RESUMO
Berberine (BBR), a natural compound extracted from a Chinese herb, has been shown to effectively attenuate insulin resistance (IR) and inflammation in the clinic. However, its ameliorative mechanism against IR is not well defined. This study is aimed at investigating the effect of BBR and protein phosphatase, Mg2+/Mn2+-dependent 1B (PPM1B) on IR. Biochemical measurements and liver histopathology were detected using the biochemical analyzer and HE staining in ZDF rats, respectively. Microarray analysis of liver tissues was performed, and differentially expressed gene (DEG) levels were examined by quantitative real-time PCR (qPCR) and Western blot. Additionally, the effect of BBR was also explored in HepG2-IR cells. The glucose oxidase method and the fluorescent glucose analog were used to detect glucose consumption and uptake, respectively. The PKA inhibitor H89, ELISA, qPCR, Western blot, and immunofluorescence staining were employed to estimate the expression levels of related signaling pathways. To evaluate the roles of PPM1B, HepG2-IR cells were stably infected with lentivirus targeting PPM1B. The administration of BBR drastically decreased the body weight, urine volume, blood glucose, blood urea nitrogen (BUN), CHOL, hepatic index levels, and pathologic changes and improved ALB levels in ZDF rats with PPM1B upregulation. Furthermore, BBR effectively improves glucose consumption, uptake, and inflammation in HepG2-IR cells. The knockdown of PPM1B expression aggravated the inflammatory response and glycometabolism disorder in HepG2-IR cells. Mechanistically, a reversal in the expression of cAMP, PKA, PPM1B, PPARγ, LRP1, GLUT4, NF-κB p65, JNK, pIKKß Ser181, IKKß, IRS-1 Ser307, IRS-1, IRS-2 Ser731, IRS-2, PI3K p85, and AKT Ser473 contributes to ameliorate IR in HepG2-IR cells with BBR treatment. Altogether, these results suggest that BBR might regulate IR progression through the regulation of the cAMP, PKA, PPM1B, PPARγ, LRP1, GLUT4, NF-κB p65, JNK, pIKKß Ser181, IKKß, IRS-1 Ser307, IRS-1, IRS-2 Ser731, IRS-2, PI3K p85, and AKT Ser473 expression in the liver.
Assuntos
Anti-Inflamatórios/farmacologia , Berberina/farmacologia , Resistência à Insulina , Insulina/metabolismo , Proteína Fosfatase 2C/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Biomarcadores , Sobrevivência Celular/efeitos dos fármacos , Biologia Computacional/métodos , AMP Cíclico/metabolismo , Diabetes Mellitus Experimental , Modelos Animais de Doenças , Metabolismo Energético/efeitos dos fármacos , Perfilação da Expressão Gênica , Inativação Gênica , Glucose/metabolismo , Células Hep G2 , Humanos , Insulina/farmacologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Camundongos Knockout , NF-kappa B/metabolismo , Proteína Fosfatase 2C/genética , RatosRESUMO
BACKGROUND Shen Qi Wan (SQW) as a well-known formula for the amelioration of kidney yang deficiency syndrome (KYDS), and it has been widely employed in traditional Chinese medicine (TCM). This study aimed to investigate the effect and underlying mechanism of SQW medicated serum on proliferation and migration in NRK-52E cells. MATERIAL AND METHODS We employed the real-time cell analysis (RTCA) system to investigate the effect of SQW medicated serum on proliferation and migration in NRK-52E cells. In addition, the migration was further investigated by using a wound-healing assay. The mRNA and protein expression level of aquaporin 1 (AQP1) of NRK-52E cells with SQW medicated serum-treated were quantified by real-time quantitative polymerase chain reaction (q-PCR) and western blot assay, respectively. Furthermore, NRK-52E cells were transfected with lentivirus AQP1-RNAi to assess migratory cell abilities in vitro. RESULTS The migratory abilities of NRK-52E cells were significantly increased after SQW medicated serum treatment (P<0.05), and no significant difference in cell proliferation. In addition, SQW medicated serum was significantly upregulated the mRNA and protein expression level of AQP1 in NRK-52E cells (P<0.05). Additionally, the in vitro metastasis test proved that knockdown of AQP1 suppressed migratory abilities according to RTCA and wound healing test while was reversed by SQW medicated serum (P<0.05). CONCLUSIONS Our study demonstrates that SQW medicated serum effectively promotes the migration of NRK-52E cells by increasing AQP1 expression, and AQP1 may be as a therapeutic target of SQW for renal injury treatment under KYDS.
Assuntos
Aquaporina 1/metabolismo , Medicamentos de Ervas Chinesas/farmacologia , Nefropatias/tratamento farmacológico , Deficiência da Energia Yang/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Aquaporina 1/biossíntese , Aquaporina 1/genética , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Rim/efeitos dos fármacos , Rim/metabolismo , Nefropatias/genética , Nefropatias/metabolismo , Nefropatias/patologia , Masculino , Terapia de Alvo Molecular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Deficiência da Energia Yang/genética , Deficiência da Energia Yang/metabolismo , Deficiência da Energia Yang/patologiaRESUMO
This study was aimed to investigate the effect and mechanism of Zhenwu Tang on AVP-V2R-AQP2 pathway in NRK-52E cells in vitro. Forty eight male SD rats were randomly divided into eight groups with 6 animals in each group. Distilled water or 22.68 g·kg⻹·d⻹ Zhenwu Tang(calculated by raw drug dosage meter) was given by gavage. Blood samples were collected by cardiac punctureï¼ and the medicated serum was centrifuged from the blood by 3 000 r·min⻹. NRK-52E cells were treated with different medicated serum or dDAVP. The condition of cell proliferation was detected by RTCA. The distribution of V2R and AQP2 in cells were detected by immunofluorescence. The expression of V2Rï¼ PKA and AQP2 were detected by Western blot and AQP2 mRNA level was detected by real-time PCR. Results showed that the level of AQP2 mRNA(P<0.01) and protein expression of V2Rï¼ PKA and AQP2(P<0.05ï¼ P<0.01ï¼ P<0.05) of Z7d group which was treated with Zhenwu Tang medicated serum for 24 h were significantly higher than that of normal rat serum group. And the expression level of V2Rï¼ p-AQP2 and AQP2(P<0.01ï¼ P<0.05ï¼ P<0.01) of Z7d+dDAVP group were significantly increased comparing to normal rat serum group. The results indicate that the applying of Zhenwu Tang medicated serum could increase the expression level of V2Rï¼ PKA and AQP2 which exist in AVP-V2R-AQP2 pathway in NRK-52Eï¼ and there is synergistic effect between Zhenwu Tang medicated serum and dDAVP. So the pathway of AVP-V2R-AQP2 may be one of the mechanism for which Zhenwu Tang regulate balance of water transportation.
Assuntos
Aquaporina 2/metabolismo , Medicamentos de Ervas Chinesas/farmacologia , Receptores de Vasopressinas/metabolismo , Transdução de Sinais , Animais , Linhagem Celular , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Rim/citologia , Masculino , RNA Mensageiro , Ratos , Ratos Sprague-DawleyRESUMO
This study was aimed to investigate the protective effect and mechanism of ß-asarone on PC12 cells injury induced byAß1â42 activated astrocytes, and provide experimental basis for ß-asarone application in the prevention and control of Alzheimer's disease (AD). Firstly, RA-h and PC12 cells were co-cultured in the special transwell chamber, and the Real time cell analysis (RTCA) system was used to real-time observe its effect on PC12 cells survival rate in the co-culture system after astrocytes injury induced by Aß1â42. The best intervention time of ß-asarone was selected according to the survival curve and parameters generated automatically. ß-asarone with different concentrations was used for intervention on astrocytes, then the changes of PC12 cells survival rate in the co-culture system were observed. Secondly, MTT assay was used to detect the effect of Aß1â42 on PC12 cells survival rate as well as the intervention effect of ß-asarone, and verify the testing results of RTCA. The levels of IL-1ß, TNF-α and BDNF in culture media of the lower chamber were detected by ELISA. The NF-κB activity and phosphorylation levels of ERK, p38 and JNK were detected by Western blot. Results showed that ß-asarone (55.5 mgâ¢L⻹) could significantly slowdown the decline of PC12 cells survival rate caused by Aß1â42-induced RA-h activation (P<0.01), significantly reduce the levels of IL-1ß, TNF-α and the phosphorylation levels of ERK, p38 and JNK in culture media of the lower chamber (P<0.01). ß-asarone(166.7 mgâ¢L⻹) could promote the release of BDNF in culture media of the lower chamber(P<0.05). These results indicated that Aß1â42 could induce RA-h activation and its release of IL-1ß, TNF-α and other inflammatory factors to aggravate the PC12 cells injury; ß-asarone could reduce the levels of IL-1ß, TNF-α, promote the release of BDNF, and inhibit the NF-κB activity as well as phosphorylation levels of ERK, p38 and JNK protein in PC12 cells.
Assuntos
Anisóis/farmacologia , Fármacos Neuroprotetores/farmacologia , Derivados de Alilbenzenos , Peptídeos beta-Amiloides , Animais , Interleucina-1beta/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Células PC12 , Fragmentos de Peptídeos , Ratos , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Recent studies confirmed that the new cell survival signal pathway of Insulin-PI3K-Akt exerted cyto-protective actions involving anti-apoptosis. This study was undertaken to investigate the potential neuroprotective effects of insulin in the pathogenesis of spinal cord injury (SCI) and evaluate its therapeutic effects in adult rats. SCI was produced by extradural compression using modified Allen's stall with damage energy of 40 g-cm force. One group of rats was subjected to SCI in combination with the administration of recombinant human insulin dissolved in 50% glucose solution at the dose of 1 IU/kg day, for 7 days. At the same time, another group of rats was subjected to SCI in combination with the administration of an equal volume of sterile saline solution. Functional recovery was evaluated using open-field walking, inclined plane tests, and motor evoked potentials (MEPs) during the first 14 days post-trauma. Levels of protein for B-cell lymphoma/leukemia-2 gene (Bcl-2), Caspase-3, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) were quantified in the injured spinal cord by Western blot analysis. Neuronal apoptosis was detected by TUNEL, and spinal cord blood flow (SCBF) was measured by laser-Doppler flowmetry (LDF). Ultimately, the data established the effectiveness of insulin treatment in improving neurologic recovery, increasing the expression of anti-apoptotic bcl-2 proteins, inhibiting caspase-3 expression decreasing neuronal apoptosis, reducing the expression of proinflammatory cytokines iNOS and COX-2, and ameliorating microcirculation of injured spinal cord after moderate contusive SCI in rats. In sum, this study reported the beneficial effects of insulin in the treatment of SCI, with the suggestion that insulin should be considered as a potential therapeutic agent.