functional parameters were negatively correlated with HbA1c levels and diabetes duration (P< 0.05). Moreover, renal functional parameters were positively correlated with HbA1c levels and diabetes duration (P<0.05). Additionally, pulmonary functional parameters were negatively correlated with renal functional parameters (P<0.05). Multiple linear regression analysis of the relationship between pulmonary functional parameters and the bilateral kidney arterial resistivity index (RI) showed that all the pulmonary functional parameters were significantly correlated with the arterial RI (P< 0.05). CONCLUSIONS: Patients displayed changes in pulmonary function and intrarenal haemodynamics during the preclinical stages of DKD. Regulating glycaemia may improve intrarenal haemodynamics in the bilateral interlobular renal arteries. Moreover, during the preclinical stages of DKD, the right kidney RI is a effective predictor of early changes in pulmonary function in adult T2DM patients. TRIAL REGISTRATION: ClinicalTrials.gov (NCT02798198); registered 8 June 2016.
Assuntos
Complicações do Diabetes/etiologia , Diabetes Mellitus Tipo 2/complicações , Hemodinâmica , Rim/fisiopatologia , Pulmão/fisiopatologia , Artéria Renal/fisiopatologia , Resistência Vascular , Biomarcadores/análise , Glicemia/análise , Estudos de Casos e Controles , Complicações do Diabetes/patologia , Feminino , Seguimentos , Taxa de Filtração Glomerular , Hemoglobinas Glicadas/análise , Humanos , Masculino , Pessoa de Meia-Idade , PrognósticoRESUMO
OBJECTIVE: The primary aim of this study is to examine the hemodynamics of retrobulbar and intrarenal in the changes of early stage of type 2 diabetes mellitus (T2DM) patients from 2000 to 2015 and to assess incidence associated with diabetic kidney disease (DKD) and diabetic retinopathy (DR). METHOD: Our study contained 60 subjects newly diagnosed of T2DM were divided into 2 groups base on the mean resistive index (RI) (≤0.7 and >0.7) of hemodynamic and to compare between-group differences of the early changes in hemodynamics of retrobulbar and intrarenal and also to conclude the incidences of diabetic kidney disease (DKD) and diabetic retinopathy (DR)subsequently with a long follow-up duration(2000-2015). First, to compare the mean RI of central retinal artery (CRA) between 2 groups. Second, to compare the mean RI of intrarenal hemodynamics in the bilateral interlobular renal arteries, renal function parameters (blood urea nitrogen (BUN), creatinine (Cr), blood glucose parameters (glycosylated hemoglobinA1c (HbA1c), fasting plasma glucose (FBG), and 2-hour postprandial blood glucose (2hPBG)), glomerular filtration rate (GFR), albumin excretion rate (AER), and urine albumin-to-creatinine ratio (UACR) between 2 groups. RESULTS: First part of our follow-up studies was to compare hemodynamic RI index of retrobulbar in years of 2000 and 2015, both renal function and blood glucose parameters were fund significantly enhanced in subject group RIs ≤0.7. Incidence of DKD and DR was notably lower in group RIs ≤0.7 than group RIs >â0.7, difference was statistically significant (Pâ<â.05). Incidence of HbA1c ≤7% was higher in group RIs ≤0.7 than group RIs >0.7, but difference was not statistically significant (Pâ>â.05). Incidence of proliferative diabetic retinopathy (PDR) was notably lower in group RIs ≤0.7 than group RIs >0.7, but the difference was not statistically significant (Pâ>â.05). Second part of our follow-up studies was to compare hemodynamic RI index of interlobular renal in years of 2000 and 2015, both renal function and blood glucose parameters were fund significantly enhanced in subject group RIs ≤0.7. Compared data of various incidences from first part of study were coherent with second part. (Incidence of DKD and DR was notably lower in group RIs ≤0.7 than group RIs >0.7, difference was statistically significant (Pâ<â.05). Incidence of HbA1c ≤7% was higher in group RIs ≤0.7 than group RIs >0.7, but difference was not statistically significant (Pâ>â.05). Incidence of PDR was notably lower in group RIs ≤0.7 than group RIs >0.7, but the difference was not statistically significant (Pâ>â.05). CONCLUSIONS: RIs of retrobulbar and interlobular renal which would serve as a good predictors for the hemodynamics changes in retrobulbar and intrarenal would assess incidence of DKD and DR during the preclinical stage in long-term range excluding renal function and HbA1c in T2DM patients.
Assuntos
Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/fisiopatologia , Nefropatias Diabéticas/epidemiologia , Nefropatias Diabéticas/fisiopatologia , Retinopatia Diabética/epidemiologia , Retinopatia Diabética/fisiopatologia , Idoso , Glicemia , Feminino , Seguimentos , Hemoglobinas Glicadas , Hemodinâmica/fisiologia , Humanos , Rim/fisiopatologia , Testes de Função Renal , Masculino , Pessoa de Meia-Idade , Artéria Renal/fisiopatologia , Artéria Retiniana/fisiopatologiaRESUMO
A novel vector pBIN-35S-GFP was constructed from the plasmids of pBIN19, pGFP, and pCHS, which included gfp gene driven by the CaMV 35S promoter. The hairy roots of Petunia hybrida were induced by wild-type Agrobacterium rhizogenes K599 harboring pBIN-35S-GFP with the frequency of 45%. The PCR results showed that rolB from K599 Ri plasmid and gfp from pBIN-35S-GFP were co-transformed into the genome of P. hybrida. The high activity of green fluo-rescence protein was detected by fluorescence microscopy. In particularly, the vector carries multiple cloning sites at both 5' and 3' of the CaMV 35S promoter, which allows easy exchange 35S promoter to study other promoter functions. In addition, there are multiple cloning sites at 5' end and one-sites of EcoRand Bsmsites at 3' end of gfp. Therefore, it supports to fusion target genes to expression fusion protein and can be replaced with any other genes of interest for genetic transforma-tion.
Assuntos
Vetores Genéticos/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Petunia/metabolismo , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Microscopia de Fluorescência , Modelos Genéticos , Petunia/genética , Raízes de Plantas/genética , Plantas Geneticamente Modificadas/genética , Plasmídeos/genética , Reação em Cadeia da Polimerase , Transformação Genética/genéticaRESUMO
BACKGROUND: The primary goals of this study were to evaluate early changes in pulmonary function and retrobulbar hemodynamics and to examine the correlation between these parameters in patients with type 2 diabetes during the preclinical stages of diabetic retinopathy. METHODS: For the single-time point measurements, 63 subjects with type 2 diabetes without diabetic retinopathy (diabetes group) and 32 healthy subjects (control group) were selected to evaluate any early changes in pulmonary function and retrobulbar hemodynamics and to examine the correlation between these parameters. In the longitudinal follow-up study, 32 subjects who were newly diagnosed with type 2 diabetes were divided into 2 groups according to their resistivity index (≤0.7 and >0.7). Early changes in pulmonary function and retrobulbar hemodynamics were studied in these groups and compared with the previous values. RESULTS: For the single-time point measurements, the fasting plasma glucose, 2-h postprandial blood glucose, glycosylated hemoglobin A1c, total cholesterol, low-density lipoprotein cholesterol, and triglyceride levels as well as the pulmonary function parameters were significantly higher in the diabetes group than in the control group. The pulmonary function parameters were negatively and significantly correlated with glycosylated hemoglobin A1c and the duration of diabetes. The retrobulbar hemodynamics were positively correlated with glycosylated hemoglobin A1c and diabetes duration; in contrast, the correlation between retrobulbar hemodynamics and glycosylated hemoglobin A1c. In the longitudinal follow-up study, the pulmonary function of the 2 groups categorized by their resistivity index levels indicated that subjects with resistivity index levels ≤0.7 showed significantly better pulmonary function, and the pulmonary function of this group showed improvement and a significantly smaller decrease. The incidence of diabetic retinopathy in the group with resistivity index levels ≤0.7 (9 of 22, 40.9%) was significantly lower than that in the group with resistivity index levels >0.7. CONCLUSIONS: Pulmonary function and retrobulbar hemodynamics changed during the preclinical stages of diabetic retinopathy. Regulating glycemia may improve retrobulbar hemodynamics in the retrobulbar arteries (ie, central retinal artery, posterior ciliary artery, and arteria ophthalmica). By detecting the retrobulbar resistivity index and the levels of glycosylated hemoglobin A1c, we could predict future changes in pulmonary function during the preclinical stages of diabetic retinopathy as well as the degree of retinopathy. (ClinicalTrials.gov registration NCT02774733.).