To explore the effect and mechanism of Dahuang Zhechong Pills(DHZCP), a classical prescription, in improving testicular aging(TA) in vivo, the authors randomly divided 24 male rats into four groups: the normal, model, DHZCP and vitamin E(VE) groups. The TA rat model was established by continuous gavage of D-galactose(D-gal). During the experiment, the rats in the DHZCP and VE groups were given DHZCP suspension and VE suspension, respectively by gavage, while those in the normal and model groups were gavaged saline separately every day. After the co-administration of D-gal and various drugs for 60 days, all rats were sacrificed, and their blood and testis were collected. Further, various indexes related to TA and necroptosis of testicular cells in the model rats were examined and investigated, which included the aging phenotype, total testicular weight, testicular index, histopathological features of testis, number of spermatogenic cells, sex hormone level, expression characteristics of reactive oxygen species(ROS) in testis, expression levels and characteristics of cyclins in testis, and protein expression levels of the key molecules in receptor-interacting serine/threonine-protein kinase 1(RIPK1)/receptor-interacting serine/threonine-protein kinase 3(RIPK3)/mixed lineage kinase domain like pseudokinase(MLKL) signaling pathway in each group. The results showed that, for the TA model rats, both DHZCP and VE improved their aging phenotype, total testicular weight, testicular index, pathological features of testis, number of spermatogenic cells, serum testosterone and follicle stimulating hormone levels, expression characteristics of ROS and protein expression levels and characteristics of P21 and P53 in testis. In addition, DHZCP and VE improved the protein expression levels of the key molecules in RIPK1/RIPK3/MLKL signaling pathway in testis of the model rats. Specifically, DHZCP was better than VE in the improvement of RIPK3. In conclusion, in this study, the authors found that DHZCP, similar to VE, ameliorated D-gal-induced TA in model rats in vivo, and its mechanism was related to reducing necroptosis of testicular cells by inhibiting the activation of RIPK1/RIPK3/MLKL signaling pathway. This study provided preliminary pharmacological evidence for the development and application of classical prescriptions in the field of men's health.
Aging ; Animals ; Drugs, Chinese Herbal ; Male ; Necroptosis ; Protein Kinases/genetics* ; Rats ; Reactive Oxygen Species/metabolism* ; Receptor-Interacting Protein Serine-Threonine Kinases/pharmacology* ; Serine/pharmacology* ; Signal Transduction ; Testis ; Threonine/pharmacology*

The aim of this study was to observe whether necroptosis is involved in the process of cardiac hypertrophy induced by pressure overload. SD rats underwent transverse abdominal aortic constriction (TAC) operation for establishing cardiac hypertrophy model. The structure and function of the left ventricle of rats were evaluated via echocardiography, left ventricular mass index, the expression of markers of cardiac hypertrophy and histological detection. Real-time PCR and Western blot were used to measure the gene and protein expression of receptor interacting protein kinase 1 and 3 (RIPK1 and RIPK3, the necroptosis markers) respectively. Four weeks after TAC operation, rat model for cardiac hypertrophy was established. The experimental data showed that the gene and protein expressions of RIPK1 and RIPK3 in the rat heart hypertrophic tissues after TAC for 4 weeks were increased significantly compared with those in the sham group. HE staining showed cardiomyocytes injury and hypertrophy in the hearts of TAC rat models. By transmission electron microscope, we observed that mitochondria of cardiomyocytes were damaged seriously in the TAC models. Treatment with losartan used, the selective antagonist of angiotensin II type I receptor could improve the cardiac function of TAC rats. Moreover, losartan treatment decreased the expression of RIPK1 and RIPK3 in heart tissues of TAC rats. The results suggest that necroptosis occurrs in the process of cardiac hypertrophy with pressure overload, and losartan could alleviate the cardiac hypertrophy and inhibit necroptosis.
Animals ; Apoptosis ; Cardiomegaly ; pathology ; Disease Models, Animal ; Echocardiography ; Heart ; physiopathology ; Losartan ; pharmacology ; Myocytes, Cardiac ; Pressure ; Protein-Serine-Threonine Kinases ; metabolism ; Rats ; Rats, Sprague-Dawley ; Real-Time Polymerase Chain Reaction ; Receptor-Interacting Protein Serine-Threonine Kinases ; metabolism

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*

OBJECTIVETo investigate the effect of small interfering RNA-mediated receptor-interacting protein kinase 1 (RIP1) knockdown on the sensitivity of human oral squamous carcinoma cells to to oxaliplatin (L-OHP)-induced apoptosis and explore a new target for clinical treatment of oral squamous carcinoma.

METHODSThe viability of human oral squamous carcinoma cell line KB exposed to different concentrations (0, 0.25, 0.5, 1, 2, 4 µmol/L) of L-OHP were detected by MTT assay. PI/Annexin V staining was used to observe cell apoptosis in naive KB cells, cell and transfected with pSH1Si-RIP1 or with the empty plasmid. Western blotting was used to detect RIP1 expression in KB cells exposed to L-OHP and in cells transfected with pSH1Si-RIP1.

RESULTSExposure to L-OHP (1µmol/L) for 24, 48, 72 h resulted in KB cell survival rates of 67.66%, 55.17%, and 41.34%, respectively, but the cell apoptosis rate was only 9.6% following a 24-h exposure. KB cells transfected with pSH1Si-RIP1 showed an apoptotic rate of 9.4%, which increased to 29.1% following L-OHP exposure. RIP1 expression was first up-regulated and then down-regulated in KB cells treated with L-OHP, and was significantly reduced after cell transfection with pSH1Si-RIP1.

CONCLUSIONSuppression of RIP1 expression increases the apoptotic rate of human oral squamous carcinoma cells, suggesting the potential of RIP1 as a new candidate target for clinical treatment of oral squamous carcinoma.

Apoptosis ; drug effects ; Carcinoma, Squamous Cell ; genetics ; pathology ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; Humans ; Mouth Neoplasms ; genetics ; pathology ; Organoplatinum Compounds ; pharmacology ; RNA Interference ; RNA, Small Interfering ; genetics ; Receptor-Interacting Protein Serine-Threonine Kinases ; genetics ; Transfection

To explore the antagonistic effect of gingerols against the inflammation induced by lectin from Pinellia ternata. In this study, ELISA method was used to determine the effect of different extracts from gingerols on the release of inflammatory factor TNF-α from macrophages induced by lectin from P. ternata. The fluorescence probe was used to determine the effect of gingerols on the changes in ROS of macrophages induced by lectin from P. ternata. The western-blot method was applied to study the effect of gingerols on the increase in expression of cell receptor interacting protein RIP3 in macrophages induced by lectin from P. ternata. The scanning electron microscope (SEM) was used to study the effect of gingerols on morphological changes in macrophages induced by lectin from P. ternata. According to the results, gingerols can significantly inhibit the release of inflammatory factor from macrophages induced by lectin from P. ternata, ROS overproduction and increase in RIP3 expression. SEM results showed that gingerols can inhibit the cytomorphosis and necrocytosis induced by lectin from P. ternata. Fresh ginger's detoxication may be related to gingerols' effects in inhibiing release of inflammatory factor, ROS overproduction and increase in RIP3 expression caused by macrophages induced by lectin from P. ternata, which are mainly inflammatory development.
Animals ; Catechols ; pharmacology ; Cells, Cultured ; Drug Antagonism ; Fatty Alcohols ; pharmacology ; Ginger ; chemistry ; Lectins ; toxicity ; Macrophages ; drug effects ; metabolism ; Male ; Mice ; Mice, Inbred ICR ; Pinellia ; chemistry ; toxicity ; Reactive Oxygen Species ; metabolism ; Receptor-Interacting Protein Serine-Threonine Kinases ; genetics ; metabolism ; Tumor Necrosis Factor-alpha ; genetics ; metabolism