Effect of Modified Shengjiangsan on Gd-IgA1 in IgA Nephropathy Rats： Based on C1GALT1/C1GALT1C1 Signaling Pathway / 中国实验方剂学杂志

ObjectiveThe effect of modified Shengjiangsan on immunoglobulin A （IgA） nephropathy was observed. The microRNA-148b （miRNA-148b）， interleukin 6 （IL-6）， core 1 beta 1，3-galactosyltransferase （C1GALT1）， molecular chaperone Cosmc （core1β3-Gal-T-specific molecular chaperone C1GALT1C1）， and galactose-deficient IgA1 （Gd-IGA1） in serum and kidney tissues of IgA nephropathy rats were detected to explore the underlying mechanism. The result is expected to lay a scientific basis for clinical application of modified Shengjiangsan in the treatment of IgA nephropathy. MethodA total of 42 SPF male SD rats were randomized into the normal group （8rats） and modeling group （34 rats） with the random number table method. After one week of adaptive feeding， rats for modeling were given bovine serum albumin （BSA， gavage）， lipopolysaccharide （LPS， injection into tail vein）， carbon tetrachloride （CCl4， subcutaneous injection）， and castor oil to induce IgA nephropathy. After modeling， two rats were randomly selected to test the modeling outcome. Then the model rats were classified into the model group， low-dose Chinese medicine group （modified Shengjiangsan，6.27 g·kg-1）， high-dose Chinese medicine group （modified Shengjiangsan，12.54 g·kg-1）， and benazepril group （10 mg·kg-1） with the random number table method， 8 in each group. The administration （gavage， once a day） lasted 4 weeks. The 24-h urinary total protein （24 h-UTP） was detected at the end of the 1st， 9th， and 13th week of the experiment. At the 14th week， after anesthesia， femoral artery blood was collected and centrifugated. The supernatant was collected to detect albumin （ALB）， aspartate aminotransferase （AST）， alanine aminotransferase （ALT）， serum creatinine （SCr）， and blood urea nitrogen （BUN）. The expression levels of IL-6 and Gd-IGA1 were determined by enzyme-linked immunosorbent assay （ELISA）. Based on hematoxylin-eosin （HE）/Masson/periodic Schiff-methenamine silver （PASM） staining， the pathological changes of renal tissues were observed. Ultrastructural changes of glomeruli were observed by transmission electron microscopy. The expression of miRNA-148b， IL-6， C1GALT1， and C1GALT1C1 was detected by immunohistochemistry. The mesangial area of the glomeruli was observed by immunofluorescence. Real-time polymerase chain reaction （Real-time PCR） was employed to determine the mRNA levels of mirNA-148b， IL-6， C1GALT1， and C1GALT1C1， and Western blot was used to detect the protein levels of IL-6， C1GALT1， and C1GALT1C1. ResultCompared with normal group， the model group showed increase in the content of 24 h-UTP， SCr， ALT， IL-6， and GD-IGA1 （P<0.05）， decrease in ALB content （P<0.05）. Moreover， rats in the model group demonstrated hyperplasia of glomerular mesangial cells， thickening of mesangial area， podocyte foot process effacement， and a large number of granular IgA immune complex in the mesangial area. In addition， the model group showed increase in the expression of IL-6 in mesangial area and podocytes， decrease in the expression of C1GALT1 and C1GALT1C1 in mesangial area and podocytes， enhanced expression of IL-6 mRNA and miRNA-148b （P<0.01）， weakened expression of C1GALT1 mRNA and C1GALT1C1 mRNA （P<0.01）， rise of IL-6 protein expression （P<0.01）， and reduction in the protein expression of C1GALT1 and C1GALT1C1 （P<0.01）. Compared with the model group， modified Shengjiangsan decreased the content of 24 h-UTP， SCr， ALT， IL-6， and Gd-IGA1 （P<0.05） and increased the content of ALB （P<0.05， P<0.01）. Moreover， with the treatment of this Chinese medicine， the pathological damage was significantly alleviated and the deposition of IgA immune complex in basement membrane was reduced. The expression of IL-6 in the mesangial area and podocytes of rats was decreased， and the expression of C1GALT1 and C1GALT1C1 in the mesangial area and podocytes of rats was increased. Moreover， the expression of IL-6 mRNA and miRNA-148b was decreased （P<0.01）， and the expression of C1GALT1 mRNA and C1GALT1C1 mRNA was increased （P<0.01）. The protein expression of IL-6 was decreased （P<0.05， P<0.01）， and the protein expression of C1GALT1 and C1GALT1C1 was enhanced （P<0.05， P<0.01）. The Chinese medicine group showed obvious dose-effect trend. ConclusionModified Shengjiangsan may reduce the expression of miRNA-148b and IL-6 in serum and kidney tissue of IgA nephropathy rats， restore the expression of C1GALT1 and C1GALT1C1， and decrease the generation of Gd-IGA1， so as to reduce renal pathological damage and proteinuria， protect the kidney protection， and finally delay the disease progression. Moreover， the effect is enhanced with the rise of dose.

Modified Shengjiangsan Inhibits Necroptosis by Mediating RIP1/RIP3/MLKL Signaling Pathway and Reduces Renal Fibrosis in Rats with Diabetic Nephropathy / 中国实验方剂学杂志

ObjectiveTo observe the mechanism of modified Shengjiangsan in necroptosis and renal fibrosis of rats with diabetic nephropathy based on receptor-interacting protein （RIP）1/RIP3/mixed lineage kinase domain-like protein （MLKL） signaling pathway. MethodSeventy-five SD rats were randomly divided into a model group， a normal group， three high， medium， and low-dose modified Shengjiangsan groups （4.365， 8.73， 17.46 g·kg-1）， and an irbesartan group （0.013 5 g·kg-1）. After 4 weeks of intragastric administration， the levels of 24 h urine protein （UTP）， tumor necrosis factor-α （TNF-α）， and interleukin-1β （IL-1β） of rats in each group were determined， as well as the changes in degree of renal pathology. Real-time quantitative polymerase chain reaction （Real-time PCR） and immunohistochemistry were used to detect the mRNA and protein expression levels of IL-1β， TNF-α， monocyte chemoattractant protein-1 （MCP-1）， transforming growth factor-β1 （TGF-β1）， and nuclear factor kappa B （NF-κB） in kidney tissues of rats. Western blot assay was used to detect the expression levels of key proteins in the RIP1/RIP3/MLKL signaling pathway. ResultAs compared with the normal group， the renal interstitial fibrosis in the model group was obvious， and the 24 h UTP， IL-1β， TNF-α levels were significantly increased （P<0.05）. In the model group， the mRNA and protein expression levels of IL-1β， TNF-α， MCP -1， TGF-β1， and NF-κB in the kidney tissues were significantly increased （P<0.05）， and protein expression levels of RIP1， RIP3， p-MLKL， and MLKL were significantly increased （P<0.05）. Compared with the model group， all modified Shengjiangsan groups and the irbesartan group improved the levels of renal interstitial fibrosis in rats to varying degrees. As compared with the model group， the 24 h UTP levels in all modified Shengjiangsan groups and the irbesartan group were decreased to varying degrees （P<0.05）， the content of IL-1β and TNF-α in the serum were decreased （P<0.05）， the mRNA and protein expression levels of IL-1β， TNF-α， MCP-1， TGF-β1， and NF-κB in renal tissues was down-regulated （P<0.05）， and the protein expression levels of RIP1， RIP3， p-MLKL， and MLKL were down-regulated （P<0.05）. ConclusionModified Shengjiangsan ameliorates renal injury of rats with diabetic nephropathy， and the mechanism may be related to the down-regulation of the RIP1/RIP3/MLKL signaling pathway， the prevention of renal tissue necroptosis， and the inhibition of renal fibrosis.

Mechanism of Modified Shengjiangsan in Treatment of IgA Nephropathy Based on Network Pharmacology， Molecular Docking， and Experimental Verification / 中国实验方剂学杂志

ObjectiveTo preliminarily predict the active components， targets， and signaling pathways of modified Shengjiangsan in the treatment of immunoglobulin A nephropathy （IgAN） based on network pharmacology， and to explore its underlying mechanism through molecular docking and experimental verification on animals. MethodThe active ingredients and related targets of modified Shengjiangsan were obtained from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform（TCMSP）， UniProt， SwissTargetPrediction， and literature review. IgAN-related targets were obtained from GeneCards and Online Mendelian Inheritance in Man （OMIM）. Cytoscape 3.9.0 was used to construct the regulation network of the related targets of Shengjiangsan and IgAN， and the protein-protein interaction （PPI） network was plotted by STRING. The common genes were analyzed for gene ontology （GO） functional annotation and Kyoto encyclopedia of genes and genomes（KEGG） pathway enrichment by Metascape. Key targets and main active ingredients were selected for molecular docking by AutoDockTools 1.5.6. The experimental model of IgAN was induced by bovine serum albumin（BSA， ig） combined with lipopolysaccharide （LPS， iv） and the complex of CCl4 and castor oil （sc） in rats. The model rats were treated with modified Shengjiangsan and benazepril hydrochloride for four weeks. The rats were sacrificed after drug administration. The levels of transforming growth factor-β1 （TGF-β1） and interleukin-6 （IL-6） in the serum and kidney tissues were detected by enzyme-linked immunosorbent assay（ELISA）， immunohistochemistry， Real-time quantitative polymerase chain reaction （Real-time PCR）， and Western blot. ResultA total of 105 active ingredients were obtained according to oral bioavailability（OB）， drug-likeness（DL）， and literature screening. There were 124 common genes and 59 core targets. Neurotrophic tyrosine receptor kinase 1 （NTRK1）， cullin-3 （CUL3）， tumor protein 53 （TP53）， epidermal growth factor receptor （EGFR）， exportin 1 （XPO1）， and other targets might be closely related to IgAN. As predicted by KEGG enrichment analysis， the treatment of IgAN with modified Shengjiangsan mainly involved the phosphoinositide 3-kinase （PI3K）/protein kinase B （Akt） signaling pathway， nuclear transcription factor-kappa B （NF-κB） signaling pathway， and cytokine-cytokine receptor interaction signaling pathway. As revealed by molecular docking， the main active ingredients in modified Shengjiangsan showed stable binding activities with NTRK1， CUL3， TP53， EGFR， and XPO1 in the core targets， indicating that it presumedly regulated inflammatory responses by affecting NTRK1， CUL3， TP53， EGFR， and XPO1 target proteins. The results of experimental verification on animals showed that the expression levels of cytokines TGF-β1 and IL-6 in the serum and kidney tissues of IgAN rats were significantly decreased by modified Shengjiangsan， suggesting that Shengjiangsan might inhibit excessive fibrosis， and inflammatory and immune responses by regulating signaling pathways such as cytokine-cytokine receptor interaction， PI3K/Akt， and NF-κB. ConclusionModified Shengjiangsan may treat IgAN through multiple targets and pathways. Its mechanism may be related to the inhibition of excessive fibrosis， and inflammatory and immune responses by affecting the expression of NTRK1， CUL3， TP53， EGFR， and XPO1 and the regulation of the cytokine-cytokine receptor interaction， PI3K/Akt， NF-κB， and other signaling pathways.

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 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 Modified Shengjiangsan on Notch Signal Pathway and Podocyte Apoptosis in MN Rats / 中国实验方剂学杂志

Objective:The purpose of this article was to observe the effect of modified Shengjiangsan on podocyte apoptosis in membranous nephropathy （MN） rats， to explore the molecular mechanism of its treatment of MN and to provide experimental basis for its clinical application. Method:The MN rat model was established by injection of cationic bovine serum albumin into the tail vein of rats. The successfully modeled rats were then randomly divided into model group （equal volume of normal saline）， modified Shengjiangsan group （27.3 g·kg^-1） and benazepril group （10 mg·kg^-1）， with corresponding drug dosage once a day for 4 weeks of continuous intervention. After drug administration， the 24-hour urine protein （UTP） was detected. Real time fluorescent quantitative polymerase chain reaction （Real-time PCR） and immunohistochemical （IHC） methods were used to detect Podocalyxin， Nephrin， Podocin， Synaptopodin mRNA and protein expression levels in rat kidney tissue. terninal-deoxynucleoitidyl transferase medsated nick and labeling （TUNEL） method was used to detect cell apoptosis rate in rat kidney tissue， and Western blot was used to detect Notch1， Hes1， B lymphoblastoma-2 （Bcl-2） associated X protein （Bax）， and Bcl-2 protein expression levels in rat kidney tissue. Result:Compared with the normal group， UTP in the model group increased significantly， renal tissue cell apoptosis increased significantly， podocyte marker proteins podocalyxin， Nephrin， Podocin， Synaptopodin mRNA and protein expression levels decreased significantly， and Notch1， Hes1， Bax protein expression increased significantly， and Bcl-2 protein expression was significantly reduced（<italic>P</italic><0.05）. Compared with the model group， UTP levels in MN rats were significantly reduced in modified Shengjiangsan and benazepril groups， with reduced rate of renal cell apoptosis， increased mRNA and protein expression levels of podocalyxin， Nephrin， Podocin， and Synaptopodin in renal tissue， decreased Notch1， Hes1， Bax protein expression， and increased Bcl-2 protein expression（<italic>P</italic><0.05）. Conclusion:Modified Shengjiangsan can inhibit the Notch signaling pathway， reduce the apoptosis of rat kidney tissue podocytes， and reduce the kidney injury of MN rats.

Effect of Modified Shengjiangsan Influences on PI3K/Akt/mTOR Signaling Pathway and Autophagy in Rats with Membranous Nephropathy / 中国实验方剂学杂志

Objective:To explore the intervention effect of modified Shengjiangsan in rats with membranous nephropathy (MN) and its related mechanism. 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, the levels of 24-hour urine protein (UTP), total cholesterol (TC), triglyceride (TG), total protein (TP), Albumin (Alb), creatinine (SCr), urea nitrogen (BUN) level was detected. we observed the pathological changes of rat kidneys by the technology of Masson staining, silver hexylamine iodate (PASM) staining and transmission electron microscopy. immunofluorescence technology was used to detect immunoglobulin （Ig）G deposition in rat kidneys. Western blot was used to detect the expression levels of key proteins in phosphatidylinositol 3-kinase/proline protein kinase B/rapamycin target protein (PI3K/Akt/mTOR) signaling pathway and autophagy marker proteins LC3 and Beclin1. Result:Compared with normal group, the UTP, serum TC and TG levels were significantly increased, TP and Alb levels were significantly reduced in model group(P<0.05). We detected the kidney pathological changes include of glomerulus enlargement, basement membrane thickening,vacuolar degeneration, pheotropin deposition, glomerular capillary loop IgG diffuse deposition, electron dense deposits of varying sizes and podocytes under the epithelium extensive integration of foot processes， the expression of p-PI3K, p-Akt and p-mTOR protein was significantly increased (P<0.05). The expression of autophagy marker proteins LC3 and Beclin1 protein decreased significantly(P<0.05). Compared with model group, the UTP, serum TC and TG levels were decreased in the benazepril group and modified Shengjiangsan group, and the TP and Alb levels were increased (P<0.05)， the histopathological changes of rat kidney were all reduced， the expression of p-PI3K, p-Akt and p-mTOR protein was significantly reduced(P<0.05)， autophagy marker proteins LC3 and Beclin1 protein expression were significantly increased. Conclusion:Modified Shengjiangsan can reduce urinary protein, reduce kidney pathological damage and delay disease progression, which is related to its inhibition of PI3K/Akt/mTOR signaling pathway and activation of renal autophagy.

Effect of Modified Shengjiangsan on Renal Protection for Membranous Nephropathy Rats and Expression of ROS / 中国实验方剂学杂志

Objective: To detect the effect of modified Shengjiangsan on the expression of reactive oxygen species(ROS) in mitochondria of renal foot cells of rats, in order to study the mechanism of modified Shengjiangsan. Method: After be fed for 7 days,the 60 SD male rats were randomly divided into four groups:blank control group, model group, positive medicine group and traditional Chinese medicine(TCM) treatment group. After establishment of the rat model of membranous nephropathy, model group, positive medicine group and TCM treatment group were treated differently. After 4 weeks, all of the rats were put to death, and the expressions of ROS, 24-hour urinary protein quantity,total cholesterol,triglyceride,total protein,albumin,urea nitrogen,creatinine were detected by Real-time fluorescence quantitative polymerase chain reaction Real-time PCR. Result: The expression of 24-hour urinary protein quantity,total cholesterol,triglyceride in positive medicine group and TCM treatment group were reduced,and the expressions of total protein,albumin in positive medicine group and TCM treatment group were reduced compared with those of model group (PPConclusion: Modified Shengjiangsan can effectively control the development of ROS in mitochondria of renal foot cells of rats, and repair the renal function of membranous nephropathy rats by recovering foot cells.