CONTEXT:Qi-dan-dihuang decoction (QDD) has been used to treat diabetic kidney disease (DKD), but the underlying mechanisms are poorly understood. OBJECTIVE:This study reveals the mechanism by which QDD ameliorates DKD. MATERIALS AND METHODS:The compounds in QDD were identified by high-performance liquid chromatography and quadrupole-time-of-flight tandem mass spectrometry (HPLC-Q-TOF-MS). Key targets and signaling pathways were screened through bioinformatics. Nondiabetic Lepr db/m mice were used as control group, while Lepr db/db mice were divided into model group, dapagliflozin group, 1% QDD-low (QDD-L), and 2% QDD-high (QDD-H) group. After 12 weeks of administration, 24 h urinary protein, serum creatinine, and blood urea nitrogen levels were detected. Kidney tissues damage and fibrosis were evaluated by pathological staining. In addition, 30 mmol/L glucose-treated HK-2 and NRK-52E cells to induce DKD model. Cell activity and migration capacity as well as protein expression levels were evaluated. RESULTS:A total of 46 key target genes were identified. Functional enrichment analyses showed that key target genes were significantly enriched in the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) and mitogen-activated protein kinase (MAPK) signaling pathways. In addition, in vivo and in vitro experiments confirmed that QDD ameliorated renal fibrosis in diabetic mice by resolving inflammation and inhibiting the epithelial-mesenchymal transition (EMT) via the p38MAPK and AKT-mammalian target of rapamycin (mTOR) pathways. DISCUSSION AND CONCLUSION:QDD inhibits EMT and the inflammatory response through the p38MAPK and AKT/mTOR signaling pathways, thereby playing a protective role in renal fibrosis in DKD.

聂晓莉教授长期从事肾脏病中医药防治研究,认为气血营卫不调是肿瘤患者基本病理状态,湿浊瘀毒痹阻肾络是铂类药急性肾损伤发病及进展的关键病机.总结出益气泄浊活血法治疗铂类化疗药物肾损伤的学术思想,研创出益肾泄浊方,收效甚佳,为中医药有效防治铂类药物肾损伤提供理论及实践依据.

Ethnopharmacological relevance: Yi-Shen-Xie-Zhuo formula (YSXZF) is a traditional Chinese medicine prescription developed from the classic prescription Mulizexie powder documented in the book of Golden Chamber Synopsis and the Buyanghuanwu Decoction recorded in the book of Correction of Errors in Medical Classics. According to our years of clinical experience, YSXZF can effectively improve qi deficiency and blood stasis in kidney disease. However, its mechanisms need further clarification.Aim of the study: Apoptosis and inflammation play key roles in acute kidney disease (AKI). The Yi-Shen-Xie-Zhuo formula, consisting of four herbs, is commonly used for treating renal disease. However, the underlying mechanism and bioactive components remain unexplored. This study aimed to investigate the protective effects of YSXZF against apoptosis and inflammation in a cisplatin-treated mouse model, and identify the main bioactive components of YSXZF. Materials and methods: C57BL/6 mice were administered cisplatin (15 mg/kg) with or without YSXZF (11.375 or 22.75 g/kg/d). HKC-8 cells were treated with cisplatin (20 mu M) with or without YSXZF (5% or 10%) for 24 h. Renal function, morphology, and cell damage were evaluated. UHPLC-MS was used to analyze the herbal components and metabolites in the YSXZF-containing serum. Results: Blood urea nitrogen (BUN), serum creatinine, serum and urine neutrophil gelatinase-associated lipocalin (NGAL) levels were clearly increased in the cisplatin-treated group. Administration of YSXZF reversed these changes; it improved renal histology, downregulated kidney injury molecule 1 (KIM-1) expression, and lowered the number of TdT-mediated dUTP-biotin nick end labeling (TUNEL)-positive cells. YSXZF significantly downregulated cleaved caspase-3 and BAX, and upregulated BCL-2 proteins in renal tissues. YSXZF suppressed increase in cGAS/STING activation and inflammation. In vitro treatment with YSXZF markedly reduced cisplatininduced HKC-8 cell apoptosis, relieved cGAS/STING activation and inflammation, improved mitochondrial membrane potential (MMP), and lowered reactive oxygen species (ROS) overgeneration. Small RNA interference (siRNA)-mediated silencing of cGAS or STING inhibited the protective effects of YSXZF. Twenty-three bioactive constituents from the YSXZF-containing serum were identified as key components.Conclusion: This is the first study to demonstrate that YSXZF protects against AKI by suppressing inflammation and apoptosis via the cGAS/STING signaling pathway.

Background: Although cisplatin (DDP) is an important clinical anti-tumor drug, its use is limited by its nephrotoxicity. How to avoid the renal injury incurred by platinum drugs and improve the clinical efficiency of platinum drugs use has become an urgent clinical problem. Previous studies have verified that Chinese medicine has definite effects on acute kidney injury (AKI). Yishen Xiezhuo formula (YSXZ) is a traditional Chinese medicine (TCM) compound which is an effective clinical drug for AKI, but its mechanism remains unclear. Methods: In our research, an AKI model was induced by DDP in human renal tubular epithelial cell (HKC) lines in the in vitro study. The mechanism of the YSXZ on cell senescence was analyzed by Cell Counting Kit-8 (CCK-8), senescence-associated beta-galactosidase (SA-beta-Gal) staining, western blot, flow cytometry, and enzyme-linked immunosorbent assay (ELISA). Network pharmacology was used to analyze the role of YSXZ against AKI. Results: Compared with the control group, the cells in the DDP intervention group were significantly senescent. Compared with DDP group, YSXZ decreased the number of SA-beta-Gal-positive senescence cells, down regulated the expression of senescence-related proteins, reduced the release of senescence-related secreted phenotypic factors, and reversed the phenomenon of cell cycle S-phase arrest. Network pharmacology and experimental studies showed that the mitogen-activated protein kinase (MAPK) signaling pathway played a central role. Conclusions: Our present results suggested that YSXZ ameliorated the development of DDP-induced AKI by attenuating renal tubular epithelial cell (RTEC) senescence via alleviating the activation of MAPK pathway.

Background Triptolide (PG490), as a triterpene dicyclic oxide has been reported to increase the generation of reactive oxygen species (ROS) and nitric oxide (NO) and induce apoptosis of RAW 264.7 cells in a dose-dependent manner. The activity of death NETs plays an important role in anti-bacterial processes in the human body. This study aimed to investigate the effect of triptolide (PG490) on neutrophil extracellular traps (NETs) formation. Methods After isolating peripheral blood neutrophils from healthy volunteers, cells were incubated with PG490 to observe and detect the level of NETs and detect the level of reactive oxygen species (ROS). The cells were cultured, stained and analyzed by fluorescence microscopy. Results Compared with the 12-myristate-13-acetate (PMA) group, the average fluorescence intensity of SYTOX Green in the PG490 + PMA group, as detected by a multifunctional microplate reader, was significantly decreased. Intracellular ROS were labeled by fluorescence, with fluorescence intensity then measured by multifunctional microplate reader and flow cytometry. The results showed that compared with the control group, the fluorescence intensity of the PMA group was significantly increased, while there was no significant difference between PMA group and PG490 + PMA group. Conclusions The production of NETs is inhibited by PG490 in vitro, which is not associated with the level of cellular ROS. This suggests that PG490in Tripterygium wilfordii Hook F can suppress related diseases.

目的 探讨小乌桂汤治疗类风湿关节炎(RA)的作用机制.方法 60只雄性DBA/1小鼠随机分为正常组8只及造模组52只.造模组采用尾根部及背部分3点皮内注射鸡Ⅱ型胶原蛋白乳化液建立胶原诱导性关节炎(CIA)小鼠模型.造模成功的40只小鼠随机分成模型组、甲氨蝶呤组、小乌桂汤低剂量组、小乌桂汤中剂量组、小乌桂汤高剂量组,每组8只.甲氨蝶呤组予0.1 mg/ml甲氨蝶呤药液2mg/kg灌胃,每周1次;小乌桂汤低、中、高剂量组分别予含生药19.5、39、75g/ (kg·d)小乌桂汤灌胃,每日1次;正常组与模型组给予生理盐水0.2 ml/10 g灌胃,每日1次.4周后评定关节炎指数评分,测量发病足厚度,观察踝关节组织病理并进行组织病理学评分;检测脾脏辅助性T细胞17 (Th17)、调节性T细胞(Treg)及血浆肿瘤坏死因子α(TNF-α)、白细胞介素6(IL-6)、白细胞介素17(IL-17)、白细胞介素1β(IL-1β)、白细胞介素10 (IL-10)的水平.结果 与模型组比较,甲氨蝶呤组及小乌桂汤中、高剂量组关节炎指数评分,发病足厚度,脾脏Th17细胞水平,血浆TNF-α、IL-6、IL-17、IL-1β水平,关节病理总评分,血管翳及骨破坏评分均显著降低,Treg细胞水平均显著升高,甲氨蝶呤组及小乌桂汤高剂量组Th17/Treg值、滑膜炎评分显著降低(P ＜0.05或P＜0.01).与甲氨蝶呤组比较,小乌桂汤低剂量组脾脏Th17细胞水平、Th17/Treg值、关节病理总评分、血管翳及骨破坏评分显著升高,小乌桂汤低、中剂量组Treg细胞水平显著降低(P＜0.01).与小乌桂汤低剂量组比较,小乌桂汤中、高剂量组Th17细胞水平,Th17/Treg值显著降低,Treg细胞水平显著升高,小乌桂汤高剂量组病理总评分、血管翳评分显著降低(P＜0.05或P＜0.01).与小乌桂汤中剂量组比较,小乌桂汤高剂量组Treg细胞水平显著升高(P＜0.01).结论 小乌桂汤对CIA小鼠具有抗炎及免疫调节作用,以高剂量效果最佳,其作用机制与调节Th17/Treg的平衡,抑制TNF-α、IL-6、IL-17、IL-1β炎症因子的表达相关.

Background: Qi-Dan-Dihuang Decoction (QDD) has been used for treating diabetic kidney disease (DKD), but the mechanisms are poorly understood. The aim of this study is to reveal the therapeutic effects and the mechanism of QDD in ameliorating DKD by network pharmacology, in vivo, and in vitro studies.Methods: The effect of QDD on body weight, fast blood glucose, oral glucose tolerance test (OGTT), 24 h urinary protein (24hU-Pro), serum creatinine (Scr), blood urea nitrogen (BUN), and pathological evaluation in kidney were investigated in vivo using C57BLKS/J db/db mice. The main active compounds of QDD, compound-disease interaction targets, and related processes and pathways were discerned by network pharmacology analysis through Chinese Medicine Systems Pharmacology Database (TCMSP) and TCM Database@Taiwan. The protein-protein interaction (PPI) network were established through STRING database. GO and KEGG pathway were used for analysis processes and pathways. Then Western blot was used to verified the predicted results. Finally, cell viability, wound healing and mainly pathway protein expression were detected in vitro using renal tubular epithelial cells HK-2 and NRK-52E cells.Results: Although QDD treatment showed no significant difference in FBG and AUC of OGTT, but had significant reduction in Scr level in C57BLKS/J db/db mice. Histopathologic results showed that QDD ameliorated the expansion of mesangial area, thickened membranes of Bowman’s capsules and basement membrane of glomerular capillaries, renal tubular epithelial cells vacuolar degeneration and reversed the glomerular and tubulointerstitial in C57BLKS/J db/db mice. For network pharmacology analysis of QDD, 143 active compounds related to 274 possible targets in QDD obtained and 117 compound-disease interaction targets were screened out combining with Genecards database. 18 key targets was excavated through network topological analysis. GO and KEGG pathway enrichment analysis showed that compound-disease interaction targets were significantly enriched in processes and pathways that are closely related to DKD. Western blot results showed that QDD significantly attenuated EMT-related proteins, p-NF-κb, IL-1β, IL-18, p-p38MAPK/p38MAPK, p-AKT/AKT, and p-mTOR/ mTOR protein expressions. Treatment with QDD could alleviate cell viability damaged, EMT process, p-NF-κb, IL-1β, IL-18, p-p38MAPK/p38MAPK, p-AKT/AKT and p-mTOR/ mTOR protein expressions by high D-gulcose.Conclusions: This study provides convincing evidence suggest that QDD protects renal fibrosis of DKD, by regulating EMT in RTECs and inflammatory response through p38MAPK and AKT/mTOR signaling pathways.

OBJECTIVE:To explore the mechanism of Xiaowugui decoction (XWGD) decoction in treating rheumatoid arthritis (RA) in mice.METHODS:Healthy male DBA/1 mice were used for CIA modeling. Twenty-five CIA mice with successful modeling and similar arthritis index (AI) scores were randomized equally into model group (CIA), methotrexate (MTX) group, and low-, medium-, and high-dose XWGD groups (0.975, 1.95, and 3.9 g/mL, respectively), with another 5 normal mice as the normal control group. The mice in normal control and CIA groups were given saline once a day, those in MTX group were given 0.1 mg/mL MTX once a week, and those in XWGD groups were treated daily via garage of XWGD containing crude drugs of different doses for 28 consecutive days. The AI score and HE staining were used to evaluate the changes in the joints of the CIA mice. The effect of XWGD on Th1, Th17, MDSC, G-MDSC and M-MDSC cells were evaluated with flow cytometry.RESULTS:Treatment with MTX and different doses of XWGD significantly decreased the AI score of the mice and relieved joint inflammation as compared with the model group (P < 0.05), and a higher dose of XWGD decoction produced a stronger therapeutic effect. Compared with those in CIA model group, the mice in MTX and XWGD treatment groups showed significantly decreased percentages of Th1, Th17 and M-MDSC cells in the spleen and increased percentages of G-MDSC cells (P < 0.01), and these changes were more conspicuous with a higher dose of XWGD. Correlation analysis showed that Th1 and Th17 cells were positively correlated with M-MDSC and negatively correlated with G-MDSC cells (P < 0.01).CONCLUSIONS:XWGD can improve joint inflammation in CIA mice by increasing the percentages of G-MDSC cells and decreasing the percentages of M-MDSC, Th1 and Th17 cells, and a high dose of XWGD can produce an equivalent therapeutic effect to methotrexate but with better safety.

Background: To study the protective effect of berberine (BBR) on cisplatin-induced acute kidney injury (AKI) and its effect on mitophagy. Methods: ( I) Male C57BL/6 mice aged 6-8 weeks were randomly divided into control group (saline), cisplatin group (cisplatin), and cisplatin + BBR (5, 10 mg/kg) groups. In the cisplatin group and BBR groups, mice were injected intraperitoneally with 15 mg/kg of cisplatin. Mice in BBR groups were given BBR at 72, 48, 24, 0.5 h before and 24, 48 h after cisplatin injection. Mice were sacrificed 72 h after cisplatin injection, and blood were collected for detecting serum creatinine (SCr) and blood urea nitrogen ( BUN) levels. Kidneys were collected for detecting protein expression levels of Kidney injury molecule 1 (KIM-1), LC3 II/LC3 I, p62, PINK 1, Parkin in the renal tissue by Western blotting. The pathological changes in renal tissues were observed using periodic acid-Schiff (PAS) staining. (II) Renal tubular epithelial cells (RTECs) were pretreated with different concentrations (1, 2, and 4 mu M) of BBR, and then incubated with cisplatin. Changes in autophagy proteins LC3 II/LC3 I, p62, PINK 1, and Parkin were detected by Western blotting, and changes in cellular reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) were detected by flow cytometry. Results: (I) Mice treated with BBR at dosage of 5 and 10 mg/kg for 6 days showed significant reduction in SCr and BUN compared to that in mice treated with cisplatin. PAS staining and immunohistochemistry showed that BBR ameliorated cisplatin-induced nephrotoxicity and reduced cisplatin-induced increase in protein expression levels of KIM-1. Compared to cisplatin-treated mice, the mice treated with BBR showed increased LC3 II/LC3 I, PINK 1, and Parkin, and decreased p62 protein expression. (II) Compared to cisplatin-incubated RTECs, cells pretreated with BBR for 24 h exhibited increased protein expressions of LC3 II/LC3 I, PINK1, and Parkin and decreased protein expression of p62. BBR reversed cellular ROS and cell MMP level induced by cisplatin. Conclusions: BBR plays a protective role in cisplatin-induced AKI by up-regulating mitophagy in RTECs.

OBJECTIVE:To investigate the effects of total glucosides of paeony (TGP) on the proliferation and activation-induced cell death of mouse T cells and the mechanism underlying the immunosuppressive effects of TGP.METHODS:Purified total T cells isolated from the spleen of C57BL/6 mice were treated with TGP at 0, 50, 100, or 200 μg/mL and stimulated by anti-CD3/ CD28. Flow cytometry was performed to detect the cell death and the proliferation of CFSE-labeled T cells. The expression of Fas/FasL mRNA was detected using RT-PCR, and flow cytometry was used to analyze the expression of Fas/FasL proteins on activated T cells. Western blotting was used to detect the expression of Bcl-2 in the cells.RESULTS:TGP treatment for 48 h significantly reduced the total number and percentage of viable T cells and dose-dependently lowered the percentage of divided T cells. TGP treatment obviously up-regulated the cellular expression of Fas mRNA, enhanced Fas expression on the surface of the T cells, and decreased the expression level of Bcl-2 protein in the cells.CONCLUSIONS:TGP significantly inhibits proliferation and promotes activation-induced cell death of mouse T cell by increasing the expression of Fas and downregulating the expression of Bcl-2.

Excessive degradation of the cartilage articular extracellular matrix (ECM) in chondrocytes has been considered as an important pathological characteristics of OA. In the present study, we demonstrate that the G protein-coupled receptor GPR39 is expressed on SW1353 chondrocytes and is significantly downregulated in response to advanced glycation end products (AGEs). Our findings show that agonism of GPR39 exerts significant protective effects against AGE-induced degradation of articular extracellular matrix. Agonism of GPR39 rescued degradation of type II collagen by decreasing expression of the collagen-degrading enzymes matrix metalloproteinase (MMP)-3 and MMP-13. Additionally, agonism of GPR39 rescued AGE-induced suppression of tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2. Agonism of GPR39 prevented degradation of aggrecan by downregulating AGE-induced expression of a disintegrin and metalloproteinase with type I thrombospondin motif (ADAMTS)-4 and ADAMTS-5. Finally, we demonstrate that the effects of GPR39 are mediated through the p38 mitogen activated protein kinase (MAPK)/nuclear factor-κB (NF-κB) cellular signaling pathway. Taken together, our findings show for the first time that targeted therapies involving GPR39 may provide a novel approach for the prevention and treatment of osteoarthritis.

目的 探讨广东人群亚健康状态与中医体质的相关性.方法 于2012-2013年对24 159人(12 ～80岁)开展大样本、多中心的横断面调查;健康状况的判定主要结合被调查者的体检报告及《亚健康评定量表》,中医体质的评估使用《中医体质分类与判定表》.结果 亚健康状态的现患率为46.0％(11 121/24 159),疾病为35.2％(8 505/24 159).偏颇体质在亚健康状态和疾病状态中所占的比例比较高,而平和质在健康人群中所占的比例最高.多元回归分析显示中医体质对健康状态有显著影响(P＜0.001),与偏颇体质比较,平和质与亚健康危险降低显著相关(OR 0.23,95％CI:0.21-0.25,P＜o.oo1);与疾病危险降低也显著相关(OR 0.11,95％CI:0.10-0.12,P＜0.001).偏颇体质与亚健康状态危险增加密切相关(P＜ 0.001),如气虚质OR 10.44 (95％CI:8.86-12.30)、阴虚质OR 4.31 (95％CI:3.71-5.01)、阳虚质OR 3.51 (95％CI:3.02-4.08)、湿热质OR 4.11 (95％CI:3.58-4.73)、痰湿质OR 7.19 (95％CI:5.88-8.81)、气郁质OR 21.17 (95％CI:15.74-28.45)、血瘀质OR 7.27(95％CI:5.71-9.25)、特禀质OR 3.68(95％CI:2.90-4.67).结论 亚健康状态与中医体质密切相关,偏颇体质是亚健康的危险因素,平和质是亚健康的保护因素.