Meihua Yan

Efficacy and safety of tangshen formula on patients with type 2 diabetic kidney disease: a multicenter double-blinded randomized placebo-controlled trial.

Background Persons with diabetes are at high risk of developing diabetic kidney disease (DKD), which is associated with high morbidity and mortality. Current drug therapies for DKD, such as angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin receptor blockers (ARBs), are not entirely satisfactory. This study aimed to evaluate the additional benefit and safety of the Chinese herbal granule Tangshen Formula (TSF) in treating DKD. Methods The study was designed as a six-center randomized, double-blind, placebo-controlled trial. From April 2007 through December 2009, 180 patients with DKD were enrolled. In addition to conventional treatment with ACEIs or ARBs, 122 participants were randomly assigned to receive TSF and 58 participants to receive placebo for 24 weeks. Primary outcome was urinary protein level, measured by urinary albumin excretion rate (UAER) for participants with microalbuminuria, 24-hour urinary protein (24h UP) for participants with macroalbuminuria. Secondary outcomes included renal function, serum lipids, quality of life, symptoms, and adverse events. Findings After 24 weeks of treatment, no statistically significant difference in UAER (TSF −19.53 μg/min compared with placebo −7.01 μg/min, with a mean difference of −12.52 μg/min; 95%CI, −68.67 to 43.63, P = 0.696) was found between TSF and placebo groups. However, TSF displayed a statistically significant decrease in 24h UP (TSF−0.21 g compared with placebo 0.36 g, with a mean difference of −0.57g; 95%CI, −1.05 to −0.09, P = 0.024). Estimated glomerular filtration rate (eGFR) was improved in both patients with microalbuminuria and macroalbuminuria, with a mean difference of 15.51 ml/min/1.73 m2 (95%CI, 3.71 to 27.31), 9.01 ml/min/1.73 m2 (95%CI, −0.10 to 18.13), respectively. Other secondary outcomes showed no statistically significant difference between groups or in the incidence of adverse events. Conclusions Based on conventional treatments, TSF appears to provide additional benefits compared with placebo in decreasing proteinuria and improving eGFR in DKD patients with macroalbuminuria. Nevertheless, further study is needed to evaluate TSF treating patients with microalbuminuria. Trial Registration Chinese Clinical Trial Registry ChiCTR-TRC-10000843

Attenuation of diabetic nephropathy by Chaihuang-Yishen granule through anti-inflammatory mechanism in streptozotocin-induced rat model of diabetics.

ETHNOPHARMACOLOGICAL RELEVANCE Traditional Chinese medical herbs have been used in China for a long time to treat different diseases. Based on traditional Chinese medicine (TCM) principle, Chaihuang-Yishen granule (CHYS) was developed and has been employed clinically to treat chronic kidney disease including diabetic nephropathy (DN). The present study was designed to investigate its mechanism of action in treatment of DN. MATERIALS AND METHODS Diabetic rats were established by having a right uninephrectomy plus a single intraperitoneal injection of STZ. Rats were divided into four groups of sham, diabetes, diabetes with CHYS and diabetes with fosinopril. CHYS and fosinopril were given to rats by gavage for 20 weeks. Samples from blood, urine and kidney were collected for biochemical, histological, immunohistochemical and molecular analyses. RESULTS Rats treated with CHYS showed reduced 24h urinary protein excretion, decreased serum TC and TG levels, but CHYS treatment did not affect blood glucose level. Glomerular mesangial expansion and tubulointerstitial fibrosis in diabetic rats were significantly alleviated by CHYS treatment. Moreover, CHYS administration markedly reduced mRNA levels of NF-κB p65 and TGF-β1, as well as decreased protein levels of NF-κB p65, MCP-1, TNF-α and TGF-β1 in the kidney of diabetic rats. CONCLUSIONS CHYS ameliorates renal injury in diabetic rats through reduction of inflammatory cytokines and their intracellular signaling.

Renoprotective effect of berberine on type 2 diabetic nephropathy in rats.

Inflammation, fibrosis, and lipid disorder are essential promoters in the pathogenesis of diabetic kidney injury in diabetes mellitus type 2. Berberine (BBR) has been reported to have beneficial effects on diabetic nephropathy, but its action mechanism is still unclear. The present study was designed to elucidate the therapeutic mechanism of BBR in a type 2 diabetic nephropathy rat model induced by a high‐fat diet and low‐dose streptozotocin injection. The diabetic rats were treated with or without BBR by gavage for 20 weeks and examined by serology, 24‐h albuminuria, histology, immunohistochemistry, and molecular analyses. Results showed that treatment with BBR significantly reduced serum levels of blood glucose and lipids, inhibited urinary excretion of albumin, and attenuated renal histological injuries in diabetic rats. Berberine treatment also inhibited renal inflammation, which was associated with inactivation of nuclear factor kappa‐light‐chain‐enhancer of activated B‐cell signalling. As a result, the upregulation of pro‐inflammatory cytokines (interleukin‐1β, tumour necrosis factor‐α) and chemokine (monocyte chemotactic protein‐1) was blocked. In addition, BBR treatment also inactivated transforming growth factor‐β/Smad3 signalling and suppressed renal fibrosis, including expression of fibronectin, collagen I, and collagen IV. The present study reveals that BBR is a therapeutic agent for attenuating type 2 diabetic nephropathy by inhibiting nuclear factor kappa‐light‐chain‐enhancer of activated B cell‐driven renal inflammation and transforming growth factor‐β/Smad3 signalling pathway.

Therapeutic Effects of Tangshen Formula on Diabetic Nephropathy in Rats

Objective Inflammation and fibrosis are essential promoters in the pathogenesis of diabetic nephropathy (DN) in type 2 diabetes. The present study examined the anti-inflammation and anti-fibrosis effect of Tangshen Formula (TSF), a traditional Chinese medicine, on DN. Research Design and Methods Protective role of TSF in DN was examined in a rat model of type 2 DN that was established by high-fat diet-fed and low-dose-streptozotocin injection. TSF was suspended in 0.5% CMC-Na solution and delivered by oral gavage at a dosage of 1.67g/Kg body weight/day. The therapeutic effects and mechanisms of TSF on diabetic kidney injury were examined. Results We found that TSF treatment for 20 weeks attenuated DN by significantly inhibiting urinary excretion of albumin and renal histological injuries. These beneficial effects were associated with an inactivation of NF-κB signaling, thereby blocking the upregulation of pro-inflammatory cytokines (IL-1β, TNFα), chemokine (MCP-1), and macrophage infiltration in the TSF-treated rats with type 2 DN. In addition, TSF treatment also inactivated TGF-β/Smad3 signaling and therefore suppressed renal fibrosis including expressions of fibronectin, collagen I, and collagen IV. Further studies revealed that the inhibitory effect of TSF on TGF-β/Smad3 and NF-κB signaling in DN was associated with inhibition of Smurf2-dependent ubiquitin degradation of Smad7. Conclusions The present study reveals that TSF has therapeutic potential for type 2 DN in rats. Blockade of NF-κB-driven renal inflammation and TGF-β/Smad3-mediated renal fibrosis by preventing the Smurf2-mediated Smad7 degradation pathway may be mechanisms through which TSF inhibits type 2 DN.

Tangshen formula attenuates hepatic steatosis by inhibiting hepatic lipogenesis and augmenting fatty acid oxidation in db/db mice

Tangshen formula (TSF), a well-prescribed traditional Chinese formula, has been used in the treatment of diabetic nephropathy. However, whether TSF ameliorates dyslipidemia and liver injury associated with diabetes remains unclear. In this study, we examined the effects of TSF on lipid profiles and hepatic steatosis in db/db mice. For this purpose, 8-week-old db/db mice were treated with TSF or saline for 12 weeks via gavage and db/m mice were used as controls. Body weight and blood glucose levels were monitored weekly and bi-weekly, respectively. Blood samples were obtained for the analysis of lipids and enzymes related to hepatic function, and liver tissues were analyzed by histology, immunohistochemistry and molecular examination. The results revealed that TSF markedly reduced body weight, liver index [liver/body weight (LW/BW)] and improved lipid profiles, hepatic function and steatosis in db/db mice. TSF induced the phosphoralation of AMP-activated protein kinase and inhibited the activity of sterol regulatory element-binding protein 1 together with the inhibition of the expression of genes involved in de novo lipogenesis (DNL) and gluconeogenesis, such as fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC), stearoyl CoA desaturase 1 (SCD1), glucose-6-phosphatase (G6pc) and phosphoenolpyruvate carboxykinase 1 (Pck1). Additionally, the silent mating type information regulation 2 homolog 1 (Sirt1)/peroxisome proliferator-activated receptor α (PPARα)/malonyl-CoA decarboxylase (MLYCD) cascade was potently activated by TSF in the liver and skeletal muscle of db/db mice, which led to enhanced fatty acid oxidation. These findings demonstrated that TSF attenuated hepatic fat accumulation and steatosis in db/db mice by inhibiting lipogenesis and augmenting fatty acid oxidation.

Tangshen formula attenuates diabetic renal injuries by upregulating autophagy via inhibition of PLZF expression

The Chinese herbal granule Tangshen Formula (TSF) has been proven to decrease proteinuria and improve estimated glomerular filtration rate (eGFR) in diabetic kidney disease (DKD) patients. However, the underlying mechanism of TSF on treatment of diabetic nephropathy (DN) remains unclear. The present study aimed to identify the therapeutic target of TSF in diabetic renal injuries through microarray-based gene expression profiling and establish its underlying mechanism. TSF treatment significantly attenuated diabetic renal injuries by inhibiting urinary excretion of albumin and renal histological injuries in diabetic (db/db) mice. We found that PLZF might be the molecular target of TSF in DN. In vivo, the db/db mice showed a significant increase in renal protein expression of PLZF and collagen III, and decrease in renal autophagy levels (downregulated LC3 II and upregulated p62/SQSTM1) compared to db/m mice. The application of TSF resulted in the downregulation of PLZF and collagen III and upregulation of autophagy level in the kidneys of db/db mice. In vitro, TSF reduced high glucose (HG)-induced cell proliferation for NRK52E cells. Further studies indicated that the exposure of NRK52E cells to high levels of glucose resulted in the downregulation of cellular autophagy and upregulation of collagen III protein, which was reversed by TSF treatment by decreasing PLZF expression. In conclusion, TSF might have induced cellular autophagy by inhibiting PLZF expression, which in turn resulted in an increase in autophagic degradation of collagen III that attenuated diabetic renal injuries.

Transcriptional Profile of Kidney from Type 2 Diabetic db/db Mice

Diabetic nephropathy (DN), a common diabetic microvascular complication, is characterized by progressive glomerular sclerosis and tubulointerstitial fibrosis. However, the underlying mechanisms involved in DN remain to be elucidated. We explored changes in the transcriptional profile in spontaneous type 2 diabetic db/db mice by using the cDNA microarray. Compared with control db/m mice, the db/db mice exhibited marked increases in body weight, kidney weight, and urinary albumin excretion. Renal histological analysis revealed mesangial expansion and thickness of the basement membrane in the kidney of the db/db mice. A total of 355 differentially expressed genes (DEGs) were identified by microarray analysis. Pathway enrichment analysis suggested that biological oxidation, bile acid metabolism, and steroid hormone synthesis were the 3 major significant pathways. The top 10 hub genes were selected from the constructed PPI network of DEGs, including Ccnb2 and Nr1i2, which remained largely unclear in DN. We believe that our study can help elucidate the molecular mechanisms underlying DN.

The decreased expression of electron transfer flavoprotein β is associated with tubular cell apoptosis in diabetic nephropathy

Tubular injury is closely correlated with the development of progressive diabetic nephropathy (DN), particularly in cases of type 2 diabetes. The apoptosis of tubular cells has been recognized as a major cause of tubular atrophy, followed by tubulointerstitial fibrosis. Electron transfer flavoprotein β (ETFβ) is known as an important electron acceptor in energy metabolism, but its role in DN was not fully understood. In the present study, we examined the expression pattern of ETFβ using diabetic kidney samples and further investigated ETFβ involvement in tubular epithelial cell (TEC) apoptosis. Human renal biopsy specimens from patients with DN as well as a spontaneous rat model of diabetes using Otsuka Long-Evans Tokushima fatty (OLETF) rats, were employed in order to examine the expression of ETFβ and cell apoptosis in kidneys during the development of DN (for the rats, at 36 and 56 weeks of age respectively). Moreover, ETFβ siRNA was used to investigate the role of ETFβ in the apoptosis of renal tubular cells. Our present results showed that the expression of ETFβ in the kidneys was progressively decreased both in patients with DN and OLETF rats, which coincided with progressive renal injury and TEC apoptosis. In addition, the in vitro study demonstrated that knockdown of ETFβ caused apoptosis in tubular cells, as proven by the increased expression of pro-apoptotic proteins and TUNEL assay. Therefore, the findings of our present study suggest that ETFβ plays an important role in renal tubular cell apoptosis during the progression of DN.

Tangshen Formula Attenuates Diabetic Nephropathy by Promoting ABCA1-Mediated Renal Cholesterol Efflux in db/db Mice

The commonly prescribed Tangshen Formula (TSF) is a traditional Chinese formulation that has been shown to reduce plasma lipid metabolism and proteinuria and improve the estimated glomerular filtration rate (eGFR) in patients with diabetic kidney disease. This study investigated the underlying mechanism whereby TSF regulates renal lipid accumulation and ameliorates diabetic renal injuries in spontaneous diabetic db/db mice and in vitro in sodium palmitate (PA)-stimulated and Abca1-SiRNA-transfected mouse tubular epithelial cells (mTECs). The results revealed that TSF treatment significantly ameliorated the renal injuries by lowering urinary albumin excretion and improving renal tissue injuries in diabetic (db/db) mice. Interestingly, the treatment with TSF also resulted in decreased cholesterol levels in the renal tissues of db/db mice, which was associated with increased expression of the peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α), the Liver X receptors (LXR), and ATP-binding cassette subfamily A member 1 (ABCA1), suggesting that TSF might attenuate diabetic kidney injury via a mechanism associated with improving cholesterol efflux in the diabetic kidney. This was investigated in vitro in mTECs, and the results showed that TSF reduced the PA-stimulated cholesterol accumulation in mTECs. Mechanistically, the addition of TSF was capable of reversing PA-induced downregulation of PGC-1α, LXR, and ABCA1 expression and cholesterol accumulation in mTECs, suggesting that TSF might act the protection via the PGC-1α-LXR-ABCA1 pathway to improve the cholesterol efflux in the renal tissues of db/db mice. This was further confirmed by silencing ABCA1 to block the promotive effect of TSF on cholesterol efflux in vitro. In conclusion, TSF might ameliorate diabetic kidney injuries by promoting ABCA1-mediated renal cholesterol efflux.

Association of EPHX2 R287Q Polymorphism with Diabetic Nephropathy in Chinese Type 2 Diabetic Patients

The aim of this study was to investigate the relationship between EPHX2 rs751141 (R287Q polymorphism) and diabetic nephropathy (DN) in Chinese type 2 diabetes (T2D). This case-control study explored the association between EPHX2 rs751141 and DN in a total of 870 Chinese T2D patients (406 T2D patients with DN and 464 T2D patients without DN). DNA was extracted from peripheral leukocytes of the patients and rs751141 was genotyped. The A allele frequency of rs751141 was significantly lower in DN patients (20.94%) compared with non-DN controls (27.8%) (P = 0.001), and the A allele of rs751141 was associated with a significantly lower risk of DN after adjustment for multiple covariates in the additive genetic model (OR = 0.68, 95% CI = 0.52–0.88, P = 0.004). Significant association between rs751141 and homocysteine (Hcy) level on the risk of DN was observed, indicating that in patients with the highest Hcy levels, the A allele showed marked association with lower risk of DN in all three genetic models. In conclusion, the A allele of exonic polymorphism in EPHX2 rs751141 is negatively associated with the incidence of DN in the Chinese T2D population, which could be modulated by Hcy level status.