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.

Effect of Modified Shengjiangsan on Renal Fibrosis in Rats with Membranous Nephropathy Based on TLR4/NF-κB Signaling Pathway / 中国实验方剂学杂志

Objective:To observe the effect of modified Shengjiangsan on renal fibrosis in rats with membranous nephropathy （MN） and to explore the mechanism of its complications of renal fibrosis. Method:Rats were injected with cationized bovine serum albumin（C-BSA）in the tail vein to establish a rat model of membranous nephropathy. The normal group，model group，modified Shengjiangsan group （27.3 g·kg^-1）and benazepril group（10 mg·kg^-1）were established in this study. Each group was given corresponding dosage of the drug once a day for 4 weeks of continuous intervention. After the administration，we observed the pathological changes of rat kidneys by the technology of Masson staining， silverhexylamine iodate （PASM） staining， transmission electron microscopy （TEM）， immunofluorescence technology （IF） was used to detect immunoglobulin（Ig）G deposition in rat kidneys. The levels of interleukin-1<italic>β</italic> （IL-1<italic>β</italic>）， interleukin-6 （IL-6）， tumor necrosis factor-<italic>α</italic> （TNF-<italic>α</italic>） in rat serum were detected by enzyme-linked immunosorbent assay （ELISA） method. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） and immunohistochemistry （IHC） were used to detect the mRNA and protein expression levels of monocyte chemotactic protein 1 （MCP-1）， intercellular adhesion molecule 1 （ICAM-1）， nuclear factor kappa B （NF-<italic>κ</italic>B）， Toll-like receptor 4 （TLR4）， plasminogen activator inhibitor 1 （PAI-1）， transforming growth factor-<italic>β</italic>₁ （TGF-<italic>β</italic>₁）， <italic>α</italic>-smooth muscle actin （<italic>α</italic>-SMΑ） and type Ⅳ Collagen （Collagen Ⅳ） in rat kidney tissues. Result:Compared with normal group， the kidney tissue of the model group was obviously fibrotic， the serum levels of IL-1<italic>β</italic>， IL-6， and TNF-<italic>α</italic> were significantly increased（<italic>P</italic><0.05）， and the expressions of MCP-1， ICAM-1， NF-<italic>κ</italic>B， TLR4， PAI-1， TGF-<italic>β</italic>₁， <italic>α</italic>-SMA and Collagen Ⅳ mRNA and protein in kidney tissue were significantly increased（<italic>P</italic><0.05）. Compared with model group， modified Shengjiangsan and benazepril significantly improved renal fibrosis in rats， reduced the levels of IL-1<italic>β</italic>， IL-6， and TNF-<italic>α</italic> in the serum of MN rats（<italic>P</italic><0.05）， down-regulated MCP-1， ICAM-1， NF-<italic>κ</italic>B， TLR4， PAI-1， TGF-<italic>β</italic>₁， <italic>α</italic>-SMA and Collagen Ⅳ mRNA and protein expression in kidney tissue（<italic>P</italic><0.05）. Conclusion:Modified Shengjiangsan can reduce the release and expression of inflammatory factors by down-regulating the TLR4/NF-<italic>κ</italic>B signaling pathway， inhibit renal fibrosis， and reduce renal damage in MN rats.

Effect of moxibustion on renal function and hypercoagulable state in patients with idiopathic membranous nephropathy of low to medium risk with spleen-kidney deficiency and blood stasis / 中国针灸

OBJECTIVE@#To compare the effect of moxibustion combined with basic treatment and simple basic treatment on the clinical symptoms, renal function and hypercoagulable state in patients with idiopathic membranous nephropathy (IMN) of low to medium risk with spleen-kidney deficiency and blood stasis.@*METHODS@#A total of 60 patients with IMN of low to medium risk with spleen-kidney deficiency and blood stasis were randomized into an observation group (30 cases, 2 cases dropped off) and a control group (30 cases, 1 case dropped off). In the control group, the conventional basic treatment of anti-hypertension, regulating blood lipid and anti-coagulation was adopted. On the basis of the control group, moxibustion was applied at Shenshu (BL 23), Pishu (BL 20), Guanyuan (CV 4), Zusanli (ST 36) and Sanyinjiao (SP 6) in the observation group, once a day, 5 days a week continuously with 2 day interval. The treatment of 6 months was required in the both groups. Before treatment and 3 and 6 months into treatment, the total TCM syndrome score, the renal function indexes (24-hour urinary protein quantity [UTP], albumin [ALB], urea nitrogen [BUN] and creatinine [Scr]), the blood coagulation indexes (fibrinogen [FIB], D-Dimer [D-D], p-selection and von Willebrand factor [vWF]), total cholesterol (TC) and triacylglycerol (TG) levels were observed, and the therapeutic efficacy was evaluated on 3 and 6 months into treatment in the two groups.@*RESULTS@#The effective rates of 3 and 6 months into treatment were 78.6% (22/28) and 89.3% (25/28) in the observation group, which were higher than 62.1% (18/29) and 75.9% (22/29) in the control group respectively (@*CONCLUSION@#Moxibustion combined with basic treatment can effectively improve the clinical symptoms, renal function and renal microcirculation in patients with idiopathic membranous nephropathy of low to medium risk with spleen-kidney deficiency and blood stasis, the therapeutic effect is superior to the simple basic treatment.

Yiqiyangyin Huoxuetongluo Prescription Reduces Renal Cell Apoptosis in Rats with Diabetic Nephropathy by Inhibiting JAK2/STAT3 Signaling Pathway / 中国实验方剂学杂志

Objective:To observe the effect of Yiqiyangyin Huoxuetongluo prescription on janus kinase 2/signal transducer and activator of transcription 3 （JAK2/STAT3） signaling pathway and cell apoptosis in rats with diabetic nephropathy （DN）， and to explore the mechanism of its intervention in DN. Method:A total of 100 SD rats were randomly divided into an experimental group （<italic>n</italic>=80） and a normal group （<italic>n</italic>=20）. The DN model was induced by high-sugar and high-fat diet combined with intraperitoneal injection of streptozotocin （STZ） in the experimental group， and confirmed by the pathological changes of kidney tissues in rats （three in each group） observed under light and electron microscopes. The model rats were randomly divided into a model group （normal saline， equal volume）， low-， medium-， and high-dose （5.775， 11.550， and 23.100 g·kg^-1） Yiqiyangyin Huoxuetongluo prescription groups， and an irbesartan group （irbesartan tablets， 0.016 g·kg^-1）. After drug intervention （<italic>i.g</italic>.， once a day for 16 consecutive weeks）， the 24-hour urine total protein （UTP）， serum total cholesterol （TC）， triglyceride （TG）， creatinine （SCr）， and blood urea nitrogen （BUN） levels of the rats were measured. Western blot was used to detect the protein expression of JAK2/STAT3 signaling pathway. Immunohistochemistry was used to determine the protein expression of B-cell lymphoma 2 （Bcl-2）， Bcl-2-associated X protein （Bax）， zonula occludens-1 （ZO-1）， and actinin-4 in rat kidney tissues. Result:Compared with the normal group， the model group exhibited elevated UTP， serum TC， TG， BUN， and SCr levels （<italic>P</italic><0.05）， severe pathological damage of rat kidney tissues， up-regulated expression of phospho-JAK2 （p-JAK2）， phospho-STAT3 （p-STAT3）， and Bax， increased renal cell apoptosis， and diminished expression of Bcl-2， ZO-1， and actinin-4 （<italic>P</italic><0.05）. Compared with the model group， the Yiqiyangyin Huoxuetongluo prescription group and the irbesartan group showed dwindled UTP， serum TC， TG， BUN， and SCr levels （<italic>P</italic><0.05）， relieved pathological damage of rat kidney tissues， down-regulated p-JAK2， p-STAT3， and Bax expression， and up-regulated expression of Bcl-2， ZO-1， and actinin-4 （<italic>P</italic><0.05）. Conclusion:Yiqiyangyin Huoxuetongluo prescription can reduce renal cell apoptosis and improve the prognosis of DN by inhibiting the activation of JAK2/STAT3 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.

Effect of Modified Shengjiangsan on Oxidative Stress and Apoptosis in MN Rats via Regulating HIF-1α/NOX4 Signaling Pathway / 中国实验方剂学杂志

Objective:To investigate the intervention effect of modified Shengjiangsan on hypoxia-inducible factor-1<italic>α </italic>（HIF-1<italic>α</italic>）/nicotinamide adenine dinucleotide phosphate oxidase 4 （NOX4） signaling pathway in membranous nephropathy （MN） rats and to explore its mechanism to reduce oxidative stress and apoptosis in renal tissues. Method:Cationized bovine serum albumin （C-BSA） was injected into the tail vein of rats to replicate the MN model. Rats were randomly divided into a model group， a modified Shengjiangsan group， and a benazepril group after modeling， and administered by gavage once a day accordingly. At the end of the 4^th week， the 24-h urine total protein （UTP）， urea nitrogen （BUN）， and serum creatinine （SCr） levels of each group were detected. Enzyme-linked immunosorbent assay（ELISA）was used to detect the levels of superoxide dismutase （SOD）， malondialdehyde （MDA）， and reactive oxygen species （ROS） in renal tissues of rats. In situ end labeling（TUNEL） staining was used to detect the cell apoptosis rate. The mRNA and protein expression levels of HIF-1<italic>α</italic> and NOX4 were detected by real-time fluorescence-based quantitative polymerase chain reaction（Real-time PCR）and Western blot， respectively. The immunohistochemistry method was used to detect the protein expression levels of B-cell lymphomas -2 （Bcl-2）， B-cell lymphomas xl （Bcl-xl）， Bcl-2 associated X protein （Bax）， Bcl-2 cell death regulator antibody （Bim）. Result:Compared with the normal group， the model group showed increased UTP （<italic>P</italic><0.05）， decreased SOD， elevated MDA and ROS （<italic>P</italic><0.05）， up-regulated mRNA and protein expression of HIF-1<italic>α</italic> and NOX4 （<italic>P</italic><0.05）， enhanced protein expression of Bax and Bim， declining protein expression of Bcl-xl and Bcl-2 （<italic>P</italic><0.05）， and increased cell apoptosis in renal tissues. Compared with the model group， the modified Shengjiangsan group and the benazepril group displayed declining UTP （<italic>P</italic><0.05）， up-regulated SOD， decreased MDA and ROS （<italic>P</italic><0.05）， down-regulated mRNA and protein expression of HIF-1<italic>α</italic> and NOX4 （<italic>P</italic><0.05）， diminished protein expression of Bax and Bim， elevated protein expression of Bcl-xl and Bcl-2 （<italic>P</italic><0.05）， and reduced cell apoptosis in renal tissues （<italic>P</italic><0.05）. Conclusion:The protective effect of modified Shengjiangsan on the kidney is presumedly achieved by reducing the oxidative stress and apoptosis in renal tissues of MN rats via inhibiting the HIF-1<italic>α</italic>/NOX4 signaling pathway.

Effect of Yishen Tongluo Formula on kidney protection and podocyte skeleton related proteins in rats with membranous nephropathy / 中草药

Objective To explore the kidney protection and possible mechanism of Yishen Tongluo Formula (YTF) in rats with membranous nephropathy (MN). Methods A total of 60 SD healthy male rats were randomly divided into 10 for normal group and 50 for MN rat model group. The MN rat model was established by tail iv cationic bovine serum albumin (C-BSA). After successful modeling, they were randomly divided into model group, benazepril group, and YTF groups at low, medium, and high doses (6.61, 13.22, and 26.44 g/kg). Rats in each group were ig administrated once daily for continuous four weeks according to the corresponding dose. At the end of administration, the 24 h urine total protein (UTP), total cholesterol (TC), total protein (TP), albumin (ALB), blood urea nitrogen (BUN), and serum creatinine (Scr) levels were measured. Immunofluorescence was used to detect the deposition of IgG immune complexes in renal tissue. The glomerular basement membrane and podocyte morphology were observed under electron microscope. Immunohistochemistry and qRT-PCR were used to detect the expression of cytoskeleton-related proteins ezrin and synaptopodin in rat kidneys. Results Compared with the model group, the UTP and TC levels in the rats in each treatment group decreased significantly (P < 0.01), and the TP and ALB levels increased significantly (P < 0.01). The middle and high dose groups of YTF were similar to the benazepril group, and the effect was better than the low dose group of YTF. There was no significant difference in the BUN and Scr among the groups. Compared with the control group, the expression of ezrin and synaptopodin mRNA in the kidney of the model group was significantly decreased (P < 0.01). Compared with the model group, the expression of ezrin and synaptopodin mRNA in the podocytes of different treatment groups was increased in different degrees (P < 0.01). The expression levels of ezrin and synaptopodin mRNA in the kidney of rats in the middle and high dose groups of YTF were similar to those in the benazepril group, which were higher than that in the low dose group of YTF. Conclusion YTF has a therapeutic effect on membranous nephropathy in rats. The mechanism may be related to the inhibition of the degradation of podocyte skeleton related proteins ezrin and synaptopodin and the maintenance of the integrity of the podocyte skeleton and foot process.

Research progress on molecular mechanism of astragaloside IV in protecting kidney / 中草药

Astragaloside IV is the most aboundant and activitve ingredient in Astragalus membranaceus. Many studies have confirmed that astragaloside IV has a renal protective effect on chronic kidney disease. After reviewing the literatures at home and abroad for the past five years, The molecular mechanism of the protective effect of astragaloside IV on kidney was reviewed from the aspects of protection of podocytes, inhibition of renal fibrosis, protection of renal tubular cells, and inhibition of mesangial cell activation. The aim of this study is to analyze the target of astragaloside IV in the treatment of chronic kidney disease, and provide a basis for the application of astragaloside IV in nephropathy.

Genetic diversity of Paris polyphylla var. yunnanensis by SSR marker / 中草药

Objective: The genetic diversity of Paris polyphylla var. yunnanensis germplasmic resources in natural populations was analyzed by simple sequence repeat marker for protection and rational utilization. Methods: A total of 115 individuals of P. polyphylla var. yunnanensis collected from five natural populations were analyzed by SSR marker for genetic diversity analysis. Results: Eight pairs of SSR primers were screened. A relatively high level of genetic diversity was revealed: The percentage of polymorphic bands was 100%, the polymorphism information content was 0.745 6. At the population level and species level, the observed number of allele was 8.425 0 and 17.750 0, respectively, the effective number of allele was 4.960 9 and 7.500 7, respectively, the observed heterozygosity was 0.295 5 and 0.294 8, respectively, the expected heterozygosity was 0.654 8 and 0.774 4, respectively, and the Shannon's information index was 1.520 1 and 2.038 6, respectively. The genetic differentiation coefficient was 0.172 8 and the gene flow was 1.196 6. Based on UPGMA cluster analysis, the five populations of P. polyphylla var. yunnanensis were divided into two clades. Conclusion: The level of genetic diversity of P. polyphylla var. yunnanensis was relatively high and there was certain genetic differentiation within and among populations. This study puts forward several suggestions for the protection and development of P. polyphylla var. yunnanensis resources, which will be helpful for protection and sustainable utilization of scientific advice and reference.