Mechanism of Modified Erchentang on COPD Inflammation Based on TNF-α/TNFR1/RIPKs Pathway / 中国实验方剂学杂志

ObjectiveBased on tumor necrosis factor alpha （TNF-α）/tumor necrosis factor receptor 1 （TNFR1）/receptor-interacting protein kinases （RIPKs） signaling pathway， this paper aims to study the effect of modified Erchentang on inflammation in rats with chronic obstructive pulmonary disease （COPD） and explore its mechanism of action. MethodA total of 60 SD rats were randomly divided into normal group， model group， high， medium， and low-dose groups （20， 10， 5 g·kg-1·d-1） of modified Erchentang， and Xiaokechuan group （3.5 mL·kg-1·d-1）， with 10 rats in each group. The COPD rat model was established by cigarette smoke combined with lipopolysaccharide （LPS）. The normal group and model group were given the same amount of normal saline for 21 days by gavage administration. The contents of TNF-α and TNFR1 in bronchoalveolar lavage fluid （BALF） of rats were detected by enzyme-linked immunosorbent assay （ELISA）. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was used to detect mRNA expressions of RIPK1， RIPK3， and mixed lineage kinase domain-like （MLKL） in the lung tissue. The protein expressions of RIPK1， RIPK3， and MLKL in the lung tissue were detected by Western blot. The pathological changes in lung tissue were observed by hematoxylin-eosin （HE） staining. ResultCompared with the normal group， the contents of TNF-α and TNFR1 in BALF of the model group were significantly increased （P<0.01）， and the mRNA and protein expression levels of RIPK1， RIPK3， and MLKL in the lung tissue were significantly increased （P<0.01）. Compared with the model group， the contents of TNF-α and TNFR1 in BALF of high， medium， and low-dose groups of modified Erchentang and Xiaokechuan group were decreased （P<0.01）. The mRNA and protein expression levels of RIPK1， RIPK3， and MLKL in the lung tissue were decreased to different degrees （P<0.05， P<0.01）. ConclusionModified Erchentang can effectively improve the inflammatory response of lung tissue in COPD rats， and the mechanism may be by inhibiting the activation of the TNF-α/TNFR1/RIPKs signaling pathway.

RBMS3 Expression and Its Correlation with Clinicopathological Features and Prognosis of Epithelial Ovarian Cancer / 中山大学学报(医学科学版)

ObjectiveTo investigate the expression of RNA binding motif single stranded interacting protein 3 （RBMS3） in epithelial ovarian cancer （EOC） tissues and its relationship with the clinicopathological features and prognosis of EOC. MethodsThe study enrolled the paraffin-embedded tissues from 110 EOC cases and 73 benign epithelial ovarian tumor cases pathologically diagnosed in the first affiliated Hospital of Bengbu Medical College from January 2015 to December 2019. By using anti-RBMS3 polyclonal antibody， the immunohistochemical staining was employed to detect RBMS3 expression in the tissues and then its correlation with the clinicopathological parameters and prognosis of EOC was analyzed. ResultsRBMS3 was expressed in both EOC and benign epithelial ovarian tumor tissues. RBMS3 expression in EOC tissues， significantly related with International Federation of Gynecology and Obstetrics （FIGO） stage， histological grade， CEA levels and survival status， was significantly lower than that in benign epithelial ovarian tumor tissues （P<0.05）. Kaplan–Meier survival curve showed that low RBMS3 expression in EOC patients was correlated with decreased progression-free survival （PFS） and overall survival （OS） （P<0.05）. Univariate analysis showed that RBMS3 expression， FIGO stage， residual lesion size， intestinal metastasis and intraperitoneal implantation were associated with OS of EOC patients （P<0.05）； multivariate analysis showed that low RBMS3 expression and intestinal metastasis were independent risk factors for poor prognosis in EOC patients （P<0.05）. ConclusionsRBMS3 is expressed at low levels in EOC tissues， which is closely related to poor prognosis of EOC patients. RBMS3 may function as a tumor suppressor gene in EOC tissues and can be used as an EOC-independent prognostic marker for targeted therapy against EOC.

Effect of Shao's five-needle therapy pretreatment on airway inflammatory response in asthmatic rats based on ROS/TXNIP/NLRP3 pathway / 中国针灸

OBJECTIVES@#To explore the possible mechanism of Shao's five-needle therapy pretreatment on relieving airway inflammatory response in asthmatic rats.@*METHODS@#Forty SPF-grade SD rats were randomly divided into a blank group, a model group, an acupuncture group, and a medication group, with 10 rats in each group. Except the blank group, asthma model was established by aerosol inhalation of ovalbumin in the other 3 groups. The rats in the acupuncture group were treated with acupuncture at "Dazhui" (GV 14) and bilateral "Feishu" (BL 13) and "Fengmen" (BL 12), with each session lasting for 20 min. Acupuncture was given before each motivating, once daily for 7 consecutive days. The rats in the medication group were treated with intraperitoneal injection of dexamethasone sodium phosphate solution before each motivating, once daily for 7 days. General situation of the rats was observed in each group; ELISA method was used to detect the levels of inflammatory cytokines interleukin (IL)-1β and IL-18 in serum; immunofluorescence staining method was performed to assess the expression of reactive oxygen species (ROS) in lung tissues; Western blot method was used to measure the protein expression of thioredoxin interacting protein (TXNIP), NOD-like receptor protein 3 (NLRP3), apoptosis-associated speck-like protein containing a CARD (ASC), and Caspase-1 in lung tissues.@*RESULTS@#The rats in the blank group exhibited normal behavior, while those in the model group showed signs of respiratory distress, ear scratching, cheek rubbing, and dysphoria. Compared with the model group, the rats in the acupuncture group and the medication group showed stable respiration and relatively agile responses. Compared with those in the blank group, the serum levels of IL-18 and IL-1β were elevated (P<0.01), the expression intensity of ROS was increased, and the protein expressions of TXNIP, NLRP3, ASC and Caspase-1 in lung tissues were increased (P<0.01) in the model group. Compared with those in the model group, the serum levels of IL-18 and IL-1β were reduced (P<0.01), the expression intensity of ROS was lowered, and the protein expressions of TXNIP, NLRP3, ASC and Caspase-1 in lung tissues were reduced (P<0.01) in the acupuncture group and the medication group. Compared with the medication group, the protein expression of ASC in lung tissue was reduced in the acupuncture group (P<0.05).@*CONCLUSIONS@#Pretreatment of Shao's five-needle therapy could alleviate airway inflammatory response in asthmatic rats by reducing ROS levels and decreasing the aggregation and activation of pathway-related proteins in the ROS/TXNIP/NLRP3 pathway, ultimately leading to decreased secretion of IL-1β and IL-18. This mechanism may contribute to the effectiveness of Shao's five-needle therapy in preventing and treating asthma.

Effects and mechanisms of wogonin on airway inflammation in rats with chronic obstructive pulmonary disease / 中国药房

OBJECTIVE To study the effects and potential mechanism of wogonin （Wog） on airway inflammation in rats with chronic obstructive pulmonary disease （COPD）. METHODS Eighty-four rats were randomly divided into control group， model group， Wog low-dose and high-dose groups （intragastric administration of 50， 100 mg/kg）， aminophylline group （positive control， intragastric administration of 2.3 mg/kg）， recombinant rat receptor-interacting protein kinase 1 [rRIPK1， receptor-interacting protein kinase 1 （RIPK1） activator] group （tail vein injection of 8 µg/kg）， and Wog high-dose+rRIPK1 group （intragastric administration of Wog 100 mg/kg+tail vein injection of rRIPK 8 µg/kg）， with 12 rats in each group. Except for control group， COPD model of other groups was induced by smoking combined with tracheal injection of lipopolysaccharide. Twenty-four hours after successful modeling， the rats were administered once a day for 4 weeks. The changes of peak inspiratory flow （PIF）， peak expiratory flow （PEF） and minute ventilation （MV），forced expiratory volume in one second（FEV1）/forced vital capacity（FVC） were measured after the last medication； the serum levels of interleukin 1β（IL-1β）， IL-6 and tumor necrosis factor-α （TNF-α） were measured by ELISA； the pathological changes of lung tissue in rats were observed； the apoptotic rate of pulmonary epithelial cells was detected. mRNA expressions of RIPK1， RIPK3 and mixed lineage kinase domain-like protein （MLKL）， and protein expressions of RIPK1， RIPK3 and p-MLKL were all detected in lung tissue of rats. RESULTS Compared with control group， PIF， PEF， MV and FEV1/FVC of model group were decreased significantly （P＜0.05）， while the levels of IL-1β， IL-6 and TNF- α were increased significantly （P＜0.05）； there was a large number of inflammatory cells infiltration in the lung tissue and bronchialwall thickening in model group； the apoptotic rate of pulmonary epithelial cells，mRNA expressions of RIPK1， RIPK3 and MLKL， protein expressions of RIPK1， RIPK3 and p-MLKL were increased significantly （P＜0.05）. Compared with model group， above indexes of rats were improved significantly in Wog low-dose and high-dose groups （P＜0.05）， and pathological injuries were alleviated significantly. The corresponding indexes of rats were worsened in rRIPK1 group （P＜0.05）， and pathological damage had further worsened. rRIPK1 significantly attenuated the inhibitory effect of high-dose Wog on airway inflammation and RIPK1/RIPK3/ MLKL pathway in COPD rats （P＜0.05）. CONCLUSIONS Wog may improve airway inflammation in COPD rats by inhibiting RIPK1/RIPK3/MLKL signal pathway.

Role of Mitophagy in Myocardial Ischemia/Reperfusion Injury and Chinese Medicine Treatment / 中国结合医学杂志

Mitophagy is one of the important targets for the prevention and treatment of myocardial ischemia/reperfusion injury (MIRI). Moderate mitophagy can remove damaged mitochondria, inhibit excessive reactive oxygen species accumulation, and protect mitochondria from damage. However, excessive enhancement of mitophagy greatly reduces adenosine triphosphate production and energy supply for cell survival, and aggravates cell death. How dysfunctional mitochondria are selectively recognized and engulfed is related to the interaction of adaptors on the mitochondrial membrane, which mainly include phosphatase and tensin homolog deleted on chromosome ten (PTEN)-induced kinase 1/Parkin, hypoxia-inducible factor-1 α/Bcl-2 and adenovirus e1b19k Da interacting protein 3, FUN-14 domain containing protein 1 receptor-mediated mitophagy pathway and so on. In this review, the authors briefly summarize the main pathways currently studied on mitophagy and the relationship between mitophagy and MIRI, and incorporate and analyze research data on prevention and treatment of MIRI with Chinese medicine, thereby provide relevant theoretical basis and treatment ideas for clinical prevention of MIRI.

Scaffold protein MAPK8IP2 expression is a robust prognostic factor in prostate cancer associated with AR signaling activity / 亚洲男科学杂志(英文版)

Mitogen-activated protein kinase-8-interacting protein 2 (MAPK8IP2) is a scaffold protein that modulates MAPK signal cascades. Although MAPK pathways were heavily implicated in prostate cancer progression, the regulation of MAPK8IP2 expression in prostate cancer is not yet reported. We assessed MAPK8IP2 gene expression in prostate cancer related to disease progression and patient survival outcomes. MAPK8IP2 expression was analyzed using multiple genome-wide gene expression datasets derived from The Cancer Genome Atlas (TCGA) RNA-sequence project and complementary DNA (cDNA) microarrays. Multivariable Cox regressions and log-rank tests were used to analyze the overall survival outcome and progression-free interval. MAPK8IP2 protein expression was evaluated using the immunohistochemistry approach. The quantitative PCR and Western blot methods analyzed androgen-stimulated MAPK8IP2 expression in LNCaP cells. In primary prostate cancer tissues, MAPK8IP2 mRNA expression levels were significantly higher than those in the case-matched benign prostatic tissues. Increased MAPK8IP2 expression was strongly correlated with late tumor stages, lymph node invasion, residual tumors after surgery, higher Gleason scores, and preoperational serum prostate-specific antigen (PSA) levels. MAPK8IP2 upregulation was significantly associated with worse overall survival outcomes and progression-free intervals. In castration-resistant prostate cancers, MAPK8IP2 expression strongly correlated with androgen receptor (AR) signaling activity. In cell culture-based experiments, MAPK8IP2 expression was stimulated by androgens in AR-positive prostate cancer cells. However, MAPK8IP2 expression was blocked by AR antagonists only in androgen-sensitive LNCaP but not castration-resistant C4-2B and 22RV1 cells. These results indicate that MAPK8IP2 is a robust prognostic factor and therapeutic biomarker for prostate cancer. The potential role of MAPK8IP2 in the castration-resistant progression is under further investigation.

Research progress of NOD2-mediated signaling pathways and relationship with autoinflammatory diseases and its inhibitors / 药学学报

Nucleotide-binding oligomerization domain containing protein 2 (NOD2) is a member of intracellular pattern recognition receptor. After being activated, it will induce the release of inflammatory factors through a series of signal cascade transduction, thus playing an important role in the innate immune response. The abnormal NOD2 signaling pathway is involved in the occurrence and development of many diseases, especially the single nucleotide polymorphisms (SNPs) of the NOD2 gene have been identified to be closely associated with autoinflammatory diseases (AIDs). Therefore, inhibitors targeting NOD2 pathway have great potential in the treatment of inflammatory immune diseases. This review presents the recent progress of NOD2 receptor-mediated signal transduction pathways and its regulation mechanisms, the relationship between NOD2 and AIDs, and the inhibitors of NOD2 pathway.

Role of ROS/TXNIP/NLRP3 signaling pathway in high glucose-induced pyroptosis of human embryonic trophoblast cells / 中华内分泌外科杂志

Objective:To investigate the changes of ROS/TXNIP/NLRP3 signaling pathway in pyroptosis of human embryonic trophoblast cells induced by high glucose.Methods:Human embryonic trophoblast cells were cultured in vitro to establish high glucose injury model, and they were randomly divided into control group, high glucose (HG) group and HG + ROS inhibitor N-acetyl-L-cysteine (HG + NAC) group. MTT assay was used to detect the cell survival rate. The level of ROS in each group was detected by dihydroethidine ROS fluorescence probe. Expression of TXNIP and NLRP3 mRNA was detected by real-time quantitative PCR (RT-qPCR). Western blot analysis was used to detect the expression levels of TXNIP, NLRP3, Caspase-1, interleukin (IL) -1β, tumor necrosis factor-α (TNF-α) and GSDMD proteins. In addition, pyroptosis was detected by flow cytometry.Results:The optimal glucose concentration for high glucose-induced injury of human embryonic trophoblast cells was 30 mmol/L. Compared with the control group (96.27±3.10) %, the survival rate of human embryonic trophoblast cells in HG group (55.44±2.15) % was significantly lower ( P<0.05), while the fluorescence intensity (ROS level) of 7 'dichlorofluorescein (DCF), the expression levels of TXNIP and NLRP3 proteins, the number of pyroptosis, expression levels of Caspase-1, GSDMD, IL-1β and TNF-α proteins were significantly higher ( P<0.05) ; Compared with HG group, the survival rate of human embryonic trophoblast cells in HG+NAC group (84.75±2.33) % was significantly higher ( P<0.05), the fluorescence intensity (ROS level) of DCF, the expression levels of TXNIP and NLRP3 proteins, the number of pyroptosis, and expression levels of Caspase-1, GSDMD, IL-1β and TNF-α proteins were significantly lower ( P<0.05) . Conclusion:Inhibition of ROS level in human embryonic trophoblast cells induced by high glucose may promote cell proliferation and reduce the occurrence of pyroptosis by inhibiting TXNIP/NLRP3 signaling pathway.

Polypeptide from Moschus Suppresses Lipopolysaccharide-Induced Inflammation by Inhibiting NF-κ B-ROS/NLRP3 Pathway / 中国结合医学杂志

OBJECTIVE@#To examine the anti-inflammatory effects and potential mechanisms of polypeptide from Moschus (PPM) in lipopolysaccharide (LPS)-induced THP-1 macrophages and BALB/c mice.@*METHODS@#The polypeptide was extracted from Moschus and analyzed by high-performance liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Subsequently, LPS was used to induce inflammation in THP-1 macrophages and BALB/c mice. In LPS-treated or untreated THP-1 macrophages, cell viability was observed by cell counting kit 8 and lactate dehydrogenase release assays; the proinflammatory cytokines and reactive oxygen species (ROS) were measured by enzyme-linked immunosorbent assay and flow cytometry, respectively; and protein and mRNA levels were measured by Western blot and real-time quantitative polymerase chain reaction (qRT-PCR), respectively. In LPS-induced BALB/c mice, the proinflammatory cytokines were measured, and lung histology and cytokines were observed by hematoxylin and eosin (HE) and immunohistochemical (IHC) staining, respectively.@*RESULTS@#The SDS-PAGE results suggested that the molecular weight of purified PPM was in the range of 10-26 kD. In vitro, PPM reduced the production of interleukin 1β (IL-1β), IL-18, tumor necrosis factor α (TNF-α), IL-6 and ROS in LPS-induced THP-1 macrophages (P<0.01). Western blot analysis demonstrated that PPM inhibited LPS-induced nuclear factor κB (NF-κB) pathway and thioredoxin interacting protein (TXNIP)/nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain containing 3 (NLRP3) inflammasome pathway by reducing protein expression of phospho-NF-κB p65, phospho-inhibitors of NF-κB (Iκ Bs) kinase α/β (IKKα/β), TXNIP, NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and pro-caspase-1 (P<0.05 or P<0.01). In addition, qRT-PCR revealed the inhibitory effects of PPM on the mRNA levels of TXNIP, NLRP3, ASC, and caspase-1 (P<0.05 or P<0.01). Furthermore, in LPS-induced BALB/c mice, PPM reduced TNF-α and IL-6 levels in serum (P<0.05 or P<0.01), decreased IL-1β and IL-18 levels in the lungs (P<0.01) and alleviated pathological injury to the lungs.@*CONCLUSION@#PPM could attenuate LPS-induced inflammation by inhibiting the NF-κB-ROS/NLRP3 pathway, and may be a novel potential candidate drug for treating inflammation and inflammation-related diseases.

Effect of Gegen Qinliantang-medicated Serum on Nonalcoholic Steatohepatitis Based on Nrf2/TXNIP Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo investigate the effect of Gegen Qinliantang （GGQLT）-medicated serum on free fatty acid （FFA）-induced nonalcoholic steatohepatitis （NASH） in vitro model of human hepatoma cells HepG2. MethodNASH model of HepG2 cells was established in vitro， and the cells were intervened with different volume fractions of GGQLT-medicated serum and resveratrol. Intracellular lipid deposition in each group was detected by oil red O staining， the level of reactive oxygen species （ROS） in each group were detected by flow cytometry， the levels of glutathione peroxidase （GSH-Px）， superoxide dismutase （SOD）， triglyceride （TG） and malondialdehyde （MDA） in each group were detected by kits. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was used to measure the mRNA expression levels of nuclear transcription factor （NF）E2-related factor 2 （Nrf2）， heme oxygenase-1 （HO-1）， quinone oxidoreductase 1 （NQO1）， Kelch-like epichlorohydrin-associated protein-1 （Keap1）， NF-κB， thioredoxin interacting protein （TXNIP）， interleukin-1β （IL-1β） in HepG2 cells of each group. The protein expression of Nrf2， TXNIP in cells of each group was detected by Western blot. ResultFFA induced large accumulation of intracellular lipids. Compared with the normal group， the activities of GSH-Px and SOD were significantly decreased （P<0.01） and the contents of TG， ROS and MDA were significantly increased （P<0.05， P<0.01） in the model group. Compared with the model group， all GGQLT groups and resveratrol group could elevate intracellular SOD activity to different degrees （P<0.05， P<0.01） and significantly reduce the levels of intracellular ROS and MDA （P<0.05， P<0.01）， GGQLD high- and medium-dose groups and resveratrol group significantly elevated GSH-Px activity （P<0.01）， GGQLD medium- and low-dose groups and resveratrol group significantly decreased TG content （P<0.05， P<0.01）. Compared with the model group， GGQLT high- and medium-dose groups and resveratrol group could significantly upregulate the mRNA expression levels of Nrf2， HO-1 and NQO1 （P<0.01）， all GGQLT groups and resveratrol group could significantly downregulate the TXNIP protein expression level， as well as significantly downregulate the mRNA expression levels of Keap1， NF-κB （P<0.05， P<0.01）. Nrf2-siRNA transfection of cells revealed that Nrf2 expression was significantly downregulated （P<0.01） in the Nrf2-siRNA group of cells by comparing with NC-siRNA group at the corresponding dose of drugs， and the inhibitory effects of GGQLT and resveratrol on TXNIP， IL-1β were attenuated. ConclusionFFA induces the production of ROS and inflammatory factors in HepG2 cells， and GGQLT can improve the anti-inflammatory and antioxidant capacities of cells， and its mechanism may be related to the regulation of Nrf2/TXNIP signaling pathway， so as to improve NASH.

Effect of Danggui Buxuetang on Podocyte Pyroptosis in Diabetic Kidney Disease Rats： An Exploration Based on TXNIP/NLRP3/GSDMD Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo observe the effect of Danggui Buxuetang on podocyte pyroptosis in diabetic kidney disease （DKD） rats and to explore the possible mechanism of its prevention and treatment of DKD and podocyte pyroptosis. MethodEight of the 50 male SD rats were randomly classified into a normal group， and the remaining 42 were fed a high-glucose and high-fat diet for six weeks and then intraperitoneally injected with 35 mg·kg-1 streptozotocin （STZ） for inducing type 2 diabetes. After successful modeling， they were randomized into the model group， low- （0.72 g·kg-1） and high-dose （1.44 g·kg-1） Danggui Buxuetang group， and irbesartan （0.017 g·kg-1） group and gavaged with the corresponding drugs， while those in the normal group and model group with an equal volume of normal saline， once per day， for 20 weeks. During the medication， the fasting blood glucose （FBG） and 24 h urine protein （24 h-UTP） were measured regularly. After administration， the pathological changes in renal tissues were observed by periodic acid-silver metheramine （PASM） staining， followed by the observation of ultrastructural changes in podocytes under the transmission electron microscope （TEM）. Serum levels of interleukin-1β （IL-1β） and interleukin-18 （IL-18） were determined by enzyme-linked immunosorbent assay （ELISA）. The DNA damage in renal tissue cells of rats was detected by in situ nick end-labeling （TUNEL） assay. The protein expression levels of thioredoxin interacting protein （TXNIP）， cysteine-dependent aspartate-directed protease-1 （Caspase-1）， and gasdermin D （GSDMD） in renal tissues of rats were detected by immunohistochemistry （IHC）， the expression levels of nucleotide binding domain like receptor protein 3 （NLRP3） and Wilms tumor protein-1 （WT-1） in podocytes by immunofluorescent （IF） staining， and the expression levels of TXNIP/NLRP3/Caspase-1/GSDMD pathway proteins and Synaptopodin in renal podocytes by Western blot. ResultCompared with the normal group， the model group exhibited increased FBG and 24 h UTP， glomerular hypertrophy， mesangial hyperplasia， increased extracellular matrix， thickened basement membrane， K-W nodules， vacuolar degeneration in renal tubular epithelial cells， foot process fusion or loss， elevated serum IL-1β and IL-18 levels and TUNEL-positive cells in renal tissue， enhanced NLRP3 but diminished WT-1 expression in podocytes， down-regulated Synaptopodin protein expression， and up-regulated TXNIP/NLRP3/Caspase-1/GSDMD protein expression （P<0.01）. Compared with the model group， Danggui Buxuetang high-dose group remarkably lowered FBG， 24-h UTP， and TUNEL-positive cells in renal tissue， improved renal histopathology and podocyte injury and loss， down-regulated NLRP3 expression in podocytes and TXNIP/NLRP3/Caspase-1/GSDMD protein expression levels， and up-regulated WT-1 expression in podocytes and Synaptopodin protein expression （P<0.05， P<0.01）. ConclusionDanggui Buxuetang inhibits podocyte pyroptosis to reduce proteinuria and delays the development of DKD possibly by regulating the TXNIP/NLRP3/GSDMD signaling pathway.

Effect of Danggui Buxuetang on Podocyte Injury in Diabetic Kidney Disease Rats： An Exploration Based on RIPK1/RIPK3/MLKL Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo observe the effect of Danggui Buxuetang on the podocyte injury and receptor-interacting protein kinase 1/receptor-interacting protein kinase3/mixed lineage kinase domain-like protein （RIPK1/RIPK3/MLKL） signaling pathway in diabetic kidney disease （DKD） ratsand to explore its possible mechanism against DKD. MethodEight of the 50 SD rats were randomly classified intoa normal group， and the remaining were fed a high-glucose and high-fat diet for six weeks and then intraperitoneally injected with 0.035 g·kg-1streptozotocin （STZ） for inducing type 2 diabetes. After successful modeling，they were randomized into the model group，high- and low-dose （1.44，0.72 g·kg-1） Danggui Buxuetang groups， and irbesartan （0.017 g·kg-1）group. After 20 weeks of drug intervention， the fasting blood glucose （FBG）， kidney index （KI），and urinary microalbumin-to-urine creatinine ratio （UACR）were detected in each group. The pathological changes in renal tissue were observed by hematoxylin-eosin （HE） staining， followed by the observation of ultrastructural changes in podocytes under the transmission electron microscope. The levels of tumor necrosis factor-α （TNF-α） and interleukin-6 （IL-6） in renal tissue of rats were determined by enzyme-linked immunosorbent assay （ELISA）， and the protein expression levels of RIPK1， RIPK3， and MLKL in rat kidney tissue by immunohistochemistry. The apoptosis rate of podocytes was detected by in situ nick end-labeling （TUNEL） assay. The mRNA expression levels of RIPK1， RIPK3， and MLKL in kidney tissue of rats were measured by real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）， and the protein expression levels of RIPK， RIPK3， and MLKL and podocyte marker Wilms tumor protein-1 （WT-1） in rat kidney tissue were assayed by Western blot. ResultCompared with the normal group， the model group exhibited elevated FBG， UACR， and KI （P<0.01）， glomerular hypertrophy， thickened basement membrane， increased extracellular matrix， mesangial hyperplasia， foot process fusion or loss， enhanced apoptosis in renal tissue， up-regulated TNF-α and IL-6 levels （P<0.01） and RIPK1/RIPK3/MLKL mRNA and protein expression （P<0.01）， and down-regulated WT-1 protein expression. Compared with the model group， Danggui Buxuetang high-dose group significantly reduced the levels of FBG， UACR， and KI， improved renal histopathology， podocyte loss， and apoptosis in renal tissue， down-regulated TNF-α and IL-6 levels and RIPK1/RIPK3/MLKL mRNA and protein expression （P<0.05， P<0.01）， and up-regulated WT-1 protein expression. ConclusionDanggui Buxuetang alleviates podocyte injury and delays the development of DKD possibly by regulating the RIPK1/RIPK3/MLKL signaling pathway.

A study on the changes and clinical value of plasma RIP3 levels in neonatal late-onset sepsis / 中国新生儿科杂志

Objective:To study the changes of plasma receptor interacting protein 3 (RIP3) levels in neonatal late-onset sepsis (LOS) and to determine its clinical value.Methods:From October 2019 to April 2021, plasma samples and clinical data of LOS infants admitted to our hospital were prospectively studied. Infants with similar gestational ages admitted for non-infectious diseases were assigned into the control group. Enzyme-linked immunoassay was used to determine plasma RIP3 levels. The clinical value of plasma RIP3 in the diagnosis and treatment of neonatal LOS were analyzed.Results:A total of 152 cases (76 in the LOS group and 76 in the control group) were included in the study. No significant differences existed in the baseline data between the two groups. A total of 226 plasma samples were collected (76 samples from the LOS group before treatment, 74 samples after treatment and 76 samples from the control group). The plasma RIP3 level of LOS group before treatment (19.9±6.3 ng/ml) was significantly higher than the control group (11.4±3.5 ng/ml) and the after treatment group (11.9±3.5 ng/ml) ( P<0.05). The plasma RIP3 level had good diagnostic value for neonatal LOS (AUC=0.884). With cut-off value of 15.5 ng/ml, the plasma RIP3 showed the best diagnostic efficacy (Youden index 0.658, sensitivity 72.4%, specificity 93.4%, positive likelihood ratio 11.0, negative likelihood ratio 0.3). Conclusions:Plasma RIP3 level is closely related with neonatal LOS and may be used for the early diagnosis and therapeutic evaluation of neonatal LOS.

Necroptosis in inflammatory bowel disease: A potential effective target / 中南大学学报(医学版)

The morbidity of inflammatory bowel diseases (IBD) is rising rapidly but no curative therapies to prevent its recurrence. Cell death is crucial to maintaining homeostasis. Necroptosis is a newly identified programmed cell death and its roles played in IBD need to be explored. Necroptosis is mediated by receptor interacting protein kinase 1 (RIPK1), RIPK3, and mixed lineage kinase domain-like protein (MLKL), which resulted in cell swelling, plasma membrane rupture, intracellular content leaking, and eventually cell death as well as the promotion of inflammation. Studies have found that inhibiting necroptosis alleviated IBD in animal models and IBD patients with an increased level of necroptosis in inflammatory tissues, indicating that necroptosis is related to the pathogenesis of IBD. However, due to the complexity in regulation of necroptosis and the involvement of multiple functions of relevant signaling molecules, the specific mechanism remains elusive. Necroptosis may play a vital regulatory role in the pathogenesis of IBD, which provides a new idea and method for further exploring the therapeutic target of IBD.

Effect of Dahuang Xiezhuo Prescription on Renal Interstitial Fibrosis of Rats with 5/6 Nephrectomy by Regulating ROS/TXNIP/NLRP3 Pathway / 中国实验方剂学杂志

ObjectiveTo observe the effect of Dahuang Xiezhuo prescription on the changes in renal pathology and reactive oxygen species （ROS）/thioredoxin-interacting protein （TXNIP）/NOD-like receptor protein 3 （NLRP3） pathway expression in the kidney tissues of rats with 5/6 nephrectomy， and to explore the mechanism of Dahuang Xiezhuo prescription in protecting renal function and delaying renal interstitial fibrosis and the possibility. MethodNinety healthy male SD rats were randomly divided into a sham operation group， a model group， low， medium， and high-dose （6.825， 13.65， 27.30 g·kg-1） Dahuang Xiezhuo prescription groups， and a Niaoduqing granule group （2.60 g·kg-1）. Except the sham operation group， 5/6 nephrectomy was used to replicate the rat model of chronic renal failure （CRF）. After modeling， each administration group was given the corresponding dose of drug suspension by intragastric administration， once a day for consecutive 8 weeks. After administration， serum creatinine （SCr） and urea nitrogen （BUN） levels and 24 h urinary protein quantification （UTP） levels were detected. Western blot assay was used to detect the protein expressions of thioredoxin （TRX）， TXNIP， and NLRP3. The protein expressions of TRX， TXNIP， NLRP3， apoptosis-associated speck-like protein containing a CARD （ASC）， transformation growth factor-β （TGF-β）， Collagen Ⅳ， α-smooth muscle actin （α-SMA）， and fibronectin （FN） were detected by immunohistochemistry. ResultAs compared with the sham operation group， serum levels of SCr， BUN， and UTP in the model group were increased （P<0.05）， TRX， TXNIP， NLRP3， ASC， TGF-β， Collagen Ⅳ， α-SMA， and FN proteins were increased （P<0.01）， and renal interstitial fibrosis significantly occurred. As compared with the model group， the levels of SCr， 24 h BUN， and UTP in the low， medium， and high-dose Dahuang Xiezhuo prescription groups and the Niaoduqing granule group were decreased to varying degrees （P<0.05）， TRX， TXNIP， NLRP3， ASC， TGF-β， Collagen Ⅳ， α-SMA， and FN were decreased （P<0.01）， and renal interstitial fibrosis was improved to varying degrees. ConclusionDahuang Xiezhuo prescription can protect renal function and delay renal interstitial fibrosis in rats with CRF.

Expression and clinical significance of receptor-interacting protein kinase-3 levels in children with mycoplasma pneumoniae pneumonia and their relationship with inflammatory factors / 中国综合临床

Objective:To explore the level of expression, clinical significance of receptor-interacting protein kinase-3(RIPK3) in the bronchoalveolar alveolar lavage fluid (BALF) of mycoplasma pneumoniae pneumonia(MPP) in children and the relationship between RIPK3 and various cytokines.Methods:Using a case-control study, 30 refractory mycoplasma pneumoniae pneumonia children treated in Children′s Hospital of Xuzhou Medical University from February 2019 to February 2021 were selected as the MRPP group, 35 mycoplasma pneumoniae pneumonia children as the RPP group.Meanwhile, 20 sex- and age-matched hospitalized children with bronchial foreign body were selected as the case-control group.In all subjects, protein levels of RIPK3 and mixed lineage kinase domain-like protein(MLKL) were determined by Western Blot.Meanwhile, levels of IL-6, IL-1β and TNF-α were determined by enzyme linked immuno sorbent assay(ELISA). Compare levels of different parameters between the three groups and analyze the correlation between levels of RIPK3 and MLKL, L-6, IL-1β, TNF-α in the BALF of MPP children using Spearman rank correlation analysis.Results:There were statistically significant differences in the levels of RIPK3, MLKL, IL-6, IL-1β and TNF-α in BALF between RMPP group, MPP group and control group (all P<0.001). Pairwise comparisons also showed statistical differences, and the RMPP group was the highest, followed by MPP group, and the control group was the lowest.The level of RIPK3 in BALF of MPP children was positively correlated with MLKL, IL-6, IL-1β and TNF-α ( r=0.711, 0.676, 0.725, 0.651, P<0.001). Linear regression analysis shows: MLKL=0.432×RIPK3. Conclusion:RIPK3 may be involved in the occurrence and development of MPP in children, and is closely related to cytokines such as IL-6, IL-1β and TNF-α.

Proline-serine-threonine phosphatase interacting protein 1-associated autoinflammatory diseases / 中华实用儿科临床杂志

In addition to the pyogenic arthritis, pyoderma gangrenosum and acne(PAPA) syndrome, autoinflammatory diseases caused by mutations in the proline-serine-threonine phosphatase interacting protein 1 (PSTPIP1) gene also include a group of clinical syndromes such as PSTPIP1-associated myeloid-related proteinemia inflammatory(PAMI) and pyoderma gangrenosum, acne, and hidradenitis suppurativa(PASH) syndrome.In this paper, the expanded spectrum and clinical characteristics of PSTPIP1 related autoinflammatory diseases were reviewed, so as to deepen clinicians′ understanding of this disease, facilitate early diagnosis, and finally improve the prognosis of patients.

Research progress of thioredoxin system / 国际生物医学工程杂志

The thioredoxin system is composed of thioredoxin (Trx), thioredoxin reductase (TR) and reduced nicotinamide adenine dinucleotide phosphate. Trx is an important antioxidant molecule that can resist cell death caused by various stresses and plays a prominent role in redox reactions. TR is a protein containing selenium (selenocysteine), mainly in three forms, i.e. TR1, TR2 and TR3. TR1 mainly distributed in the cytoplasm, TR2 mainly distributed in the mitochondria, and TR3 mainly distributed in the testes. TR can regulate cell growth and apoptosis. After the cell becomes cancerous, the expression of TR increases to promote cell growth and metastasis. Trx system is closely related to neurodegenerative diseases, parasitic infections, acquired immunodeficiency syndrome, rheumatoid arthritis, hypertension, myocarditis and so on. The Trx system can remove the reactive oxygen species (ROS) in the body, keep the inside and outside of the cell in a balanced state, and it interacts with the thioredoxin interacting protein (TXNIP), which plays an important role in the regulation of glucose metabolism and tumor treatment. The Trx system is an important target for drug treatment of many diseases. In this paper, the research progress of the thioredoxin system was reviewed.

Essential Oil from Alpiniae Zerumbet Fructus Alleviates High Glucose-induced HUVEC Injury via TXNIP Signaling Pathway / 中国实验方剂学杂志

Objective:To study the protective effect of essential oil from Alpiniae Zerumbet Fructus （EOAZF） against high glucose （HG）-induced injury of human umbilical vein endothelial cells （HUVECs） <italic>in vitro</italic>， so as to provide experimental evidence for the treatment of diabetes-induced cardiovascular diseases with EOAZF. Method:The cells were divided into the normal group， model group （25 mmol·L^-1 glucose）， positive control group （100 mg·L^-1 vitamin C）， and the low- （0.25 μg·L^-1）， medium- （1 μg·L^-1）， and high-dose （4 μg·L^-1） EOAZF groups. The HUVECs were damaged by HG. The secretion amounts of malondialdehyde （MDA）， nitric oxide （NO）， and endothelin-1 （ET-1） in HUVECs of different groups were measured to assess the protective effect of EOAZF against HG-induced injury. The effects of EOAZF on the apoptosis and reactive oxygen species （ROS） generation of HUVECs damaged by HG were detected by Annexin V-fluorescein isothiocyanate/propidium iodide （Annexin V-FITC/PI） staining and dichloro-dihydro-fluorescein diacetate （DCFH-DA） assay. The protein and mRNA expression levels of thioredoxin interacting protein （TXNIP） and thioredoxin 1 （Trx-1） were determined by Western blot and Real-time polymerase chain reaction （Real-time PCR）， followed by the measurement of total intracellular Trx-1 activity with insulin disulfide reduction method. Result:The comparison with the control group revealed that the proliferation of HUVECs in the model group was significantly inhibited and their shape was damaged. Compared with the model group， EOAZF protected HUVECs against HG-induced injury in a concentration-dependent manner. The secretion amounts of MDA and ET-1 （<italic>P</italic><0.05） in the model group were increased in contrast to those in the control group， while the NO level was decreased （<italic>P</italic><0.01）. Compared with the model group， EOAZF at all the three concentrations， especially at 4 μg·L^-1， obviously reduced the secretion of MDA and ET-1 （<italic>P</italic><0.05）， but elevated NO after HG induction （<italic>P</italic><0.05）. The cell apoptosis assay and ROS detection results demonstrated that the apoptosis and ROS level in the model group were higher than those in the control group （<italic>P</italic><0.01）. Compared with the model group， EOAZF at 4 μg·L^-1significantly lowered the ROS level and apoptosis （<italic>P</italic><0.05） of HUVECs damaged by HG. The Western blot assay and Trx-1 activity detection uncovered that the protein and mRNA expression levels of TXNIP in the model group were significantly up-regulated as compared with those in the control group （<italic>P</italic><0.05）， whereas the Trx-1 activity was decreased （<italic>P</italic><0.01）. Compared with the model group， EOAZF at 4 μg·L^-1significantly down-regulated the mRNA and protein （<italic>P</italic><0.05） expression levels of TXNIP and enhanced the total Trx-1 activity （<italic>P</italic><0.05） in HUVECs， thus suppressing the oxidative stress. Conclusion:EOAZF exerts the protective effects against HG-induced injury in HUVECs by improving the endothelial function and reducing intracellular ROS and apoptosis. Its efficacy in anti-oxidative stress may be related to the down-regulation of mRNA and protein expression levels of TXNIP and the enhancement of Trx-1 activity.

Receptor-interacting Protein Kinase 3 Promotes Osteoarthritis-related Changes by Upregulating Integrin Subunit Beta 3 / 中国生物化学与分子生物学报

Osteoarthritis (OA) is the most common chronic disabling joint disease, and currently there is no effective treatment for the cause. Necroptosis plays a key role in many diseases, and receptor-interacting protein kinase 3 (RIP3) is a key regulator during necroptosis process. Studies have shown that the expression level of RIP3 was significantly upregulated in human and mouse OA degenerative cartilage tissues, suggesting the occurrence of necroptosis. However, the specific pathophysiological role of RIP3 in cartilage is still unclear. This study intends to sequence and analyze the transcriptome of chondrocytes before and after RIP3 overexpression, and explore the specific functional mechanism of RIP3 in OA pathogenesis. RNA sequencing results showed that overexpression of RIP3 induced upregulation of 244 genes and downregulation of 277 genes in chondrocytes. Sixteen candidate target genes were screened out by constructing gene co-expression network for further verification at mRNA level, and the results suggested that RIP3 had the most significant inductive effect on the expression of phosphoinositide-3kinase, regulatory subunit 5 (Pik3r5), integrin subunit beta 3 (Itgb3) and MYB proto-oncogene like 2 (Mybl2). Results from CCK-8 and lactate dehydrogenase activity analysis showed that silencing the expression of Itgb3 by siRNA significantly rescued chondrocyte viability decline and necroptosis induced by RIP3, and it also inhibited the upregulating effect of RIP3 on the expression of catabolism-related genes Mmp1, Mmp13 and Il6, as well as the downregulating effect of RIP3 on the expression of anabolism-related genes Acan, Col2a1 and Sox9. This study has demonstrated that RIP3 promotes chondrocyte necrosis and cartilage matrix metabolism disorders by upregulating the expression of Itgb3 in chondrocytes, and ultimately leads to cartilage degeneration. These findings provided potential novel targets for the clinical treatment of OA, and further clarified the pathophysiological significance of necroptosis.