Expression of TRPC6 in Renal Cortex and Hippocampus of Mouse during Postnatal Development

TRPC6, a member of the TRPC family, attracts much attention from the public because of its relationship with the disease. In both the brain and kidney, TRPC6 serves a variety of functions. The aim of the present study was to observe the expression and effects of TRPC6 in renal cortex and hippocampus during early postnatal development of the mouse. In the present study, immunohistochemistry and Western blotting were used to detect the expression of TRPC6 in the mouse kidney and hippocampus of postnatal day 1, 3, 5, 7, 14, 21, 28 and 49 (P1, P3, P5, P7, P14, P21, P28 and P49). Results showed that the expression of TRPC6 was increased in the mouse hippocampus, and there was a significant increase between P7 and P14 during the postnatal development. Meanwhile, the expression of TRPC6 was also detected in glomerulus and tubules, and a decreased expression was found during postnatal maturation of mouse renal cortex. From these in vivo experiments, we concluded that the expression of TRPC6 was active in the developing mouse kidney cortex, and followed a loss of expression with the development of kidney. Meanwhile, an increased expression was found in the hippocampus with the development. Together, these data suggested that the developmental changes in TRPC6 expression might be required for proper postnatal kidney cortex development, and played a critical role in the hippocampus during development, which formed the basis for understanding the nephrogenesis and neurogenesis in mice and provided a practically useful knowledge to the clinical and related research

Triptolide Inhibited Cytotoxicity of Differentiated PC12 Cells Induced by Amyloid-Beta₂₅₋₃₅ via the Autophagy Pathway.

Evidence shows that an abnormal deposition of amyloid beta-peptide25-35 (Aβ25-35) was the primary cause of the pathogenesis of Alzheimer's disease (AD). And the elimination of Aβ25-35 is considered an important target for the treatment of AD. Triptolide (TP), isolated from Tripterygium wilfordii Hook.f. (TWHF), has been shown to possess a broad spectrum of biological profiles, including neurotrophic and neuroprotective effects. In our study investigating the effect and potential mechanism of triptolide on cytotoxicity of differentiated rat pheochromocytoma cell line (the PC12 cell line is often used as a neuronal developmental model) induced by Amyloid-Beta25-35 (Aβ25-35), we used 3-(4, 5-dimethylthiazol-2-yl)-2, 5- diphenyltetrazolium bromide (MTT) assay, flow cytometry, Western blot, and acridine orange staining to detect whether triptolide could inhibit Aβ25-35-induced cell apoptosis. We focused on the potential role of the autophagy pathway in Aβ25-35-treated differentiated PC12 cells. Our experiments show that cell viability is significantly decreased, and the apoptosis increased in Aβ25-35-treated differentiated PC12 cells. Meanwhile, Aβ25-35 treatment increased the expression of microtubule-associated protein light chain 3 II (LC3 II), which indicates an activation of autophagy. However, triptolide could protect differentiated PC12 cells against Aβ25-35-induced cytotoxicity and attenuate Aβ25-35-induced differentiated PC12 cell apoptosis. Triptolide could also suppress the level of autophagy. In order to assess the effect of autophagy on the protective effects of triptolide in differentiated PC12 cells treated with Aβ25-35, we used 3-Methyladenine (3-MA, an autophagy inhibitor) and rapamycin (an autophagy activator). MTT assay showed that 3-MA elevated cell viability compared with the Aβ25-35-treated group and rapamycin inhibits the protection of triptolide. These results suggest that triptolide will repair the neurological damage in AD caused by deposition of Aβ25-35 via the autophagy pathway, all of which may provide an exciting view of the potential application of triptolide or TWHF as a future research for AD

Localization of TRPC6 in mouse hippocampus during the postnatal development.

<p>The immunohistochemical staining showed that TRPC6 was expressed in all regions of the hippocampus at P1 (B), P3 (C), P5 (D), P7 (E), P14 (F) and P21 (G). The negative control image was shown in (A). Scale bar, 200 µm. In suit hybridization of P1 (H) and P5 (I) brain sections showed the expression of TRPC6 in hippocampal neurons. Scale bar, 100 µm. The corresponding linear diagram of relative fluorescent intensity in glomeruli was shown in (J). Data were presented as means±S.D. n = 6/group. *<i>P</i><0.05, **<i>P</i><0.01 <i>vs.</i> adjacent age group.</p

Localization of TRPC6 in mouse renal cortex during postnatal development.

<p>Immunohistochemical staining of TRPC6 showed the expression of TRPC6 in the mouse renal cortex at P1 (B), P3 (C), P5 (D), P7 (E), P14 (F), P21 (G), P28 (H), and P49 (I). The negative control image was from the renal cortex, in which the primary antibody was a species-appropriate IgG (A). The corresponding linear diagram of relative fluorescent intensity in glomeruli was shown in (J). The sections from P1 to P5 showed that TRPC6 mostly expressed in comma-shaped body, S-shaped body and renal corpuscles of stage III (B-D). After P7 the expression was found in renal corpuscles and it was weakly positive (E-F). During the development, tubules were all observed. Scale bar, 100 µm. Data were presented as means±S.D. n = 6/group. *<i>P</i><0.05, **<i>P</i><0.01 <i>vs.</i> adjacent age group.</p

The immunoblot analysis of TRPC6 in mouse hippocampus during the development.

<p>Total lysates of tissues treated as indicated blotted with antibodies to TRPC6 at the indicated ages (A). Quantitative determination of TRPC6 expression showed an abrupt increase between P7 and P14 in mouse renal cortex during the development, and expression levels remained higher into adulthood (B). The expression of TRPC6 was normalized to β-actin expression. Data were presented as means±S.D. *<i>P</i><0.05, **<i>P</i><0.01 <i>vs.</i> adjacent age group.</p

Immunoblot analysis of EPOR in mouse kidney cortex during the development.

<p>The position of 68 kDa molecular size was the expression pattern of EPOR in renal cortex at the indicated ages. β-actin was used as a consult. When the expression of EPOR was normalized to β-actin, the level was found decreased with development.</p

Cytotoxicity induced by Aβ_25–35 in differentiated PC12 cells.

<p>Cells were treated with different concentrations (5, 10, 20 μmol/L) of Aβ_25–35 for 24 hours. The cell viability was measured by MTT assay. Data represents the mean ± S.E.M. n = 6/group. *<i>P</i> < 0.05 vs. control group.</p

Expression of LC3 in differentiated PC12 cells.

<p>Morphological evaluation of autophagy in differentiated PC12 cells by immunofluorescence was acquired by confocal microscopy. The immunohistochemical staining showed that LC3 was expressed in the control group (cells treated with culture medium), 10 μmol/L Aβ_25–35 group, 10 μmol/L Aβ_25–35+10⁻¹⁰ mol/L triptolide group and 10⁻¹⁰ mol/L triptolide group. Scale bar, 10 μm. Fig b was the corresponding linear diagram of relative fluorescent intensity. Data were presented as the means ±mean ± S.E.M. n = 7/group. *<i>P</i> < 0.05, **<i>P</i> < 0.01 vs. control group. ^##<i>P</i> < 0.01 vs. 10 μmol/L Aβ_25–35 group.</p

Overexpression of MicroRNA-16 Alleviates Atherosclerosis by Inhibition of Inflammatory Pathways

Background. Our previous study demonstrated that the expression of miR-16 was downregulated in the cell and animal models of atherosclerosis (AS), a main contributor to coronary artery disease (CAD). Overexpression of miR-16 inhibited the formation of foam cells by exerting anti-inflammatory roles. These findings indicated miR-16 may be an anti-atherogenic and CAD miRNA. The goal of this study was to further validate the expression of miR-16 in CAD patients and explore its therapeutic roles in an AS animal model. Methods. A total of 40 CAD patients and 40 non-CAD people were prospectively registered in our study. The AS model was established in ApoE-/- mice fed a high-fat diet. The model mice were randomly treated with miR-16 agomiR (n=10) or miR-negative control (n=10). Hematoxylin-eosin staining was conducted for histopathological examination in thoracic aorta samples. ELISA and immunohistochemistry were performed to determine the expression levels of inflammatory factors (IL-6, TNF-α, MCP-1, IL-1β, IL-10, and TGF-β). qRT-PCR and western blotting were carried out to detect the mRNA and protein expression levels of PDCD4, miR-16, and mitogen-activated protein kinase pathway-related genes. Results. Compared with the normal control, miR-16 was downregulated in the plasma and peripheral blood mononuclear cell of CAD patients, and its expression level was negatively associated with IL-6 and the severity of CAD evaluated by the Gensini score, but positively related with IL-10. Injection of miR-16 agomiR in ApoE-/- mice reduced the formation of atherosclerotic plaque and suppressed the accumulation of proinflammatory factors (IL-6, TNF-α, MCP-1, and IL-1β) in the plasma and tissues but promoted the secretion of anti-inflammatory factors (IL-10 and TGF-β). Mechanism analysis showed overexpression of miR-16 might downregulate target mRNA PDCD4 and then activate p38 and ERK1/2, but inactivate the JNK pathway. Conclusions. Our findings suggest miR-16 may be a potential diagnostic biomarker and therapeutic target for atherosclerotic CAD

Localization of EPOR during mouse kidney cortex postnatal development.

<p>The negative control was shown in (A, B). The expression of EPOR in the mouse kidney cortex could be detected at P7 (C). Suspected Immature corpuscle could be observed at P7 (D). The expression of EPOR was higher at tubule part, and the expression at corpuscle part was almost undetectable at P14 (G). The expression of EPOR was detected in the kidney cortex at P7 (E), P14 (F), P21 (H), P28 (I), P35 (J), P42 (K) and mature mouse (L).</p