Effect and Mechanism of Yishen Tongluo Prescription on miR-514a-5p/TNFSF15 Signaling Pathway in MN Rats / 中国实验方剂学杂志

ObjectiveTo explore the molecular mechanism of Yishen Tongluo prescription in inhibiting the apoptosis of glomerular podocytes in rats with membranous nephropathy （MN） based on the miR-514a-5p/tumor necrosis factor superfamily member 15 （TNFSF15） signaling pathway. MethodEighty SD rats were pre-immunized and injected with cationized bovine serum albumin （C-BSA） into the tail vein for inducing MN， and the successfully modeled MN rats were randomly divided into the model group， high-， middle-， and low-dose （26.44， 13.22， 6.61 g·kg-1） Yishen Tongluo prescription groups， and benazepril （10 mg·kg-1） group， with 10 rats in each group， and another 20 healthy rats were classified into the normal group. Rats in each group were gavaged with the corresponding drugs， once a day， for four successive weeks. After the administration， the 24-hour urine total protein （UTP） level， serum total cholesterol （TC）， triglyceride （TG）， albumin （ALB）， creatinine （SCr）， and urea nitrogen （BUN） levels were measured. The miR-514a-5p and TNFSF15 mRNA expression levels in the rat kidney tissue were detected by real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）， and the expression levels of podocyte marker proteins Nephrin， Podocin， Podocalyxin， Synaptopodin， TNFSF15， and podocyte apoptosis-related proteins B lymphocytoma-2 （Bcl-2）-related X protein （Bax）， Bcl-2-associated death promoter （BAD） protein， and B-cell lymphoma-extra large （Bcl-XL） by immunohistochemistry （IHC）. Western blot was used to detect the expression levels of TNFSF15， Bax， BAD， Bcl-2， and BCL-XL in the rat kidney tissue. The apoptosis rate of rat kidney tissue was measured using the in situ end labeling method （Tunnel）. ResultCompared with the normal group， the level of miR-514a-5p in the kidney tissue was significantly reduced （P<0.05）， and the TNFSF15 mRNA expression was significantly increased （P<0.05）. The expression levels of podocyte marker proteins Nephrin， Podocin， Podocalyxin， and Synaptopodin were down-regulated （P<0.05）. The protein expression levels of TNFSF15， Bax， and BAD were increased （P<0.05）， whereas the Bcl-2 and Bcl-XL protein expression levels were decreased （P<0.05）. The number of apoptotic cells diminished significantly （P<0.05）. Compared with the model group， the level of miR-514a-5p in the kidney tissue was significantly increased （P<0.05）， while the level of TNFSF15 mRNA was significantly decreased （P<0.05）. The expression levels of podocyte marker proteins Nephrin， Podocin， podocalyxin， and Synaptopodin were up-regulated （P<0.05）， whereas the TNFSF15， Bax， and BAD protein expression levels were down-regulated （P<0.05）. Bcl-2 and Bcl-XL protein expression levels rose （P<0.05）. The number of apoptotic cells significantly decreased （P<0.05）. ConclusionYishen Tongluo prescription reduces the apoptosis of rat kidney podocytes and alleviates the kidney injury of MN rats through the miR-514a-5p/TNFSF15 signaling pathway.

Renal Protection of Yishen Tongluo Prescription in Rats with Membranous Nephropathy and Its Influence on AMPK/mTOR/ULK1 Signaling Pathway / 中国实验方剂学杂志

Objective:To observe the effects of Yishen Tongluo prescription （YTP） on autophagy-related proteins in rats with membranous nephropathy （MN） and explore its possible molecular mechanism in protecting the kidney. Method:Twenty of 80 Sprague-Dawley （SD） rats were randomly selected as the normal control， and the rest rats were pre-immunized and injected with cationized bovine serum albumin （C-BSA） through the tail vein to induce MN. The SD rats that were successfully modeled were randomized into the model group， benazepril hydrochloride group （10 mg·kg^-1）， and low- （6.61g·kg^-1）， medium- （13.22 g·kg^-1）， and high-dose （26.44 g·kg^-1） YTP groups， and administered with the corresponding drugs by gavage， once a day， for four consecutive weeks. Then the changes in such quantitative indicators as plasma albumin （ALB）， triglyceride （TG）， total cholesterol （TC）， serum creatinine （SCr）， blood urea nitrogen （BUN）， and 24-hour urinary total protein （UTP） were detected， followed by hematoxylin and eosin （HE） staining， Masson's trichrome staining， and periodic Schiff-methenamine （PASM） staining for observing the pathological changes in kidney under the transmission electron microscope （TEM）. The deposition of immunoglobulin G （IgG） and complement 3 （C3） in the glomerulus was detected by fluorescence immunoassay. The expression levels of autophagy marker proteins Beclin-1， microtubule-associated protein light chain 3Ⅱ （LC3Ⅱ）， and p62 were measured by immunohistochemistry （IHC）， and those of related proteins in the adenosine monophosphate-activated protein kinase / mechanisic target of rapamycin/Unc-51-like kinase 1 （AMPK/mTOR/ULK1） signaling pathway were determined by Western blot assy. Result:Compared with the normal group， the model group exhibited significantly increased UTP （<italic>P</italic><0.01） and serum TG and TC （<italic>P</italic><0.01）， decreased ALB （<italic>P</italic><0.01）， disordered glomerular structure， enlarged volume， thickened basement membrane， vacuolated renal tubules， excessively deposited collagen fibers and fuchsinophilic proteins， extensively fused podocyte foot processes， and diffusely deposited IgG and C3 in glomerular capillary loops. Besides， the expression levels of Beclin-1， LC3II， and phosphorylated AMPK （p-AMPK） decreased （<italic>P</italic><0.01）， while those of p62， phosphorylated mTOR （p-mTOR）， and phosphorylated ULK1 （p-ULK1） increased （<italic>P</italic><0.01）. The comparison with the model group revealed that the TG， TC， and UTP levels in the low-， medium-， and high-dose YTP groups and the benazepril hydrochloride group were reduced to varying degrees （<italic>P</italic><0.05， <italic>P</italic><0.01）， whereas the ALB level was increased （<italic>P</italic><0.01）. There was no statistically significant difference in SCr or BUN level. The pathological damages were alleviated. The expression levels of Beclin-1， LC3Ⅱ， and p-AMPK were up-regulated （<italic>P</italic><0.05， <italic>P</italic><0.01）， while those of p62， p-mTOR， and p-ULK1 were down-regulated （<italic>P</italic><0.05， <italic>P</italic><0.01）. Conclusion:YTP protects the kidney of rats with MN possibly by regulating related proteins in the AMPK/mTOR/ULK1 signaling pathway and activating the autophagy.