Puerarin protects rat brain against ischemia/reperfusion injury by suppressing autophagy via the AMPK-mT OR-ULK1 signaling pathway

Puerarin suppresses autophagy to alleviate cerebral ischemia/reperfusion injury, and accumulating evidence indicates that the AMPKm TOR signaling pathway regulates the activation of the autophagy pathway through the coordinated phosphorylation of ULK1. In this study, we investigated the mechanisms underlying the neuroprotective effect of puerarin and its role in modulating autophagy via the AMPK-m TOR-ULK1 signaling pathway in the rat middle cerebral artery occlusion model of cerebral ischemia/reperfusion injury. Rats were intraperitoneally injected with puerarin, 50 or 100 mg/kg, daily for 7 days. Then, 30 minutes after the final administration, rats were subjected to transient middle cerebral artery occlusion for 90 minutes. Then, after 24 hours of reperfusion, the Longa score and infarct volume were evaluated in each group. Autophagosome formation was observed by transmission electron microscopy. LC3, Beclin-1 p62, AMPK, m TOR and ULK1 protein expression levels were examined by immunofluorescence and western blot assay. Puerarin substantially reduced the Longa score and infarct volume, and it lessened autophagosome formation in the hippocampal CA1 area following cerebral ischemia/reperfusion injury in a dose-dependent manner. Pretreatment with puerarin（50 or 100 mg/kg） reduced Beclin-1 expression and the LC3-II/LC3-I ratio, as well as p-AMPK and p S317-ULK1 levels. In comparison, it increased p62 expression. Furthermore, puerarin at 100 mg/kg dramatically increased the levels of p-m TOR and p S757-ULK1 in the hippocampus on the ischemic side. Our findings suggest that puerarin alleviates autophagy by activating the APMK-m TOR-ULK1 signaling pathway. Thus, puerarin might have therapeutic potential for treating cerebral ischemia/reperfusion injury.

Hepatitis C virus core protein-induced miR-93-5p upregulation inhibits interferon signaling pathway by targeting IFNAR1

AIM To investigate the mechanism by which hepatitis C virus(HCV) core protein-induced mi R-93-5 p up-regulation regulates the interferon(IFN) signaling pathway.METHODS HCV-1 b core protein was exogenously expressed in Huh7 cells using pc DNA3.1(+) vector. The expression of mi R-93-5 p and interferon receptor 1(IFNAR1) was measured using quantitative reverse transcriptionpolymerase chain reaction and Western blot. The protein expression and phosphorylation level of STAT1 were evaluated by Western blot. The overexpression and silencing of mi R-93-5 p and IFNAR1 were performed using mi R-93-5 p agomir and antagomir, and pc DNA3.1-IFNAR1 and IFNAR1 si RNA, respectively. Luciferase assay was used to identify whether IFNAR1 is a target of mi R-93-5 p. Cellular experiments were also conducted.RESULTS Serum mi R-93-5 p level was increased in patients with HCV-1 b infection and decreased to normal level after HCV-1 b clearance, but persistently increased in those with pegylated interferon-α resistance, compared with healthy subjects. Serum mi R-93-5 p expression had an AUC value of 0.8359 in distinguishing patients with pegylated interferon-α resistance from those with pegylated interferon-α sensitivity. HCV-1 b core protein increased mi R-93-5 p expression and induced inactivation of the IFN signaling pathway in Huh7 cells. Furthermore, IFNAR1 was identified as a direct target of mi R-93-5 p, and IFNAR1 restore could rescue mi R-93-5 p-reduced STAT1 phosphorylation, suggesting that the mi R-93-5 p-IFNAR1 axis regulates the IFN signaling pathway.CONCLUSION HCV-1 b core protein-induced mi R-93-5 p up-regulation inhibits the IFN signaling pathway by directly targeting IFNAR1, and the mi R-93-5 p-IFNAR1 axis regulates STAT1 phosphorylation. This axis may be a potential therapeutic target for HCV-1 b infection.

SphK1-S1P信号通路的激活参与糖尿病大鼠肾损伤的研究

目的观察鞘氨醇激酶1-1-磷酸鞘氨醇(SphK1-S1P)信号通路在链脲佐菌素(STZ)诱导的糖尿病大鼠肾脏中的激活情况。方法观察STZ诱导的糖尿病大鼠肾脏中SphK1-S1P信号通路的激活情况。分别检测大鼠肾脏功能及病理指标。采用液质联用分析SphK1活性及S1P含量。并采用免疫组织化学、Real-time PCR和Western blot法检测了SphK1的表达。结果 STZ诱导的糖尿病大鼠血糖、肾重体质量比、24h尿蛋白显著升高,糖尿病大鼠肾脏系膜区肥大,细胞外基质扩展。同时,糖尿病大鼠肾脏中SphK1-S1P信号通路被激活,表现为SphK1表达和活性增加,以及S1P生成增加。结论糖尿病大鼠肾脏中存在SphK1-S1P信号通路的激活,可能参与了糖尿病大鼠肾损伤过程。

益肾解毒通络胶囊联合氯沙坦钾对糖尿病肾病患者SphK1-S1P信号通路的影响

目的 研究糖尿病肾病(DN)患者采用益肾解毒通络胶囊、氯沙坦钾联合治疗的临床效果。方法 选取2016年3月至2018年5月本院DN患者104例,按随机数字表法分观察组(n=52)与对照组(n=52)。对照组以氯沙坦钾治疗,观察组以益肾解毒通络胶囊、氯沙坦钾联合治疗。对比两组疗效、不良反应及治疗前、治疗3个疗程后鞘氨醇激酶1(Sphk1)活性、血糖水平[空腹血糖(FPG)、餐后2 h血糖(2hPG)]、肾功能[血清胱抑素C(CysC)水平、尿白蛋白/肌酐(UACR)值]。结果 (1)疗效:观察组总有效率为90.38%(47/52),较对照组的75.00%(39/52)高(P<0.05);(2)中医证候积分:治疗3个疗程后两组中医证候均不同程度改善,且观察组中医证候积分低于对照组(P<0.05);(3)Sphk1活性:治疗3个疗程后两组Sphk1活性均显著改善,且观察组低于对照组(P<0.05);(4)血糖水平:治疗3个疗程后两组FPG、2hPG均无显著变化,组间对比,差异无统计学意义(P >0.05);(5)肾功能:治疗3个疗程后两组血清CysC水平、UACR值均显著改善,且观察组低于对照组(P<0.05);(6)不良反应:两组均未出现头晕、恶心、皮疹、心慌等不良反应。结论 DN患者采用益肾解毒通络胶囊、氯沙坦钾联合治疗效果显著,能有效调节Sphk1-S1P通路,改善临床症状及肾功能,且安全性高。

Effects of Cigu Xiaozhi Formula on miR-378a-3p Expression and Hh Signaling Pathway in TGF-β1 Induced LX2 Cells

[Objectives]To observe the effects of Cigu Xiaozhi Formula on miR-378a-3p expression and Hh signaling pathway in TGF-β1 induced and activated LX2 cells.[Methods]Cells were divided into control group,induction group,drug-containing serum group,miR-378a-3p inhibitor group,and miR inhibitor NC group.CCK-8 method was used to detect the cell viability of each group,and flow cytometry was used to detect the apoptosis rate of each group.RT-qPCR was used to detect the expression of miR-378a-3p in each group s cells,and RT-qPCR and Western blot were used to detect mRNA and protein expression of Shh,Gli1,Gli2,Col-I,andα-SMA in each group s cells.[Results]Compared with the control group,the cell viability and expression of Shh,Gli1,Gli2,Col-I,andα-SMA mRNA and protein in induction group increased(P<0.01),while the expression of miR-378a-3p decreased(P<0.01).Compared with the induction group,the cell viability and expression of Shh,Gli1,Gli2,Col-I,α-SMA mRNA andα-SMA and Gli2 protein decreased in drug-containing serum group(P<0.05),while cell apoptosis rate and miR-378a-3p expression increased(P<0.01).In miR-378a-3p inhibitor group,cell viability and the expression of Shh,Gli1,Gli2,Col-I,α-SMA mRNA and Gli1,Gli2,α-SMA protein increased(P<0.05,P<0.01),while the apoptosis rate and miR-378a-3p expression decreased(P<0.05,P<0.01).[Conclusions]Cigu Xiaozhi Formula containing serum can upregulate miR-378a-3p expression and downregulate the expression of Gli2 andα-SMA in TGF-β1 induced LX2 cells,thereby inhibiting the activation of LX2 cells and exerting the effects of anti liver fibrosis.

MiR-146a-5p targeting SMAD4 and TRAF6 inhibits adipogenensis through TGF-β and AKT/mTORC1 signal pathways in porcine intramuscular preadipocytes

Background: Intramuscular fat(IMF) content is a vital parameter for assessing pork quality. Increasing evidence has shown that microRNAs(miRNAs) play an important role in regulating porcine IMF deposition. Here, a novel miRNA implicated in porcine IMF adipogenesis was found, and its effect and regulatory mechanism were further explored with respect to intramuscular preadipocyte proliferation and differentiation.Results: By porcine adipose tissue miRNA sequencing analysis, we found that miR-146a-5p is a potential regulator of porcine IMF adipogenesis. Further studies showed that miR-146a-5p mimics inhibited porcine intramuscular preadipocyte proliferation and differentiation, while the miR-146a-5p inhibitor promoted cell proliferation and adipogenic differentiation. Mechanistically, miR-146a-5p suppressed cell proliferation by directly targeting SMAD family member 4(SMAD4) to attenuate TGF-β signaling. Moreover, miR-146a-5p inhibited the differentiation of intramuscular preadipocytes by targeting TNF receptor-associated factor 6(TRAF6) to weaken the AKT/mTORC1 signaling downstream of the TRAF6 pathway.Conclusions: MiR-146a-5p targets SMAD4 and TRAF6 to inhibit porcine intramuscular adipogenesis by attenuating TGF-β and AKT/mTORC1 signaling, respectively. These findings provide a novel miRNA biomarker for regulating intramuscular adipogenesis to promote pork quality.

Oxidized low-density lipoprotein receptor 1:a novel potential therapeutic target for intracerebral hemorrhage

Oxidized low-density lipoprotein receptor 1(OLR1)is upregulated in neurons and participates in hypertension-induced neuronal apoptosis.OLR1 deletion exerts protective effects on cerebral damage induced by hypertensive-induced stroke.Therefore,OLR1 is likely involved in the progress of intracerebral hemorrhage.In this study,we examined the potential role of OLR1 in intracerebral hemorrhage using a rat model.OLR1 small interfering RNA(10μL;50 pmol/μL)was injected into the right basal ganglia to knock down OLR1.Twenty-four hours later,0.5 U collagenase type VII was injected to induce intracerebral hemorrhage.We found that knockdown of OLR1 attenuated neurological behavior impairment in rats with intracerebral hemorrhage and reduced hematoma,neuron loss,inflammatory reaction,and oxidative stress in rat brain tissue.We also found that silencing of OLR1 suppressed ferroptosis induced by intracerebral hemorrhage and the p38 signaling pathway.Therefore,silencing OLR1 exhibits protective effects against secondary injury of intracerebral hemorrhage.These findings suggest that OLR1 may be a novel potential therapeutic target for intracerebral hemorrhage.

中医药抑制SphK1-S1P通路治疗糖尿病肾病研究进展

糖尿病肾病的发生、发展与其细胞信号转导通路的失调有着密切的联系,中医药以其低毒性、多靶点的优势在调控信号通路方面有着独特的功效。黄连素、虎杖苷、姜黄素、马钱苷等中药单体、提取物及中药复方均可以通过抑制SphK1/S1P信号通路,来拮抗糖尿病肾纤维化的进程。就目前研究而言:①对单味中药及中药有效成分提取物的研究,不能合理地解释中药多靶点调控细胞信号通路的机制;②中药复方成分复杂、组分不明、结构不清等因素限制了体外实验的细胞学和分子生物学水平研究;③各信号通路之间的关系错综复杂,研究不易开展。今后需更加系统地研究如何发挥中医药低毒性、多靶点的优势,阐明其调控机理,以更好地指导临床。

Role and related mechanism of S1P / S1P1 signal pathway during post conditioning of hypertrophic cardiomyocytes

Objective To study the role and mechanism of sphingosine-phosphate(S1P)/sphingosine-1-phosphate receptor 1(S1P1)signal pathway during post conditioning of hypertrophic cardiomyocytes.Methods Neonatal rat cardiomyocytes were isolated and cultured,then stimulated by norepinephrine(NE)to induce cardiomyocytes hypertrophy.Using tri-gas incubator to create

Sphingosine kinase 1 promotes growth of glioblastoma by increasing inflammation mediated by the NF-κB/IL-6/STAT3 and JNK/PTX3 pathways

Glioblastoma(GBM)is the most challenging malignant tumor of the central nervous system because of its high morbidity,mortality,and recurrence rate.Currently,mechanisms of GBM are still unclear and there is no effective drug for GBM in the clinic.Therefore,it is urgent to identify new drug targets and corresponding drugs for GBM.In this study,in silico analyses and experimental data show that sphingosine kinase 1(SPHK1)is up-regulated in GBM patients,and is strongly correlated with poor prognosis and reduced overall survival.Overexpression of SPHK1 promoted the proliferation,invasion,metastasis,and clonogenicity of GBM cells,while silencing SPHK1 had the opposite effect.SPHK1 promoted inflammation through the NF-κB/IL-6/STAT3 signaling pathway and led to the phosphorylation of JNK,activating the JNK-JUN and JNK-ATF3 pathways and promoting inflammation and proliferation of GBM cells by transcriptional activation of PTX3.SPHK1 interacted with PTX3 and formed a positive feedback loop to reciprocally increase expression,promote inflammation and GBM growth.Inhibition of SPHK1 by the inhibitor,PF543,also decreased tumorigenesis in the U87-MG and U251-MG SPHK1 orthotopic mouse models.In summary,we have characterized the role and molecular mechanisms by which SPHK1 promotes GBM,which may provide opportunities for SPHK1-targeted therapy.

The functional analysis of transiently upregulated miR-101 suggests a “braking” regulatory mechanism during myogenesis

Skeletal muscle differentiation is a highly coordinated process that involves many cellular signaling pathways and microRNAs(miRNAs).A group of muscle-specific miRNAs has been reported to promote myogenesis by suppressing key signaling pathways for cell growth.However,the functional role and regulatory mechanism of most non-muscle-specific miRNAs with stage-specific changes during differentiation are largely unclear.Here,we describe the functional characterization of miR-101a/b,a pair of non-muscle-specific miRNAs that show the largest change among a group of transiently upregulated miRNAs during myogenesis in C2C12 cells.The overexpression of miR-101a/b inhibits myoblast differentiation by suppressing the p38/MAPK,Interferon Gamma,and Wnt pathways and enhancing the C/EBP pathway.Mef2a,a key protein in the p38/MAPK pathway,was identified as a direct target of miR-101a/b.Interestingly,we found that the long non-coding RNA(lncRNA)Malat1,which promotes muscle differentiation,interacts with miR-101a/b,and this interaction competes with Mef2a mRNA to relieve the inhibition of the p38/MAPK pathway during myogenesis.These results uncovered a“braking”role in differentiation of transiently upregulated miRNAs and provided new insights into the competing endogenous RNA(ceRNA)regulatory mechanism in myoblast differentiation and myogenesis.

Advance in the Study of the Mechanisms Regulated by Sphingosine-1-Phosphate

Sphingosine-1-phosphate（S1P） is a bioactive lipid messenger in the cells that regulate gene expression and NF-κB signal pathway through unknown mechanisms.Recently,Cheng Luo,associate professor of DDDC in Shanghai Institute of Materia Medica,whose project was funded by the National Natural Science Foundation of China,joined in a research team led by Professor Sarah Spiegel of Virginia Commonwealth University.The team continuously made significant breakthroughs in understanding the regulation mechanism of Sphingosine-1- Phosphate.In September 2009,in a paper published on SCIENCE magazine（Science 2009, 325：1254-7）,they firstly demonstrated that S1P is a physiologically important regulator of histone deacetylases（HDACs）,HDACs are direct intracellular targets of S1P.Furthermore,they identified the mechanism that S1P regulates gene expression through regulating the activity of HDACs.In June 24th,2010,in another paper to be published on NATURE magazine（Nature 2010,June 24th,advance online publication,（doi：10.1038/ nature09128）） which reports the regulation of NF-κB signaling pathway by S1P.They demonstrate that S1P is the missing cofactor for TRAF2（tumour-necrosis factor receptor-associated factor 2） and indicate a new paradigm for the regulation of lysine-63- linked poly-ubiquitination.The study also highlight the key role of SphK1 and its product S1P in TNF-αsignalling and the canonical NF-κB activation pathway, and then play crucial role in inflammatory,antiapoptotic and immune processes.The identification of new mechanisms fay which S1P regulates gene expression and TNF and NF-κB signaling pathway will light up the road to develop novel inhibitors that might be useful for treatment of cancer and in- flammatory diseases.