Previous studies have shown that high blood glucose-induced chronic microinflammation can cause inflammatory podocyte injury in patients with diabetic kidney disease(DKD). Therein, necroptosis is a new form of podocyte death that is closely associated with renal fibrosis(RF). To explore the effects and mechanisms in vivo of total flavones of Abelmoschus manihot(TFA), an extract from traditional Chinese herbal medicine Abelmoschus manihot for treating kidney diseases, on podocyte necroptosis and RF in DKD, and to further reveal its scientific connotation with multi-pathway and multi-target, the authors randomly divided all rats into four groups: a namely normal group, a model group, a TFA group and a rapamycin(RAP) group. After the modified DKD rat models were successfully established, four group rats were given double-distilled water, TFA suspension and RAP suspension, respectively by gavage every day. At the end of the 4th week of drug treatment, all rats were sacrificed, and the samples of their urine, blood and kidneys were collected. And then, the various indicators related to podocyte necroptosis and RF in the DKD model rats were observed, detected and analyzed, respectively. The results indicated that, general condition, body weight(BW), serum creatinine(Scr), urinary albumin(UAlb), and kidney hypertrophy index(KHI) in these modified DKD model rats were both improved by TFA and RAP. Indicators of RF, including glomerular histomorphological characteristics, fibronectin(FN) and collagen type Ⅰ(collagen Ⅰ) staining extent in glomeruli, as well as the protein expression levels of FN, collagen Ⅰ, transforming growth factor-β1(TGF-β1) and Smad2/3 in the kidneys were improved respectively by TFA and RAP. Podocyte damage, including foot process form and the protein expression levels of podocin and CD2AP in the kidneys was improved by TFA and RAP. In addition, tumor necrosis factor-α(TNF-α)-mediated podocyte necroptosis in the kidneys, including the morphological characteristics of podocyte necroptosis, the extent and levels of the protein expression of TNF-α and phosphorylated mixed lineage kinase domain like pseudokinase(p-MLKL) was improved respectively by TFA and RAP. Among them, RAP had the better effect on p-MLKL. More importantly, the activation of the receptor interacting serine/threonine protein kinase 1(RIPK1)/RIPK3/MLKL signaling axis in the kidneys, including the expression levels of its key signaling molecules, such as phosphorylated receptor interacting serine/threonine protein kinase 1(p-RIPK1), p-RIPK3, p-MLKL and cysteinyl aspartate specific proteinase-8(caspase-8) was improved respectively by TFA and RAP. Among them, the effect of TFA on p-RIPK1 was superior. On the whole, in this study, the authors demonstrated that TFA alleviates podocyte necroptosis and RF in DKD through inhibiting the activation of the TNF-α-mediated RIPK1/RIPK3/MLKL signaling axis in diabetic kidneys. The authors' findings provide new pharmacological evidence to reveal the scientific connotation of TFA in treating RF in DKD in more depth.
Humans ; Rats ; Animals ; Diabetic Nephropathies/drug therapy* ; Abelmoschus ; Flavones/pharmacology* ; Podocytes ; Tumor Necrosis Factor-alpha/metabolism* ; Necroptosis ; Receptor-Interacting Protein Serine-Threonine Kinases/metabolism* ; Fibrosis ; Threonine/pharmacology* ; Collagen/metabolism* ; Serine/pharmacology* ; Diabetes Mellitus/drug therapy*

This study aimed to explore the effects and mechanisms of total flavones of Abelmoschus manihot(TFA), the extracts from traditional Chinese medicine indicated for kidney diseases, on insulin resistance(IR) and podocyte epithelial-mesenchymal transition(EMT) in diabetic kidney disease(DKD), and further to reveal the scientific connotation. Thirty-two rats were randomly divided into a normal group, a model group, a TFA group, and a rosiglitazone(ROS) group. The modified DKD model was induced in rats by methods including high-fat diet feeding, unilateral nephrectomy, and streptozotocin(STZ) intraperitoneal injection. After modeling, the rats in the four groups were given double-distilled water, TFA suspension, and ROS suspension correspondingly by gavage every day. At the end of the 8th week of drug administration, all rats were sacrificed, and the samples of urine, blood, and kidney tissues were collected. The parameters and indicators related to IR and podocyte EMT in the DKD model rats were examined and observed, including the general condition, body weight(BW) and kidney weight(KW), the biochemical parameters and IR indicators, the protein expression levels of the key signaling molecules and structural molecules of slit diaphragm in the renal insulin receptor substrate(IRS) 1/phosphatidylinositol 3-kinase(PI3K)/serine-threonine kinase(Akt) pathway, foot process form and glomerular basement membrane(GBM) thickness, the expression of the marked molecules and structural molecules of slit diaphragm in podocyte EMT, and glomerular histomorphological characteristics. The results showed that for the DKD model rats, both TFA and ROS could improve the general condition, some biochemical parameters, renal appearance, and KW. The ameliorative effects of TFA and ROS were equivalent on BW, urinary albumin(UAlb)/urinary creatinine(UCr), serum creatinine(Scr), triglyceride(TG), and KW. Secondly, they could both improve IR indicators, and ROS was superior to TFA in improving fast insulin(FIN) and homeostasis model assessment of insulin resistance(HOMA-IR). Thirdly, they could both improve the protein expression levels of the key signaling molecules in the IRS1/PI3K/Akt pathway and glomerulosclerosis in varying degrees, and their ameliorative effects were similar. Finally, both could improve podocyte injury and EMT, and TFA was superior to ROS. In conclusion, this study suggested that podocyte EMT and glomerulosclerosis could be induced by IR and the decreased activation of the IRS1/PI3K/Akt pathway in the kidney in DKD. Similar to ROS, the effects of TFA in inhibiting podocyte EMT in DKD were related to inducing the activation of the IRS1/PI3K/Akt pathway and improving IR, which could be one of the scientific connotations of TFA against DKD. This study provides preliminary pharmacological evidence for the development and application of TFA in the field of diabetic complications.
Rats ; Animals ; Diabetic Nephropathies/drug therapy* ; Proto-Oncogene Proteins c-akt/metabolism* ; Phosphatidylinositol 3-Kinases/metabolism* ; Abelmoschus/chemistry* ; Podocytes ; Rats, Sprague-Dawley ; Epithelial-Mesenchymal Transition ; Flavones/pharmacology* ; Insulin Resistance ; Reactive Oxygen Species ; Diabetes Mellitus

Renal tubular injury in patients with diabetic kidney disease(DKD) may be accompanied by glomerular and microvascular diseases. It plays a critical role in the progression of renal damage in DKD, and is now known as diabetic tubulopathy(DT). To explore the multi-targeted therapeutic effects and pharmacological mechanisms in vivo of total flavones of Abelmoschus manihot(TFA), an extract from traditional Chinese medicine for treating kidney disease, in attenuating DT, the authors randomly divided all rats into four groups: a normal control group(normal group), a DT model group(model group), a DT model+TFA-treated group(TFA group) and a DT model+rosiglitazone(ROS)-treated group(ROS group). The DT rat model was established based on the DKD rat model by means of integrated measures. After successful modeling, the rats in the four groups were continuously given double-distilled water, TFA suspension, and ROS suspension, respectively by gavage every day. After 6 weeks of treatment, all rats were sacrificed, and the samples of their urine, blood, and kidneys were collected. The effects of TFA and ROS on various indicators related to urine and blood biochemistry, renal tubular injury, renal tubular epithelial cell apoptosis and endoplasmic reticulum stress(ERS), as well as the activation of the protein kinase R-like endoplasmic reticulum kinase(PERK)-eukaryotic translation initiation factor 2α(eIF2α)-activating transcription factor 4(ATF4)-C/EBP homologous protein(CHOP) signaling pathway in the kidney of the DT model rats were investigated. The results indicated that hypertrophy of renal tubular epithelial cells, renal tubular hyperplasia and occlusion, as well as interstitial extracellular matrix and collagen deposition occurred in the DT model rats. Moreover, significant changes were found in the expression degree and the protein expression level of renal tubular injury markers. In addition, there was an abnormal increase in tubular urine proteins. After TFA or ROS treatment, urine protein, the characteristics of renal tubular injury, renal tubular epithelial cell apoptosis and ERS, as well as the activation of the PERK-eIF2α-ATF4-CHOP signaling pathway in the kidney of the DT model rats were improved to varying degrees. Therein, TFA was superior to ROS in affecting the pathological changes in renal tubule/interstitium. In short, with the DT model rats, this study demonstrated that TFA could attenuate DT by multiple targets through inhibiting renal tubular ERS-induced cell apoptosis in vivo, and its effect and mechanism were related to suppressing the activation of the PERK-eIF2α-ATF4-CHOP signaling pathway in the kidney. These findings provided preliminary pharmacological evidence for the application of TFA in the clinical treatment of DT.
Rats ; Animals ; Abelmoschus ; Reactive Oxygen Species/metabolism* ; Flavones/pharmacology* ; Endoplasmic Reticulum Stress ; Diabetic Nephropathies/drug therapy* ; Apoptosis ; Diabetes Mellitus

Athletes ; Exercise ; Flavones/pharmacology* ; Humans ; Hydrocortisone/blood* ; Resistance Training ; Stress, Physiological/drug effects*

The anti-inflammatory active ingredients of Zhi-Shi-Zhi-Zi-Chi-Tang (ZSZZCT), a traditional Chinese medicine formula, were predicted and identified using an approach based on activity index, LC-MS, semi-preparative LC and NMR. Firstly, the whole extract of ZSZZCT was analyzed using liquid chromatography-quadrupole time of flight-mass spectrometry (LC-Q-TOF-MS) and liquid chromatography - ion trap mass spectrometry (LC-IT-MS), 79 constituents were detected and 39 constituents were identified unambiguously or tentatively. Subsequently, the whole extract of the formula was separated into multiple components and the activity index method was used to calculate index values of the 79 constituents by integrating the chemical and pharmacological information of multiple components. Four polymethoxyl flavones were predicted as the major active constituents according to the activity index values. Furthermore, three polymethoxyl flavones were prepared using the strategy with semi-preparative LC guided by LC-MS, and their anti-inflammatory activities were validated. The results show that three polymethoxyl flavones with higher positive index values, i.e., 3, 5, 6, 7, 8, 3', 4'-heptamethoxyflavone, 3-hydroxynobiletein and tangeretin had significant anti-inflammatory effects. In conclusion, the predicted results indicated that the activity index method is feasible for the accurate prediction of active constituents in TCM formulae.
Animals ; Anti-Inflammatory Agents ; chemistry ; isolation & purification ; pharmacology ; Drugs, Chinese Herbal ; chemistry ; pharmacology ; Flavones ; chemistry ; isolation & purification ; pharmacology ; Lipopolysaccharides ; toxicity ; Macrophages ; drug effects ; metabolism ; Medicine, Chinese Traditional ; Mice ; Molecular Structure ; Nitric Oxide ; metabolism ; Plant Extracts ; chemistry ; pharmacology ; Plants, Medicinal ; chemistry ; RAW 264.7 Cells

OBJECTIVETo explore function and related molecular mechanism of osteopractic total flavone (OTF) on tendon healing in rats.

METHODSTen male rats aged for 8 weeks were collected and weighted from 180 to 220 g. Tendon stem cells were cultivated, the third tendon stem cells were used for experiment. OTP treated with 0, 0.1, 1, 10 ng/ml were added into tendon stem cells, and expression change of ALP, Runx2, OCN, VEGF, P-S6, P-4E/BP1 were detected after 14 days. Forty male rats aged for 8 weeks (weighted 180 to 220 g) were established extra-articular tendon-bone transplanting healing model, and divided into experimental group and control group. Experimental group were treated with OTF(100 mg·kg⁻¹·d⁻¹), while control group was treated by normal saline with the same volume. Tendon-bone healing degree were detected by biomechanical testing at 3 and 6 weeks after surgery, histological detection were applied to detect tendon-bone healing and number of new vessles.

RESULTSAfter treated by OTP, ALP staining and active index detection showed there were statistical differences among 0, 0.1, 1, 10 ng/ml group. After 14 days' cultivation, western blotting results showed mTOR downstream marker protein P-S6 protein expression were gradually increased with increase of density of OTP, expression of P-4E/BP1 was reduced, while expression of Runx2, OCN, VEGF were increased. Biological detection results showed that there was no significant difference in mechanical strength between experimental group(0.78±0.05) N/mm and control group (0.51±0.02) N/mm at 3 weeks after surgery, while mechanical strength in experimental group (1.36±0.09) N/mm was higher than control group (1.01±0.08) N/mm at 6 weeks after surgery. Histological results showed maturity of tendon-bone surface cell were higher at 3 and 6 weeks in experimental group, sharpey fiber growth more density, calcification extent of mesenchyme was high, and new bone, vessels were increased.

CONCLUSIONSOTF could promote osteogenic differentiation of tendon stem cells through mTOR signaling in vitro, and stimulate tendon-bone healing in bone tunnel and enhance connection quality between tendon and bone.

Animals ; Biomechanical Phenomena ; Bone Transplantation ; Cell Differentiation ; Cells, Cultured ; Flavones ; pharmacology ; Male ; Osteogenesis ; Rats ; Stem Cells ; cytology ; TOR Serine-Threonine Kinases ; metabolism ; Tendons ; cytology ; transplantation ; Wound Healing

OBJECTIVETo investigate the effect of total flavone of haw leaves (TFHL) on the expression of nuclear factor erythroid-2 related factor (Nrf2) and other related factors in nonalcoholic steatohepatitis (NASH) rats induced by high-fat diet and then to further discuss the mechanism of TFHL's prevention against NASH.

METHODSHigh-fat diet was fed to 40 rats to establish the NASH model. Then model rats were intragastrically administrated with 40, 80, 160 mg/(kg•day) TFHL, respectively. The pathological changes of liver tissues in NASH rats were detected by oil red O and hematoxylin-eosin (HE) stainings. The expression of Nrf2 in rat liver was examined through immunohistochemistry. The level of 8-iso-prostaglandin F2α in serum was detected through enzyme linked immunosorbent assay (ELISA). The mRNA and protein levels of Nrf2 and other related factors in liver tissue were measured by real-time reverse transcriptionpolymerase chain reaction and western blot.

RESULTSLipid deposition, hepatic steatosis, focal necrosis in lobular inflammation and ballooning degeneration were emerged in livers of NASH rats. The 8-iso-prostaglandin F2α in the serum of NASH rats increased significantly compared with the control group (P<0.05). The mRNA of Nrf2, hemeoxyenase1 (HO-1) and the mRNA and protein levels of quinine oxidoreductase (NQO1) in NASH rats liver tissue showed a striking increase, while the mRNA levels of Keap1, r-glutamylcysteine synthethase (rGCS) and glutathione S-transferase (GST) were significantly decreased compared with the control group (P<0.05). After TFHL treatment, 8-iso-prostaglandin F2α level in serum significantly decreased, and Nrf2 mRNA and protein levels in hepatocytes nucleus enhanced compared with the model group (P<0.05 or 0.01). Meanwhile the Keap1 mRNA, the mRNA and protein levels of HO-1, NQO1 antibody, rGCS antibody, GST increased after TFHL treatment (P<0.05 or 0.01).

CONCLUSIONSNrf2 and other related factors were involved in development of NASH, and they also served as an important part in its occurrence. By regulating expression of Nrf2 and other related factors, TFHL may play a role in antioxidative stress and prevention of NASH.

Animals ; Cell Nucleus ; drug effects ; metabolism ; Crataegus ; chemistry ; Dinoprost ; metabolism ; Flavones ; pharmacology ; therapeutic use ; Lipids ; chemistry ; Liver ; drug effects ; metabolism ; pathology ; NF-E2-Related Factor 2 ; genetics ; metabolism ; Non-alcoholic Fatty Liver Disease ; drug therapy ; genetics ; pathology ; Phytotherapy ; Plant Leaves ; chemistry ; RNA, Messenger ; genetics ; metabolism ; Rats, Sprague-Dawley

OBJECTIVETo explore the protective effect and mechanism of total flavones of Bidens pilosa L. (TFB) on IgA1 induced injury of venous endothelial cells in Henoch-Schönlein purpura (HSP) children patients. METHODS Human umbilical venous endothelial cells (HUVECs) were taken as subject. They were intervened by normal IgA1 and HSP children patients' serum IgA1, and added with different concentrations TFB at the same time. Then they were divided into the blank control group, the normal control group, the HSP IgA1 group, and HSP IgA1 plus TFB (1.0, 0.5, 0.25 mg/mL) groups. Levels of TNF-α and IL-8 in supernate were detected by ELISA. The NO level was detected by nitrate reductase method. mRNA and protein expressions of NF-κB and ICAM-1 in HUVECs were detected by fluorescent quantitative PCR and Western blot respectively.

RESULTSCompared with the normal control group and the blank control group, levels of IL-8, TNF-α, and NO all significantly increased in the HSP group (P < 0.05). Compared with the HSP group, levels of IL-8, TNF-α, and NO significantly decreased after intervention of TFB (1.0 and 0.5 mg/mL; P < 0.05, P < 0.01). Results of fluorescent quantitative PCR and Western blot showed, as compared with the blank control group and the normal control group, mRNA and protein expressions of NF-κB and ICAM-1 in HSP children patients' serum IgA1 induced venous endothelial cells significantly increased with statistical difference (P < 0.05, P < 0.01). Compared with the HSP group, mRNA and protein expressions of NF-KB and ICAM-1 were obviously down-regulated after intervention of TFB (1.0, 0.5, 0.25 mg/mL), with statistical difference (P < 0.05, P < 0.01).

CONCLUSIONTFB could protect vascular damage by inhibiting in vivo high expression of NF-κB, reducing the production of IL-8, TNF-α, and NO in vascular endothelial cells of HSP children patients.

Bidens ; chemistry ; Child ; Flavones ; pharmacology ; Human Umbilical Vein Endothelial Cells ; drug effects ; Humans ; Immunoglobulin A ; blood ; Intercellular Adhesion Molecule-1 ; metabolism ; Interleukin-8 ; metabolism ; NF-kappa B ; metabolism ; Nitric Oxide ; metabolism ; Purpura, Schoenlein-Henoch ; blood ; RNA, Messenger ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism

This research established an HPLC method for determination of six C-Glycoside flavones of warer-soluble total flavonoids from Isodon lophanthoides var. gerardianus (Benth.) H. Hara, and studied the antitumor activity of the warer-soluble total flavonoids. The HPLC system consisted of Kromasil 100-5 C18 (4.6 mm x 250 mm, 5 microm) column and a solution system of methanol, acetonitrile and 0.5% formic acid gradient elution at a flow rate of 0. 8 mL x min(-1) and the wavelength of detector was at 334 nm. The column temperature was 25 degrees C. The antitumor activity of water-soluble flavonoids was assayed using HepG2 cell as the tested cell. The linear ranges of vicenin II, vicenin III, isoschaftoside, schaftoside, vitexin, 6, 8-di-C-a-L-arabinosylapigenin were 0.25-2.53, 0.12-1.20, 0.37-3.69, 0.16-1.63, 0.19-1.92, 0.14-1.42 microg, respectively. The average recoveries (n = 6) were 99.6% (RSD 0.87%), 100.2% (RSD 2.0%), 99.6% (RSD 1.8%), 97.9% (RSD 1.5%), 98.8% (RSD 1.2%), 98.6% (RSD 1.2%), respectively. After exposure in 24, 48, 72 h, the total flavonoids showed inhibitory effect on the proliferation of HepG2 cells with IC50 as the evaluation index, the IC50 values of 1.89, 1.71, 1.51 g x L(-1), respectively. The method is quick, simple and accurate with good re- producibility, and can be used for determination of vicenin II, vicenin III, isoschaftoside, schaftoside, vitexin, 6, 8-di-C-a-L-arabino- sylapigenin in the warer-soluble total flavonoids from L lophanthoides var. gerardianus. The warer-soluble total flavonoids from L lophanthoides have inhibitory effect on the proliferation of HepG2 cells.
Antineoplastic Agents, Phytogenic ; analysis ; pharmacology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Chromatography, High Pressure Liquid ; Drugs, Chinese Herbal ; analysis ; pharmacology ; Flavones ; analysis ; pharmacology ; Humans ; Isodon ; chemistry ; Monosaccharides ; analysis ; pharmacology

Cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-dependent chloride channel, plays key roles in fluid secretion in serous epithelial cells. Previously, we identified two polymethoxylated flavonoids, 3',4',5,5',6,7-hexamethoxyflavone (HMF) and 5-hydroxy-6,7,3',4'-tetramethoxyflavone (HTF) which could potentiate CFTR chloride channel activities. The present study was aimed to investigate the potentiation effects of HMF and HTF on CFTR Cl(-) channel activities by using a cell-based fluorescence assay and the short circuit Ussing chamber assay. The results of cell-based fluorescence assay showed that both HMF and HTF could dose-dependently potentiate CFTR Cl(-) channel activities in rapid and reversible ways, and the activations could be reversed by the CFTR blocker CFTRinh-172. Notably, HMF showed the highest affinity (EC50 = 2 μmol/L) to CFTR protein among the flavonoid CFTR activators identified so far. The activation of CFTR by HMF or HTF was forskolin (FSK) dependent. Both compounds showed additive effect with FSK and 3-Isobutyl-1-methylx (IBMX) in the activation of CFTR, while had no additive effect with genistein (GEN). In ex vivo studies, HMF and HTF could stimulate transepithelial Cl(-) secretion in rat colonic mucosa and enhance fluid secretion in mouse trachea submucosal glands. These results suggest that HMF and HTF may potentiate CFTR Cl(-) channel activities through both elevation of cAMP level and binding to CFTR protein pathways. The results provide new clues in elucidating structure and activity relationship of flavonoid CFTR activators. HMF might be developed as a new drug in the therapy of CFTR-related diseases such as bronchiectasis and habitual constipation.
Animals ; Colforsin ; Colon ; metabolism ; Cystic Fibrosis Transmembrane Conductance Regulator ; drug effects ; Flavones ; physiology ; Flavonoids ; pharmacology ; Genistein ; Intestinal Mucosa ; metabolism ; Mice ; Rats