preparing paraffin sections of kidney and peyer&#39;s patch isolated from an igan murine model for histological and immunohistochemical analyses

<meta charset="utf8"></head><body>Tratamento da Nefropatia por Imunoglobulina A (NIgA) com Dioscina em Modelo Murino

IgA Nephropathy (IgAN) is the most common type of primary glomerulonephritis, for which there is no specific treatment, and it remains a significant cause of end-stage renal disease1. Although the pathogenesis of IgAN is still not fully understood, the &#34;multi-hit hypothesis&#34; is generally accepted and supported by a large body of clinical and experimental research evidence2. The pathogenesis of IgAN involves activating B cells and producing Galactose-deficient IgA1 (Gd-IgA1)3. The increase in circulating Gd-IgA1 due to the excessive proliferation and activation of IgA-secreting cells during the mucosal immune response is a critical link in the pathogenesis of IgAN4,5,6. As the central place for the proliferation and activation of B lymphocyte phenotype conversion to IgA-secreting cells, Peyer&#39;s patch is the primary source of IgA secretion, closely related to the occurrence and development of IgAN7,8. In addition, the proliferation of IgA1-secreting cells, as well as expression of Core 1&#946;-1,3-galactosyltransferase(C1GalT1) and C1GalT1-specific molecular chaperone (Cosmc), were associated with abnormal glycosylation of IgA1, which causes GD-IgA1 production in IgAN patients6,9.
Clinical study on IgAN treatment with herbal medicine has progressed in recent years. Yiqi Qingjie Formula is an essential formula for treating IgAN by the Department of Nephrology of Guang&#39;anmen Hospital. The previous study of our group found that Gd-IgA1 decreased in the serum of IgAN patients after treatment with Yiqi Qingjie Formula. As one of the most used herbs in the Yiqi Qingjie Formula, Dioscoreae Nipponicae Rhizoma (DNR) is the dried rhizome of Dioscorea Nipponica Makino, which has various functions such as regulating immunity, suppressing inflammation, relieving cough and asthma10,11. Several scholars treated IgAN with DNR and achieved good results12,13,14. As the main active ingredient in DNR15, Dioscin (DIO) lowers uric acid, inhibits fibrosis, inhibits inflammatory response, and anti-oxidative stress16,17. Therefore, DIO may have a novel action mechanism to inhibit the cellular secretion of excessive Gd-IgA1 and exert specific kidney protection effects. Still, no study has been reported on DIO&#39;s action mechanism for treating IgAN.
To explore the potential therapeutic mechanism of DIO on IgAN and provide a new method for the treatment of IgAN, we carried out experiments for the therapeutic effects of DIO on IgAN in vivo and in vitro.

<meta charset="utf8"></head><body>Autor em destaque: Investigando o potencial da dioscina ativa medicinal fitoterápica chinesa no tratamento da nefropatia por IgA

Alívio da nefropatia por IgA mediada por dioscina, inibindo a ativação de células B in vivo e diminuindo a produção de IgA1 deficiente em galactose in vitro

Preparação de Seções de Parafina de Rim e Adesivo de Peyer Isolados de um Modelo Murino de IgAN para Análises Histológicas e Imuno-Histoquímicas

preparing-paraffin-sections-kidney-peyer-s-patch-isolated-from-an

Research

JoVE Journal

Medicine

Preparing Paraffin Sections of Kidney and Peyer&#39;s Patch Isolated from an IgAN Murine Model for Histological and Immunohistochemical Analyses

231 visualizações. Para começar a preparar as seções de parafina do rim e o adesivo de Peyer isolados de camundongos modelo de neuropatia por IgA devidamente anestesiados, primeiro fixe um adesivo de Peyer ou tecidos renais de três milímetros de espessura com paraformaldeído a 4% por 24 horas. Em seguida, desidrate-os usando um gradiente de etanol e xileno antes de lavá-los com água. Mergulhe os tecidos em cera por duas horas antes de selá-los e congelá-los.Em ...

Vídeo: Preparação de Seções de Parafina de Rim e Adesivo de Peyer Isolados de um Modelo Murino de IgAN para Análises Histológicas e Imuno-Histoquímicas

Dioscin Mediated IgA Nephropathy Alleviation by Inhibiting B Cell Activation In Vivo and Decreasing Galactose-Deficient IgA1 Production In Vitro

The increase of circulating galactose-deficient IgA1 (Gd-IgA1) is caused by excessive activation of IgA-positive secretory cells in the process of mucosal immune responses, which is a critical link in the pathogenesis of IgA nephropathy (IgAN). Peyer&#39;s patch, the prominent place where B lymphocytes are transformed into IgA-secreting plasma cells, is the primary source of IgA. In addition, the lower expression of core 1&#946;-1,3-galactosyltransferase (C1GalT1) and its molecular chaperone, C1GalT1-specific molecular chaperone (Cosmc), is related to abnormal glycosylation of IgA1 in IgAN patients. Our clinical experience shows that Dioscoreae Nipponicae Rhizoma&#39;s (DNR) herbal medicine can relieve proteinuria and hematuria and improve renal function in IgAN patients. Dioscin (DIO) is one of the main active ingredients of DNR, which has various pharmacological activities. This study explores DIO&#39;s possible mechanism in treating IgAN.The IgAN model mouse was established by mucosal immune induction. The mice were divided into the control, model, and DIO gavage groups. The glomerular IgA deposition in mice, renal pathological changes, and B cell markers CD20 and CXCR5 expression in Peyer&#39;s patch were detected by immunofluorescence and immunohistochemistry. After lipopolysaccharide (LPS) stimulation, DIO&#39;s effects on DAKIKI cells proliferation, IgA and Gd-IgA1 secretion, C1GalT1, and Cosmc expression were studied by cell counting kit-8 (CCK-8) assay, enzyme-linked immunosorbent assay (ELISA) test, quantitative real-time polymerase chain reaction (QRT-PCR), and western blotting (WB). In in vivo studies, IgA deposition accompanied by glomerular mesangial hyperplasia and increased expression of CD20 and CXCR5 in Peyer&#39;s patch in the IgAN model mouse was alleviated by DIO. In vitro studies showed 0.25 &#181;g/mL to 1.0 &#181;g/mL DIO inhibited LPS-induced DAKIKI cell proliferation, IgA and Gd-IgA1 secretion, and up-regulated the mRNA and protein expression of C1GalT1 and Cosmc. This study demonstrates that DIO may reduce Gd-IgA1 production by inhibiting excessive activation of IgA-secreting cells and up-regulating C1GALT1/Cosmc expression.

This study provides experimental data for treating immunoglobulin A nephropathy (IgAN) with Dioscin (DIO), the active ingredient of Dioscoreae Nipponicae Rhizoma (DNR), and a paradigm for studying herbal medicine's effects and underlying mechanisms in vivo and in vitro.

The increase of circulating galactose-deficient IgA1 (Gd-IgA1) is caused by excessive activation of IgA-positive secretory cells in the process of mucosal ...