The innate immune system is critical for clearing infection, and is tightly regulated to avert excessive tissue damage. Nod1/2-Rip2 signaling, which is essential for initiating the innate immune response to bacterial infection and ER stress, is subject to many regulatory mechanisms. In this study, we found that LRRK2, encoded by a gene implicated in Crohn's disease, leprosy and familial Parkinson's disease, modulates the strength of Nod1/2-Rip2 signaling by enhancing Rip2 phosphorylation. LRRK2 deficiency markedly reduces cytokine production in macrophages upon Nod2 activation by muramyl dipeptide (MDP), Nod1 activation by D-gamma-Glu-meso-diaminopimelic acid (iE-DAP) or ER stress. Our biochemical study shows that the presence of LRRK2 is necessary for optimal phosphorylation of Rip2 upon Nod2 activation. Therefore, this study reveals that LRRK2 is a new positive regulator of Rip2 and promotes inflammatory cytokine induction through the Nod1/2-Rip2 pathway.
Animals ; Cytokines ; genetics ; immunology ; HEK293 Cells ; Humans ; Immunity, Innate ; genetics ; Inflammation ; genetics ; immunology ; Leucine-Rich Repeat Serine-Threonine Protein Kinase-2 ; genetics ; immunology ; Mice ; Mice, Knockout ; Nod1 Signaling Adaptor Protein ; genetics ; immunology ; Nod2 Signaling Adaptor Protein ; genetics ; immunology ; Phosphorylation ; genetics ; immunology ; Receptor-Interacting Protein Serine-Threonine Kinase 2 ; genetics ; immunology ; Receptor-Interacting Protein Serine-Threonine Kinases ; genetics ; immunology ; Signal Transduction ; genetics ; immunology

The aim of this study is to construct specific shRNA expressing plasmids, and to observe their effects on H9c2 cardiomyocytes injury induced by hypoxia/reoxygenation (H/R). RIPK1 and RIPK3 are the key kinases mediating the process of necroptosis. Using recombinant DNA technology, we inserted the synthetic shRNA into pSUPER vector to construct RIPK1-shRNA or RIPK3-shRNA plasmid respectively. We transfected H9c2 cardiomyocytes with the two shRNA plasmids respectively, before we treated them with H/R stimulation. Then, we measured the relevant genes and proteins by real-time PCR and Western blot. Meanwhile,we detected the markers of necroptosis and cardiomyocytes injury. The results showed that inhibition of ripk1 or ripk3 gene expression by its specific shRNA might protect the cardiomyocytes injury induced by H/R stimulation.
Animals ; Apoptosis ; Cell Hypoxia ; Cell Line ; Gene Expression ; Myocytes, Cardiac ; pathology ; Protein-Serine-Threonine Kinases ; genetics ; metabolism ; RNA, Small Interfering ; Rats ; Real-Time Polymerase Chain Reaction ; Receptor-Interacting Protein Serine-Threonine Kinases ; genetics ; metabolism ; Transfection

OBJECTIVE: To investigate the expression and clinical significance of receptor interacting protein serine-threonine kinases 1 (RIPK1) in the nucleus pulposus of patients with lumbar disc herniation (LDH). METHODS: Nucleus pulposus tissue specimens of 40 patients with LDH patients underwent surgical treatment from January 2016 to January 2018 as the case group, and nucleus pulposus tissue specimens of 30 patients with lumbar spine fracture underwent surgical treatment at the same time as the control group. The expression of RIPK1 mRNA and protein of receptor interaction were detected by polymerase chain reaction (PCR) and Western blot, respectively. The expression of RIPK1 protein in the nucleus pulposus were detected by immunohistochemical staining. The concentrations of RIPK1 and tumor necrosis factor-α (TNF-α) in nucleus pulposus were detected by ELISA method. The relationship between the concentrations of RIPK1, TNF-α in nucleus pulposus and the Pearce grade of LDH patients was analyzed by one-way ANOVA. The correlation between RIPK1 and TNF-α was analyzed by Pearson. RESULTS: RIPK1 was weakly positively expressed in nucleus pulposus of control group, and RIPK1 protein was positively or strongly positively expressed in case group. The expression of RIPK1 mRNA in nucleus pulposus of case group was higher than that of control group ( CONCLUSION The expression levels of RIPK1 mRNA and protein in the intervertebral disc tissues of LDH patients are higher than those of normal intervertebral disc tissues, and increased with the increase of Pearce grade, which may be an important factor involved in LDH inflammatory disease.
Humans ; Intervertebral Disc/metabolism* ; Intervertebral Disc Degeneration ; Intervertebral Disc Displacement/genetics* ; Nucleus Pulposus ; Receptor-Interacting Protein Serine-Threonine Kinases/genetics* ; Tumor Necrosis Factor-alpha/metabolism*

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*

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

OBJECTIVETo investigate the mRNA expressions of the TNF adapter proteins, including TNF receptor-associated death domain protein (TRADD), Fas-associated death domain protein (FADD), receptor-interacting protein 1 (RIP-1) and TNF receptor-associated factor-2 (TRAF-2) in peripheral blood mononuclear cells (PBMCs) of lupus nephritis (LN) patients of various TCM asthenia syndromes. Methods Fifty-one inpatients with LN were differentiated according to TCM syndrome differentiation, 13 cases of yin-deficiency with inner heat syndrome (A); 26 cases of both qi-yin deficiency syndrome (B), 12 cases of Pi-Shen yang-deficiency syndrome (C). Peripheral venous blood samples from the 51 LN patients and 17 healthy subjects were collected to separate PBMCs. The mRNA expressions of TNF adapter molecules (TRADD, FADD, RIP-1 and TRAF-2), as well as Caspase-3 and interleukin-1beta (IL-1beta) were analyzed by quantitative real-time PCR and the differences among them were compared.

RESULTS(1) As compared with the healthy subjects, expression of TRADD mRNA in patients of syndrome A, B and C was lowered to 0.54, 0.32, and 0.38-fold, respectively (P < 0.05, P < 0.01), showing insignificant difference among the three syndromes; (2) FADD mRNA lowered to 0.79, 0.62, and 0.72-fold respectively, only with significance shown in syndrome B (P < 0.05); (3) RIP-1 mRNA lowered to 0.79, 0.50, and 0.60-fold respectively with significance shown in syndrome B and C (P < 0.01, P < 0.05), and insignificant difference was shown among the three syndromes; (4) TRAF-2 lowered to 0.70, 0.52, and 0.50-fold respectively (P < 0.01, P < 0.01, P = 0.07), significance shown in syndrome B and C (P < 0.01), but with insignificant difference among the three; (5) Caspase-3 elevated in all patients of the three syndromes (all P < 0.01); (6) IL-1beta in syndrome A was apparently lower ed to the normal range and also lower than that in the other two syndromes (both P < 0.05).

CONCLUSIONSExpressions of TRADD, FADD, RIP-1 and TRAF-2 mRNA decreased in all the patients of various TCM asthenia syndromes, the decrement in patients of syndrome B and C was lesser than that in syndrome A. These abnormal low expressions of signal proteins might be the substantial bases for asthenia syndromes of LN patients, and the apoptotic signal mediated by them may involve in the formation of asthenia syndrome in LN.

Adaptor Proteins, Signal Transducing ; genetics ; metabolism ; Adolescent ; Adult ; Case-Control Studies ; Child ; Fas-Associated Death Domain Protein ; genetics ; metabolism ; Female ; Humans ; Leukocytes, Mononuclear ; metabolism ; Lupus Nephritis ; blood ; Male ; Medicine, Chinese Traditional ; Middle Aged ; RNA, Messenger ; genetics ; metabolism ; Receptor-Interacting Protein Serine-Threonine Kinases ; genetics ; metabolism ; TNF Receptor-Associated Death Domain Protein ; genetics ; metabolism ; TNF Receptor-Associated Factor 2 ; genetics ; metabolism ; Tumor Necrosis Factor-alpha ; blood ; metabolism ; Yang Deficiency ; blood ; Yin Deficiency ; blood ; Young Adult

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