ObjectiveTo observe the effect of Buyang Huanwutang on ferroptosis in diabetic kidney disease （DKD） mice and explore the protective effect and mechanism of Buyang Huanwutang on the kidneys of DKD mice. MethodSeventy-three C57BL/6 mice were randomly divided into model group （63 mice） and normal group （10 mice）. After the model group mice were fed with high-sugar and high-fat diets for six weeks， they were injected with streptozotocin （STZ） of 50 mg·kg^-1 intraperitoneally for five days for preparing the diabetes model and then fed with high-sugar and high-fat diets for eight weeks. When the mice showed positive urine protein， the DKD model was successfully prepared. DKD mice were randomly divided into model group （n = 10）， rosiglitazone （7.05 × 10^-4 g·kg^-1） group （n = 9）， Buyang Huanwutang low-dose （3.21 g·kg^-1） group （n = 9）， middle-dose （6.41 g·kg^-1） group （n = 10）， and high-dose （12.82 g·kg^-1） group （n = 10） for gavage. The normal group and model group were given the same volume of normal saline once a day for eight weeks. Fasting blood glucose （FBG）， 24 h urinary protein （24 h-UTP）， and renal weight index （KI） were measured after administration. Hematoxylin-eosin （HE） staining， Periodic acid Schiff （PAS） staining， and Masson staining were used to observe the pathological changes in mouse kidneys. Western blot was used to detect the protein expressions of long-chain acyl-CoA synthetase 4 （ACSL4）， member 11 of solute carrier family 7 （SLC7A11）， glutathione peroxidase 4 （GPX4）， ferritin heavy chain （FTH-1）， and transferrin receptor 1 （TFR-1） in mouse kidneys. The activities of glutathione （GSH） and contents of malondialdehyde （MDA） and 4-hydroxynonenal （4-HNE） were measured. The expression level of reactive oxygen species （ROS） in mouse kidneys was detected by fluorescence probe labeling. ResultCompared with the normal group， KI， FBG， and 24 h-UTP in the model group increased significantly， and mesangium in the glomerulus proliferated. The basement membrane thickened， and glycogen particles were deposited around the glomerulus. FTH-1 expression decreased， while TFR-1 and ROS expressions increased. MDA and 4-HNE increased， but GSH activity decreased. ACSL4 expression increased， and SLC7A11 and GPX4 expressions decreased （P<0.01）. Compared with the model group， in Buyang Huanwutang and rosiglitazone groups， KI and 24 h-UTP decreased， and FBG showed a downward trend， but there was no statistical significance. Pathological damage of kidney tissue was improved to different degrees， FTH-1 expression increased， and TFR-1 and ROS expressions decreased. MDA and 4-HNE contents decreased， and GSH activity increased. ACSL4 expression decreased， and SLC7A11 and GPX4 expressions increased （P<0.05， P<0.01）. ConclusionBuyang Huanwutang can alleviate the pathological damage of kidney tissue in DKD mice， and its mechanism is related to the regulation of ferroptosis.

ObjectiveTo observe the effect of serum containing Buyang Huanwutang on podocyte injury induced by high glucose in mice， and to explore its potential mechanism. MethodsMouse podocytes （MPC5） were cultured in vitro， and the optimal intervention concentration and time of serum containing Buyang Huanwutang were screened. Cells were divided into normal group （5.5 mmol·L^-1 glucose）， isotonic group （5.5 mmol·L^-1 glucose+24.5 mmol·L^-1 Mannitol）， high glucose group （30 mmol·L^-1 glucose）， blank serum group （30 mmol·L^-1 glucose+20% blank serum） and Buyang Huanwutang（30 mmol·L^-1 glucose+10% serum containing）. FerroOrange fluorescent probe was used to detect the level of iron （Fe²⁺） in cells. The level of reactive oxygen species （ROS） in cells was detected by Hoechst staining. The levels of glutathione （GSH） and malondialdehyde （MDA） in cells were detected by the kit. Enzyme-linked immunosorbent assay （ELISA） was used to determine the level of 4-hydroxynonenal （4-HNE）. Western blot was used to detect Desmin， podocyte hole membrane protein （Nephrin）， Podocalyxin， long-chain acyl-CoA synthetase 4（ACSL4）， member 11 of solute carrier family 7 （SLC7A11）， glutathione peroxidase 4（GPX4）， nuclear transcription factor E₂-related factor 2（Nrf2） and heme oxygenase -1（HO-1）. The mRNA expression levels of Desmin， Nephrin， Podocalyxin， ACSL4， SLC7A11， GPX4， Nrf2 and HO-1 were detected by real-time fluorescence quantitative polymerase chain reaction. ResultsCompared with the normal control group， the expressions of Desmin and ACSL4， the levels of Fe²⁺ and ROS， and the contents of MDA and 4-HNE in the high glucose group increased significantly， and the expressions of GSH， Nephrin， Podocalyxin， SLC7A11， GPX4， Nrf2 and HO-1 decreased（P<0.05， P<0.01）. There was no significant change in the above results in isotonic group. Compared with the high glucose group， the expressions of Desmin and ACSL4， the levels of Fe²⁺ and ROS， and the contents of MDA and 4-HNE in Buyang Huanwutang-containing serum group and Fer-1 group decreased significantly， and the expressions of GSH， Nephrin， Podocalyxin， SLC7A11， GPX4， Nrf2 and HO-1 increased（P<0.05， P<0.01）. There was no significant change in the above results in blank serum group. ConclusionBuyang Huanwutang medicated serum can alleviate podocyte injury induced by high glucose， and its mechanism is related to regulating Nrf2/HO-1 pathway and inhibiting ferroptosis.

ObjectiveTo observe the effects of Danggui Buxuetang on the mitochondrial fission and apoptosis of podocytes in the rat model of diabetic kidney disease （DKD） and to explore the protective effect and mechanism of Danggui Buxuetang on DKD rats. MethodSD rats were randomized into a modeling group （n=65， fed with a high-sugar and high-fat diet） and a normal group （n=10， fed with a normal diet）. After 6 weeks， the modeled rats were injected intraperitoneally with streptozotocin （STZ） for the modeling of type 2 diabetes mellitus （T2DM）. Sixty T2DM rats were randomized into model， irbesartan （0.014 g·kg^-1）， and low-， medium-， and high-dose （0.36， 0.72， 1.44 g·kg^-1， respectively） Danggui Buxuetang groups and administrated with corresponding drugs by gavage for 16 weeks. The levels of fasting blood glucose （FBG） and 24 h urinary protein （24 h UTP） were determined at the end of the 16th week. The pathological changes of the renal tissue were observed by hematoxylin-eosin and Masson staining. The mitochondrial ultrastructure of rat podocytes was observed by transmission electron microscopy. The level of reactive oxygen species （ROS） in the renal tissue of rats was measured by the fluorescent probe labeling of dihydroethidium （DHE）. The apoptosis of renal cells was detected by terminal-deoxynucleotidyl transferase-mediated dUTP nick end labeling （TUNEL）. The expression levels of Synaptopodin， Podocin， and cleaved caspase-3 in renal podocytes were detected by the immunohistochemical method （IHC）. Western blot was employed to determine the protein levels of A-kinase anchoring protein 1 （AKAP1）， phosphorylated dynamin-related protein 1 （p-Drp1）， mitofusin-2 （Mfn2）， B-cell lymphoma-2 （Bcl-2）， and Bcl-2-associated X protein （Bax）. ResultCompared with the control group， the model group showed elevated levels of FBG and 24 h UTP， mesangial hyperplasia， basement membrane thickening， mitochondrial swelling， mitochondrial crista breakage and disorder， and increased ROS and TUNEL-positive cells. In addition， the model group showcased down-regulated expression of Synaptopodin and Podocin， increased expression of cleaved Caspase-3， up-regulated protein levels of AKAP1 and p-Drp1 ， and down-regulated protein levels of Mfn2 and Bcl-2/Bax （P<0.01）. Compared with the model group， high-dose Danggui Buxuetang lowered the levels of FBG and 24 h UTP， alleviated the pathological injuries of the renal tissue and the mitochondrial injury of podocytes， decreased ROS and TUNEL-positive cells， promoted the expression of Synaptopodin and Podocin， inhibited the expression of cleaved Caspase-3， down-regulated the protein levels of AKAP1 and p-Drp1， and up-regulated the protein levels of Mfn2 and Bcl-2/Bax （P<0.05， P<0.01）. ConclusionDanggui Buxuetang may inhibit mitochondrial fission and apoptosis of podocytes and reduce urine protein by regulating the AKAP1/Drp1 pathway， thereby delaying the progression of DKD.

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 1^st， 9^th， and 13^th 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.

ObjectiveTo explore the preparation method of a rat model of adenine-induced chronic renal failure complicated with cardiovascular disease by investigating the effect of different time points of adenine gastric lavage on general vital signs， biochemical indicators， and cardiac and renal tissue structure and function of model rats. MethodRats in the model group were administered adenine at 150 mg·kg^-1·d^-1 by gavage for 16 weeks， while those in the normal group were given an equal volume of 0.5% carboxymethyl cellulose sodium solution by gavage. At weeks 5， 13， 17， 24-hour urinary protein quantification （24 h-UTP）， biochemical indicators， cardiac ultrasound， and changes in cardiac and renal tissue structure and function were measured in both the model and normal groups. Blood pressure was measured at weeks 5 and 13 in both groups. Weekly changes in body weight were recorded， and general conditions of the rats were observed daily. Result① Compared with the normal group， the model group showed a significant decrease in body weight （P<0.05）. ② Rats in the model group exhibited a significant increase in urine volume， and proteinuria appeared at week 13. ③ Compared with the normal group， the model group showed significant differences in triglyceride （TG）， total cholesterol （TC）， creatinine （Cr）， blood urea nitrogen （BUN）， blood potassium， and blood phosphorus at week 5 （P<0.05）， which increased gradually over time. At week 17， uric acid levels were significantly elevated （P<0.05）， and blood calcium levels were reduced at the end of week 17 （P<0.01）. ④ Compared with the normal group， the model group showed a significant increase in blood pressure at week 5 （P<0.05）， which progressively worsened. ⑤ There was no statistically significant difference in left ventricular wall thickness between the model and normal groups at week 5， but a significant difference was observed at week 13 （P<0.05）. ⑥ Fibrosis appeared in the kidneys of rats in the model group at week 5 and gradually worsened， while obvious fibrosis occurred around the cardiovascular system at week 13 as compared with the results in the normal group. ⑦ In the proximal tubular epithelial cells of the model group， there was an increasing presence of high-density rhomboid needle-shaped crystals， damaged cell membrane integrity， increased cell spacing， increased lysosomes， increased mitochondrial proliferation， denser mitochondrial cristae， and outer mitochondrial membrane. ⑧ Compared with the rats in the normal group， rats in the model group exhibited depressed spirits， significantly reduced activity， hunched posture， dry fur， pale ears and toes， swollen cheeks， increased nocturnal urination， and dark and viscous blood. ConclusionAdenine by gavage at 150 mg·kg^-1·d^-1 for 12 weeks can be used to establish a rat model of chronic renal failure complicated with cardiovascular disease， which can be used for the prevention and treatment research on chronic renal failure and its associated cardiovascular complications. The syndrome of adenine-induced rat model of chronic renal failure belongs to the deficiency of spleen and kidney， turbidity and stasis obstruction， and can be used to study the mechanisms of warming and tonifying the spleen and kidney， resolving stasis， and eliminating turbidity in the treatment of chronic renal failure.

ObjectiveTo explore the underlying mechanism of modified Zhenwutang in delaying renal interstitial fibrosis in chronic renal failure （CRF） by observing the effects of modified Zhenwutang on the expression of angiotensin Ⅱ （Ang Ⅱ）， angiotensin Ⅱ type 1 receptor （AT1R）， nicotinamide adenine dinucleotide phosphate （NADPH） oxidase 4 （NOX4）， transforming growth factor-β₁ （TGF-β₁）， type I collagen （COL1A1）， and type Ⅲ collagen （COL3A1） in the serum and renal tissues of adenine-induced CRF rats. MethodFifty male SPF-grade SD rats were randomly divided into a normal group （n=10） and an experimental group （n=40） using a random number table. After one week of adaptive feeding， the experimental CRF model was established in rats by administering adenine at 150 mg·kg^-1·d^-1 orally. Three rats from each group were randomly selected to evaluate the model induction. After successful modeling， rats in the experimental group were randomly divided into a model group， low-， medium， and high-dose modified Zhenwutang groups， and a benazepril hydrochloride group， with six rats in each group. The rats were orally administered the corresponding drugs once daily for four weeks. At the end of the first week， 13^th week， and 17^th week of the experiment， 24 hour urinary protein quantification （24 h-UTP） was measured. At the end of the 17th week， the rats were euthanized， and blood samples were collected from the abdominal aorta for the measurement of total protein （TP）， albumin （ALB）， creatinine （Cr）， and blood urea nitrogen （BUN） in the serum. Enzyme-linked immunosorbent assay （ELISA） was used to measure the expression levels of serum Ang Ⅱ. Hematoxylin-eosin （HE） staining and Masson's trichrome staining were performed to observe the pathological changes in renal tissues. Immunohistochemistry （IHC） was performed to observe the expression of AT1R， NOX4， TGF-β₁， COL1A1， and COL3A1. Real-time fluorescence-based quantitative polymerase chain reaction （Real-time PCR） was used to observe the mRNA expression levels of AT1R， NOX4， and TGF-β₁. Western blot was conducted to measure the protein expression levels of AT1R， NOX4， and TGF-β₁. Result① Compared with the normal group， the model group showed a significant increase in 24 h-UTP （P<0.01）. The levels of Cr and BUN in the model group were significantly higher （P<0.01）， while the levels of TP and ALB were significantly lower （P<0.01）. The serum Ang Ⅱ level in the model group was significantly elevated （P<0.01）. The model group exhibited widening of the renal glomerular mesangial space， necrotic glomeruli， increased interstitial width with extensive inflammatory cell infiltration， brownish precipitates blocking the renal tubular lumens， irregular renal tubules， and significant deposition of collagen fibers in the renal interstitium. Additionally， the collagen fibers around the renal vessels， outside the parietal layer of the renal sacs， glomerular basement membrane， and tubular basement membrane increased significantly. The expression of AT1R and NOX4 in the glomeruli and renal tubules of the model group was significantly enhanced， and TGF-β₁ expression also significantly increased in the renal tubules. The expression of COL1A1 and COL3A1 in the renal interstitium significantly increased. The mRNA expression of AT1R and TGF-β₁ in the model group significantly increased （P<0.01）， while NOX4 mRNA expression significantly decreased （P<0.01）. The protein expression of AT1R， NOX4， and TGF-β₁ was significantly enhanced （P<0.01）. ② Compared with the model group， modified Zhenwutang significantly reduced 24h-UTP （P<0.01）， decreased levels of Cr and BUN （P<0.01）， increased levels of TP and ALB （P<0.01）， reduced serum Ang Ⅱ level （P<0.01）， alleviated renal pathological damage， reduced expression of AT1R， NOX4， TGF-β₁， COL1A1， and COL3A1 in the glomeruli， renal tubules， and renal interstitium， reduced mRNA expression of AT1R and TGF-β₁ （P<0.01）， increased NOX4 mRNA expression （P<0.01）， and weakened protein expression of AT1R， NOX4， and TGF-β₁ （P<0.01）. The modified Zhenwutang groups showed a significant dose-effect trend. ConclusionModified Zhenwutang may delay renal interstitial fibrosis in CRF rats by reducing the expression of Ang Ⅱ， AT1R， NOX4， and TGF-β₁ in the serum and renal tissues， thereby alleviating renal pathological damage， reducing proteinuria， protecting renal function， and delaying the progression of CRF. The modified Zhenwutang group exhibited a dose-effect trend.

ObjectiveBy observing the effect of modified Zhenwutang on the expression of superoxide dismutase 1（SOD1）， malondialdehyde（MDA）， advanced oxidation protein product（AOPP）， nuclear factor kappa-B（NF-κB） p65，p-p65，IL-1β， TNF-α in serum and renal tissue of adenine-induced chronic renal failure rats and the pathology of heart and kidney tissue， the possible mechanism of modified Zhenwutang delaying the progression of chronic renal failure complicated with heart disease was discussed. MethodFifty SPF male SD rats were divided into normal group 10 and model group 40 according to the random number table method. After one week of adaptive feeding， the experimental chronic renal failure complicated with cardiovascular disease rat model was established by intragastric administration of adenine 150 mg·kg^-1·d^-1. After the model was completed， 3 rats in the normal group and the model group were randomly selected to detect whether the model was successful. After successful modeling， the rats in the model group were divided into model group ， modified Zhenwutang low-dose group ， modified Zhenwutang medium-dose group， modified Zhenwutang high-dose group and Benazepril hydrochloride group according to the random number table method， with 6 rats in each group. Drugs were administered once a day for 4 weeks. At the end of the 17^th week of the experiment， 24-hour urinary total protein（24 h-UTP） and urine creatinine（UCr）were detected. At the end of the 17^th week， the rats in each group were anesthetized and the abdominal aorta was taken. After centrifugation， the supernatant was taken to detect triglyceride（TG）， total cholesterol（TC）， serum calcium（Ca）， serum potassium， serum phosphate， serum creatinine（Scr）， blood urea nitrogen（BUN）； the expression levels of serum AOPP， IL-1β and TNF-α were detected by enzyme linked immunosorbent assay（ELISA）. The pathological changes of heart and kidney tissues were observed by hematoxylin-eosin（HE）/Masson method. The ultrastructural changes of proximal renal tubules were observed by transmission electron microscopy . The kidney tissue expressions of SOD1， MDA， AOPP， NF-κB p65，p-p65，IL-1β and TNF-α were observed by immunohistochemistry. The kidney tissue expression levels of SOD1， NF-κB p65， IL-1β and TNF-α mRNA were observed by real-time polymerase chain reaction（Real-time PCR）. The kidney tissue expression levels of SOD1， MDA， NF-κB p65 and p-p65 were detected by Western blot. Result①Compared with the normal group， the experimental rats in the model group showed an increase in 24-hour UTP （P<0.01）and a decrease in UCr（P<0.01）. The experimental rats in the model group showed an increase in Cr， BUN， TG， TC， serum phosphate， and serum potassium（P<0.01）.The levels of AOPP， IL-1β and TNF-α in serum of rats in the model group were significantly increased（P<0.01）. In the model group， the glomerular balloon space was significantly widened， the renal interstitium was significantly widened with a large number of inflammatory cell infiltration， a large number of renal tubular lumens were blocked by brown deposits， and there were a large number of collagen fiber deposition in the renal interstitium. The collagen fibers around the renal vessels， outside the capsule wall of the renal capsule wall， glomerular basement membrane and renal tubular basement membrane were significantly increased， and the cardiac muscle fibers were significantly thickened. There was a small amount of inflammatory cell infiltration around the blood vessels， and a large number of collagen fibers around the cardiac vessels and between the myocardial cells. In the model group， high-density diamond-shaped needle-like crystals were observed in the proximal renal tubular epithelial cells of rats， with increased lysosomes， mitochondrial proliferation， mitochondrial cristae and dense mitochondrial outer membrane. The left ventricular diastolic wall thickness and systolic wall thickness of the experimental rats in the model group was increased in proximal renal tubular epithelial cells and their nuclei.In the model group， the expression of MDA， AOPP， NF-κB p65，p-p65 IL-1β and TNF-α in proximal renal tubular epithelial cells was significantly increased（P<0.01）， the expression of p-p65 in the nucleus of proximal renal tubular epithelial cells was significantly increased（P<0.01）， and the expression of SOD1 in proximal renal tubular epithelial cells was significantly decreased（P<0.01）. The kidney tissue expression of NF-κB p65， IL-1β and TNF-α mRNA in the model group was increased（P<0.01）， and the expression of SOD1 mRNA was decreased（P<0.01）. The kidney tissue expression of SOD1 protein in the model group was significantly decreased（P<0.01）. The kidney tissue expression of MDA， NF-κB p65 and p-p65 protein was increased （P<0.01）. ② Compared with the model group， after the intervention of modified Zhenwutang， 24 h-UTP was decreased （P<0.01）and UCr was increased（P<0.01）. Cr， BUN， TG， TC， serum phosphate， serum potassium was decreased （P<0.01）. Serum AOPP， IL-1β and TNF-α levels were decreased（P<0.01）. Cardiac and Renal pathological damage was reduced； mitochondrial damage in proximal renal tubules was reduced； the expression of MDA， AOPP， NF-κB p65， IL-1β， TNF-α in proximal renal tubular epithelial cells was decreased （P<0.01）， the expression of p-p65 in the nucleus of proximal renal tubular epithelial cells was significantly decreased （P<0.01）， and the expression of SOD1 in proximal renal tubular epithelial cells was significantly increased （P<0.01）. The kidney tissue expression of NF-κB p65， IL-1β， TNF-α mRNA was decreased （P<0.01）， and the expression of SOD1 mRNA was increased（P<0.01）. The kidney tissue expression of SOD1 protein was significantly increased （P<0.01）， and the expression of MDA， NF-κB p65 and p-p65 protein was decreased （P<0.01）. The Chinese medicine group showed a significant dose-effect trend. ConclusionModified Zhenwutang may reduce the production of oxidative stress and mitochondrial damage in proximal renal tubular epithelial cells， thereby reducing oxidative stress products and inhibiting the release of inflammatory factors caused by the activation of NF-κB signaling pathway， reducing the damage to heart and kidney tissues and functions， and delaying the progression of chronic renal failure complicated with heart disease， and the traditional Chinese medicine group has a dose-effect trend.

ObjectiveTo preliminarily predict the active ingredients， targets， and signaling pathways of modified Zhenwutang in the treatment of chronic renal failure （CRF） based on network pharmacology and explore its potential mechanism for delaying disease progression through molecular docking and animal experiments. MethodThe effective ingredients and targets of modified Zhenwutang were obtained from the HERB database. The targets related to CRF were obtained from the GeneCards. The intersection target genes were obtained using Venny 2.1 software and a protein-protein interaction （PPI） network was constructed using the STRING. The core targets for treating CRF with modified Zhenwutang were screened using Cytoscape 3.9.1 software. The intersection genes were analyzed using Metascape database for gene ontology （GO） analysis and Kyoto Encyclopedia of Genes and Genomes （KEGG） enrichment analysis. Molecular docking validation was performed using AutoDockTools 1.5.6 software for the key targets and active ingredients. An experimental CRF model was established in rats by administering adenine via gavage for 12 weeks， followed by intervention with modified Zhenwutang and benazepril hydrochloride for four weeks. After treatment， the rats were euthanized， and immunohistochemistry （IHC）， immunofluorescence （IF）， real-time quantitative polymerase chain reaction （Real-time PCR）， and western blot were performed to detect the expression levels of prolyl hydroxylase domain-containing proteins 1 （PHD1）， prolyl hydroxylase domain-containing proteins 2 （PHD2）， hypoxia-inducible factor-1α （HIF-1α）， and α-smooth muscle actin （α-SMA） in the renal tissues of the rats. ResultA total of 426 drug target genes of modified Zhenwutang were obtained from the HERB database. A total of 2 698 target genes related to CRF were obtained from the GeneCards database. There were 154 intersection genes between the drug and the disease. Eight core targets were identified， including albumin （ALB）， protein kinase B1 （Akt1）， tumor necrosis factor （TNF）， interleukin-6 （IL-6）， insulin （INS）， vascular endothelial growth factor A （VEGFA）， tumor protein p53 （TP53）， and interleukin-1β （IL-1β）， which might be closely related to the treatment of CRF with modified Zhenwutang. KEGG enrichment analysis predicted that the main mechanism of modified Zhenwutang in treating CRF involved lipid and atherosclerosis， HIF-1 signaling pathway， cell apoptosis， and nuclear factor kappa B （NF-κB） signaling pathway. Molecular docking results showed that the ingredients of modified Zhenwutang had stable binding activity with the core targets ALB， Akt1， TNF， IL-6， INS， VEGFA， TP53， and IL-1β， which may regulate inflammation and cell apoptosis by affecting the target proteins. The animal model validation results demonstrated that modified Zhenwutang could reduce the expression levels of HIF-1α and α-SMA in the renal tissues of CRF rats， increase the expression levels of PHD1 and PHD2， alleviate renal tissue hypoxia injury， reduce myofibroblast formation， and slow down the progression of CRF in rats. ConclusionModified Zhenwutang may improve renal tissue hypoxia， inhibit cell transdifferentiation， cell apoptosis/necroptosis， and inflammation by affecting the expression of target proteins such as ALB， Akt1， TNF， IL-6， INS， VEGFA， TP53， and IL-1β， as well as regulating the HIF-1 signaling pathway， thus delaying the progression of CRF.

ObjectiveTo observe the modulatory effect of modified Zhenwutang on the interleukin-6 （IL-6）， matrix metallopeptidase-9（MMP-9）， type Ⅳ collagen（COL-Ⅳ） in rats with chronic renal failure （CRF） and to investigate the potential mechanism of its treatment of CRF. MethodFifty male SD rats were randomly divided into a modeling group of 40 rats and a normal group of 10 rats， and the modeling group was prepared by continuous adenine gavage for 12 weeks. After successful modelling， the modelling group was divided into the model group， the low dose （7.2 g·kg^-1·d^-1） group， the medium dose （14.4 g·kg^-1·d^-1） group， the high dose （28.8 g·kg^-1·d^-1） group and the Benadryl hydrochloride （10 mg·kg^-1·d^-1） group for gavage according to the random number table method， In the normal group and the model group， equal volume of distilled water was administered by gavage for 4 weeks. After the administration， the levels of blood creatinine （SCr）， blood urea nitrogen （BUN） and 24 h urine protein （24 h-UTP） were measured， the levels of serum IL-6 were measured by enzyme linked immunosorbent assay（ELISA）. Immunohistochemistry （IHC） was used to detect intercellular cell adhesion molecule-1 （ICAM-1）， IL-6， MMP-9， and other molecules in the rat kidney. The expression of ICAM-1 mRNA， IL-6 mRNA， MMP-9 mRNA and COL-Ⅳ mRNA in rat kidney tissues was measured by Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）. The expression levels of ICAM-1， IL-6， MMP-9 and COL-Ⅳ in rat kidney tissues were measured by Western blot. ResultCompared with the normal group， the levels of SCr， BUN and 24 h-UTP were significantly increased in the model group （P<0.01）； the serum IL-6 level was significantly increased （P<0.01）， the tubular lumen was dilated with atrophy， the tubular epithelial cells were necrotic， swollen and vacuolated， the interstitium was infiltrated by a large number of inflammatory cells and collagen fibers were deposited， the levels of IL-6， ICAM-1 and COL-Ⅳ were strongly positive in the tubular interstitium of the model group （P<0.01）， The levels of ICAM-1 mRNA， IL-6 mRNA and COL-Ⅳ mRNA were significantly increased （P<0.01） and MMP-9 mRNA was significantly decreased （P<0.05） in the model rats. ICAM-1， IL-6 and COL-Ⅳ protein expression was significantly increased （P<0.01） and MMP-9 protein expression was significantly increased （P<0.01） in the renal tissue， and MMP-9 protein expression was significantly decreased （P<0.01）. Compared with the model group， the 24 h-UTP， SCr and BUN levels of rats were significantly reduced after treatment with modified Zhenwutang （P<0.01）， the serum IL-6 level was significantly decreased （P<0.01）， the renal lesions of rats were significantly improved and collagen fiber deposition was reduced； the expression of IL-6， ICAM-1 and COL-Ⅳ in renal tubules and interstitium was weakened， and MMP-9 in ICAM-1 mRNA， IL-6 mRNA and COL-Ⅳ mRNA levels were significantly reduced （P<0.01） and MMP-9 mRNA levels were significantly increased （P<0.05）， ICAM-1， IL-6 and COL-Ⅳ protein expression was significantly reduced （P<0.01） and MMP-9 protein expression was significantly The expression of ICAM-1， IL-6 and COL-Ⅳ proteins was significantly decreased （P<0.01） and MMP-9 protein expression was significantly increased （P<0.01）. ConclusionModified Zhenwutang may regulate the IL-6/MMP-9/COL-Ⅳ signaling pathway， thereby reducing proteinuria， improving renal function， reducing renal pathological damage and delaying the progression of CRF interstitial fibrosis.

Different fragments of SARS-CoV-2 nucleocapsid (N) protein were expressed and purified, and a fluorescence immunochromatography method for detection of SARS-CoV-2 total antibody was established. The effect of different protein fragments on the performance of the method was evaluated. The N protein sequence was analyzed by bioinformatics technology, expressed in prokaryotic cell and purified by metal ion affinity chromatography column. Different N protein fragments were prepared for comparison. EDC reaction was used to label fluorescence microsphere on the synthesized antigen to construct sandwich fluorescence chromatography antibody detection assay, and the performance was systemically evaluated. Among the 4 prepared N protein fragments, the full-length N protein (N419) was selected as the optimized coating antigen, N412 with 0.5 mol/L NaCl was used as the optimal combination; deleting 91-120 amino acids from the N-terminal of N412 reduced non-specific signal by 87.5%. the linear range of detection was 0.312-80 U/L, the limit of detection was 0.165 U/L, and the accuracy was more than 95%. A fluorescence immunochromatographic detection method for analysis of SARS-CoV-2 total antibody was established by pairing N protein fragments. The detection result achieved 98% concordance with the commercially available Guangzhou Wanfu test strip, which is expected to be used as a supplementary approach for detection of SARS-CoV-2. The assay could also provide experimental reference for improving the performance of COVID-19 antibody detection reagents.
Antibodies, Viral ; COVID-19 ; Chromatography, Affinity ; Fluorescent Antibody Technique ; Humans ; Microspheres ; SARS-CoV-2 ; Sensitivity and Specificity