Suppressing high mobility group box-1 release alleviates morphine tolerance via the adenosine5'-monophosphate-activated protein kinase/heme oxygenase-1 pathway

Opioids,such as morphine,are the most potent drugs used to treat pain.Long-term use results in high tolerance to morphine.High mobility group box-1(HMGB1) has been shown to participate in neuropathic or inflammatory pain,but its role in morphine tolerance is unclear.In this study,we established rat and mouse models of morphine tolerance by intrathecal injection of morphine for 7 consecutive days.We found that morphine induced rat spinal cord neurons to release a large amount of HMGB1.HMGB1 regulated nuclear factor κB p65 phosphorylation and interleukin-1β production by increasing Toll-like receptor 4receptor expression in microglia,thereby inducing morphine tolerance.Glycyrrhizin,an HMGB1 inhibito r,markedly attenuated chronic morphine tole rance in the mouse model.Finally,compound C(adenosine 5’-monophosphate-activated protein kinase inhibitor) and zinc protoporphyrin(heme oxygenase-1 inhibitor)alleviated the morphine-induced release of HMGB1 and reduced nuclear factor κB p65 phosphorylation and interleukin-1β production in a mouse model of morphine tolerance and an SH-SY5Y cell model of morphine tole rance,and alleviated morphine tolerance in the mouse model.These findings suggest that morphine induces HMGB1 release via the adenosine 5’-monophosphate-activated protein kinase/heme oxygenase-1 signaling pathway,and that inhibiting this signaling pathway can effectively reduce morphine tole rance.

p21-activated kinase signalling in pancreatic cancer: New insights into tumour biology and immune modulation

Pancreatic cancer is one of the most aggressive and lethal malignancies worldwide, with a very poor prognosis and a five-year survival rate less than 8%. This dismal outcome is largely due to delayed diagnosis, early distant dissemination and resistance to conventional chemotherapies. Kras mutation is a well-defined hallmark of pancreatic cancer, with over 95% of cases harbouring Kras mutations that give rise to constitutively active forms of Kras. As important down-stream effectors of Kras, p21-activated kinases(PAKs) are involved in regulating cell proliferation, apoptosis, invasion/migration and chemo-resistance. Immunotherapy is now emerging as a promising treatment modality in the era of personalized anti-cancer therapeutics. In this review, basic knowledge of PAK structure and regulation is briefly summarised and the pivotal role of PAKs in Kras-driven pancreatic cancer is highlighted in terms of tumour biology and chemoresistance. Finally, the involvement of PAKs in immune modulation in the tumour microenvironment is discussed and the potential advantages of targeting PAKs are explored.

Group Ⅱ p21-activated kinases as therapeutic targets in gastrointestinal cancer

P21-activated kinases(PAKs) are central players in various oncogenic signaling pathways. The six PAK family members are classified into group Ⅰ(PAK1-3) and group Ⅱ(PAK4-6). Focus is currently shifting from group Ⅰ PAKs to group Ⅱ PAKs. Group Ⅱ PAKs play important roles in many fundamental cellular processes, some of which have particular significance in the development and progression of cancer. Because of their important functions, group Ⅱ PAKs have become popular potential drug target candidates. However, few group Ⅱ PAKs inhibitors have been reported, and most do not exhibit satisfactory kinase selectivity and "drug-like" properties. Isoform- and kinase-selective PAK inhibitors remain to be developed. This review describes the biological activities of group Ⅱ PAKs, the importance of group Ⅱ PAKs in the development and progression of gastrointestinal cancer, and smallmolecule inhibitors of group Ⅱ PAKs for the treatment of cancer.

Gene polymorphisms of interleukin-28, p21-activated protein kinases 4, and response to interferon-α based therapy in Chinese patients with chronic hepatitis B

Background Peg-lnterferon-a treatment is expensive and associated with considerable adverse effects, selection of patients with the highest probability of response is essential for clinical practice. The objective of this study was to assess the relationship between the gene polymorphisms of interleukin-28 （IL-28）, p21-activated protein kinase 4 （PAK4） and the response to interferon treatment in chronic hepatitis B patients. Methods Two hundred and forty interferon-naive treatment HBeAg seropositive chronic hepatitis B patients were enrolled in the present prospective nested case-control study. Peripheral blood samples were collected, including 92 with favorable response and 148 without response to the interferon treatment. Rs8099917, rs12980602, and rs9676717 SNP was genotyped using matrix assisted laser desorption/ionization time of flight mass spectrometry （MALDI-TOF MS）. Results IL-28 genotype was not associated with response to interferon treatment （OR for GT/GG vs. TT, 0.881 （95% CI 0.388-2.002）; P=0.762; OR for CT/CC vs. TT, 0.902 （95% CI 0.458-1.778）; P=-0.766）. Rs9676717 in PAK4 genotype was independently associated with the response （OR for CT/CC vs. TT, 0.524 （95% CI 0.310-0.888）; P=0.016）. When adjusting for age, gender, smoking, drinking, levels of hepatitis B virus DNA, and alanine aminotransferase （ALT）, rs9676717 genotype TT appeared to be associated with a higher probability of response for interferon treatment （OR, 0.155 （95% CI 0.034-0.700）; P=0.015）. Conclusion Genotype TTfor rs9676717 in PAK4 gene and no drinking may be predictive of the interferon-a treatment success.

Targeting P21-activated kinase suppresses proliferation and enhances chemosensitivity in T-cell lymphoblastic lymphoma

T-cell lymphoblastic lymphoma(T-LBL)is a highly aggressive non-Hodgkin lymphoma with a poor prognosis.P21-activated kinase(PAK)is a component of the gene expression-based classifier that can predict the prognosis of T-LBL.However,the role of PAK in T-LBL progression and survival remains poorly understood.Herein,we found that the expression of PAK1 was significantly higher in T-LBL cell lines(Jurkat,SUP-T1,and CCRF-CEM)compared to the human T-lymphoid cell line.Moreover,PAK2 mRNA level of 32 relapsed T-LBL patients was significantly higher than that of 37 cases without relapse(P=.012).T-LBL patients with high PAK1 and PAK2 expression had significantly shorter median RFS than those with low PAK1 and PAK2 expression(PAK1,P=.028;PAK2,P=.027;PAK1/2,P=.032).PAK inhibitors,PF3758309(PF)and FRAX597,could suppress the proliferation of T-LBL cells by blocking the G1/S cell cycle phase transition.Besides,PF could enhance the chemosensitivity to doxorubicin in vitro and in vivo.Mechanistically,through western blotting and RNA sequencing,we identified that PF could inhibit the phosphorylation of PAK1/2 and downregulate the expression of cyclin D1,NF-κB and cell adhesion signaling pathways in T-LBL cell lines.These findings suggest that PAK might be associated with T-LBL recurrence and further found that PAK inhibitors could suppress proliferation and enhance chemosensitivity of T-LBL cells treated with doxorubicin.Collectively,our present study underscores the potential therapeutic effect of inhibiting PAK in T-LBL therapy.

Cross-talk between Smad4 and P38 Proteins in Non-small Cell Lung Cancer

Objective： Impaired signal transduction is associated with tumorigenesis and progression of various kinds of human cancers. Transforming growth factor （TGF）-beta/Smad and ras-mitogen activated protein kinase （MAPK） are two major signal transduction pathways for adjusting cell proliferation and differentiation. Little is known about TGF-beta/Smad4 in non-small cell lung cancer （NSCLC）. Hereby, we investigated the expression of Smad4 in NSCLC, its correlation with MAPK proteins （including p38, ERK1 and JNK1 proteins） and their clinical significance in NSCLC. Methods： The expressions of Smad4, p38, ERK1 and JNK1 were detected at protein level with Western blotting and immunohistochemistry, at transcription level with RT-PCR. Statistical analysis was performed for the comparisons of expressions of Smad4, p38, ERK1 and JNK1, and their correlation with various clinicopathological parameters and the prognosis of NSCLC. Results： The levels of protein and mRNA expression of Smad4 in lung cancer tissues were significantly lower than in normal tissues （P〈0.05）. All these four proteins were associated with TNM staging. There was a strongly negative correlation between p38 and Smad4. Expressions of Smad4, p38 and JNK1, as well as tumor differentiation and staging were significantly correlated with the prognosis of NSCLC by univariate analysis. By multivariate analysis, only Smad4, p38, tumor differentiation and staging were correlated with the prognosis. Taken together, the negative expression of p38 and positive expression of Smad4 were associated with a better prognosis of NSCLC. Conclusion： Smad4 could be of vital importance for the initiation and development of NSCLC. The expression of Smad4 might be inhibited by p38, supporting a cross-talk between main proteins of TGF-beta/Smad and ras-MAPK signal transduction pathways. Smad4 and p38 could be possible prognostic factors for NSCLC.

Activation of p38 mitogen-activated protein kinase contribute to BMP4-induced alkaline phosphatase expression in MC3T3-E1 preosteoblast

Bone morphogenetic proteins （BMPs） induce ectopic bone formation and promote osteoblast differentiation. It has been documented that Smad transcriptional factors function as primary mediators of BMPs activity. Receptor-regulated Smad （Smad1, 5, 8） could be phosphorylated by activated BMPR-I and form complex with Smad4. The Smad complex translocates to the nucleus and regulate target gene transcription. Recently, several reports suggested that Mitogen-Activated Protein Kinase （MAPK） signaling pathways could be initiated downstream of the BMP receptor complex. Alkaline phosphatase （ALP） is an early marker of osteoblast differentiation Both ALP activity and its mRNA expression level could be increased by BMP4 treatment. Previously, we demonstrated that mutation of ERK1/2 phosphorylation sites in Smad5 partially rescued Smad transcriptional activity. However, fibroblast growth factor2-suppressed ALP activity could not be rescued similarly by introduction of Smad5 mutant in MC3T3-E1. These results prompted us to further evaluate the effect of BMP4-stimulated Smad transcriptional activity on ALP expression in this study.

蛋白激酶C抑制剂对SACC-83系P_(21)^(ras)、c-myc表达的影响

本实验采用ＰＫＣ抑制剂Ｓｔａｕｒｏｓｐｏｒｉｎｅ作用于ＳＡＣＣ－８３系，观察对Ｐ２１ｒａｓ、ｃ－ｍｙｃ表达的影响．结果表明，Ｓｔａｕｒｏｐｏｒｉｎｅ可明显降低Ｈ－ｒａｓ、ｃ－ｍｙｃ表达程度（ｐ＜０．０１），这提示ＰＫＣ对ｒａｓ、ｃ－ｍｙｃ基因表达具有调控作用，ＰＫＣ抑制剂可能通过调控癌基因的表达来表现抗肿瘤作用的．

The p21-activated kinases in neural cytoskeletal remodeling and related neurological disorders

The serine/threonine p21-activated kinases(PAKs),as main effectors of the Rho GTPases Cdc42 and Rac,represent a group of important molecular switches linking the complex cytoskeletal networks to broad neural activity.PAKs show wide expression in the brain,but they differ in specific cell types,brain regions,and developmental stages.PAKs play an essential and differential role in controlling neural cytoskeletal remodeling and are related to the development and fate of neurons as well as the structural and functional plasticity of dendritic spines.PAK-mediated actin signaling and interacting functional networks represent a common pathway frequently affected in multiple neurodevelopmental and neurodegenerative disorders.Considering specific small-molecule agonists and inhibitors for PAKs have been developed in cancer treatment,comprehensive knowledge about the role of PAKs in neural cytoskeletal remodeling will promote our understanding of the complex mechanisms underlying neurological diseases,which may also represent potential therapeutic targets of these diseases.

蜀羊泉散加味联合同步放化疗对中晚期宫颈癌患者血清CYFRA21-1、SCC-Ag、TK1、HE4的影响

目的 观察蜀羊泉散加味联合同步放化疗对中晚期宫颈癌患者疗效、血清指标及生存的影响。方法 选取2015年1月—2019年12月在医院就诊的中晚期宫颈癌患者120例,采用随机数字表法分为观察组和对照组各60例,对照组应用常规放化疗方法,观察组应用蜀羊泉散加味联合同步放化疗治疗,治疗4个疗程后进行疗效评价,比较两组患者治疗前后卡氏生活质量(karnofsky performance status, KPS)评分、疼痛视觉模拟(visual analogue scale, VAS)评分及血清细胞角质蛋白19片段抗原21-1(cytokeratin 19 fragment antigen 21-1,CYFRA21-1)、鳞状上皮细胞癌抗原(squamous cell carcinoma antigen, SCC-Ag)、胸苷激酶1(thymidine kinase 1,TK1)、人附睾蛋白4(human epididymis protein 4,HE4)水平,并比较不良反应和生存情况。结果 治疗后,观察组总有效率及生存率显著高于对照组(P<0.05),恶心呕吐、白细胞减少及血小板下降发生率显著低于对照组(P<0.05);治疗后,两组VAS评分及血清CYFRA21-1、SCC-Ag、TK1、HE4水平均较治疗前降低,KPS评分较治疗前升高(P<0.05);治疗后,观察组血清CYFRA21-1、SCC-Ag、TK1、HE4水平均低于对照组,KPS评分高于对照组(P<0.05)。结论 蜀羊泉散加味联合同步放化疗对中晚期宫颈癌疗效显著,可有效减轻患者疼痛,提高患者生存率及生活质量,降低血清CYFRA21-1、SCC-Ag、TK1、HE4水平,具有一定临床应用价值。

Activation of Rac1-PI3K/Akt is required for epidermal growth factorinduced PAK1 activation and cell migration in MDA-MB-231 breast cancer cells

Epidermal growth factor （EGF） may increase cell motility, an event implicated in cancer cell invasion and metastasis. However, the underlying mechanisms for EGF-induced cell motility remain elusive. In this study, we found that EGF treatment could activate Ras-related C3 botulinum toxin substrate 1 （Racl）, PI3K/Akt and p21- actived kinase （PAK1） along with cell migration. Ectopic expression of PAK1 K299R, a dominant negative PAK1 mutant, could largely abolish EGF-induced cell migration. Blocking PI3K/Akt signalling with LY294002 or Akt siRNA remarkably inhibited both EGF-induced PAK1 activation and cell migration. Furthermore, expression of dominant-negative Racl （T17N） could largely block EGF-induced PI3K/Akt-PAK1 activation and cell migration. Interestingly, EGF could induce a significant production of ROS, and N-acetyl-L-cysteine, a scavenger of ROS which abolished the EGF-induced ROS generation, cell migration, as well as activation of PI3K/Akt and PAK, but not Racl. Our study demonstrated that EGF-induced cell migration involves a cascade of signalling events, including activation of Racl, generation of ROS and subsequent activation of PI3K/Akt and PAK1.

Ⅰ类PAKs在神经退行性疾病的研究进展

P21活化的蛋白激酶(p21-activated kinases,PAKs)是Rho家族中GTP酶重要效应物,已证实对细胞繁殖和生存有重要意义。研究发现PAKs与神经元树突发育、细胞骨架的形成以及在细胞信号转导等方面起重要作用。本研究对Ⅰ类PAKs在神经退行性疾病中作用研究进展作一综述。

Antitumor synergism between PAK4 silencing and immunogenic phototherapy of engineered extracellular vesicles

Immunotherapy has revolutionized the landscape of cancer treatment.However,single immunotherapy only works well in a small subset of patients.Combined immunotherapy with antitumor synergism holds considerable potential to boost the therapeutic outcome.Nevertheless,the synergistic,additive or antagonistic antitumor effects of combined immunotherapies have been rarely explored.Herein,we established a novel combined cancer treatment modality by synergizing p21-activated kinase 4(PAK4)silencing with immunogenic phototherapy in engineered extracellular vesicles(EVs)that were fabricated by coating M1 macrophage-derived EVs on the surface of the nano-complex cores assembled with si RNA against PAK4 and a photoactivatable polyethyleneimine.The engineered EVs induced potent PAK4 silencing and robust immunogenic phototherapy,thus contributing to effective antitumor effects in vitro and in vivo.Moreover,the antitumor synergism of the combined treatment was quantitatively determined by the Compu Syn method.The combination index(CI)and isobologram results confirmed that there was an antitumor synergism for the combined treatment.Furthermore,the dose reduction index(DRI)showed favorable dose reduction,revealing lower toxicity and higher biocompatibility of the engineered EVs.Collectively,the study presents a synergistically potentiated cancer treatment modality by combining PAK4 silencing with immunogenic phototherapy in engineered EVs,which is promising for boosting the therapeutic outcome of cancer immunotherapy.

沉默PAK4基因对胶质瘤细胞U251侵袭与迁移能力的影响

目的观察沉默P21活化酶4(p-21 activated kinase 4, PAK4)基因对神经胶质瘤细胞U251侵袭与迁移能力的影响。方法构建稳定沉默PAK4基因的U251细胞系,shRNA-U251(转染空载组作为对照即shNC组);应用免疫荧光实验检测PAK4在胶质瘤细胞系U251中的表达;应用细胞划痕愈合实验与Transwell侵袭实验比较两组细胞的迁移与侵袭能力的变化;通过Western blotting实验检测沉默PAK4基因后U251细胞系中基质金属蛋白酶MMP2与MMP9的表达改变。结果 PAK4蛋白主要表达于胶质瘤细胞系U251细胞核与细胞质; shRNA-U251组细胞划痕愈合率明显降低; shRNA-U251组细胞进入小室下壁的数量明显减少,shRNA-U251组细胞MMP2与MMP9蛋白的表达水平显著下调。结论沉默PAK4基因下调神经胶质瘤细胞U251的迁移与侵袭能力与降低基质金属蛋白酶MMP2、MMP9的表达相关。

Pollen Typhae Total Flavone Inhibits Endoplasmic Reticulum Stress-Induced Apoptosis in Human Aortic-Vascular Smooth Muscle Cells through Down-Regulating PERK-eIF2α-ATF4-CHOP Pathway

Objective: To test the hypothesis that the inhibition of endoplasmic reticulum(ER) stress-induced apoptosis in oxidized low-density lipoproteins(ox-LDL)-induced human aortic-vascular smooth muscle cells(HA-VSMCs) was associated with suppression of the protein kinase RNA-like ER kinase(PERK)-eukaryotic translation initiation factor 2α(e IF2α)-activating transcription factor 4(ATF4)-CCAAT/enhancer binding protein homologous protein(CHOP) signaling pathway by Pollen Typhae total flavone(PTF). Methods: Primary HA-VSMCs were cultured and identified. The cultured HA-VSMCs were randomized into 5 groups, including a normal control group, an ox-LDL group(70 μg/m L high ox-LDL), an HPTF group(70 μg/m L high ox-LDL+500 μg/m L PTF), an MPTF group(70 μg/m L high ox-LDL+250 μg/m L PTF), and a LPTF group(70 μg/m L high ox-LDL+100 μg/m L PTF) in the first part;and a normal control group, an ox-LDL group(70 μg/mL high ox-LDL), an MPTF group(70 μg/m L high ox-LDL+250 μg/m L PTF), a sh RNA group(transducted with PERK shRNA lentiviral particles), a scramble shRNA group(transducted with control shRNA lentiviral particles), an MPTF+ox-LDL+shRNA group(250 μg/mL PTF+70 μg/mL high ox-LDL+PERK shRNA lentiviral particles) and an ox-LDL+shRNA group(70 μg/mL high ox-LDL+PERK shRNA lentiviral particles) in the second part. The protein expression levels of ER-associated apoptosis proteins were detected by Western blot, and their m RNA expression levels were detected by quantitative real-time reverse transcription-polymerase chain reaction. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay was applied to test cel viability, and the level of apoptosis was monitored by flow cytometry. Results: The MTT assay and flow cytometry showed that the ox-LDL group had a significant increase in apoptosis, which was attenuated in PTF treatment groups and sh RNA groups. Moreover, the ox-LDL group had increased protein and m RNA levels of binding immunoglobulin protein and ER-associated apoptosis proteins, such as PERK, e IF2α, ATF4 and CHOP, which were attenuated in PTF treatment groups and sh RNA groups. Conclusions: The apoptosis induced by ox-LDL had a strong relation to ER stress. The protective effect of PTF on ER stressinduced apoptosis was associated with inhibition of the PERK-eIF2α-ATF4-CHOP pathway, which might be a potential therapeutic strategy for enhancing the stability of atherosclerotic plaques.

DDIT4 promotes gastric cancer proliferation and tumorigenesis through the p53 and MAPK pathways

Background:Gastric cancer(GC)is one of the most common malignancies worldwide,particularly in China.DNA damage-inducible transcript 4(DDIT4)is a mammalian target of rapamycin inhibitor and is induced by various cellular stresses;however,its critical role in GC remains poorly understood.The present study aimed to investigate the poten-tial relationship and the underlying mechanism between DDIT4 and GC development.Methods:We used western blotting,real-time polymerase chain reaction,and immunohistochemical or immunoflu-orescence to determine DDIT4 expression in GC cells and tissues.High-content screening,cell counting kit-8 assays,colony formation,and in vivo tumorigenesis assays were performed to evaluate cell proliferation.Flow cytometry was used to investigate cell apoptosis and cell cycle distribution.Results:DDIT4 was upregulated in GC cells and tissue.Furthermore,downregulating DDIT4 in GC cells inhibited proliferation both in vitro and in vivo and increased 5-fluorouracil-induced apoptosis and cell cycle arrest.In contrast,ectopic expression of DDIT4 in normal gastric epithelial cells promoted proliferation and attenuated chemosensitivity.Further analysis indicated that the mitogen-activated protein kinase and p53 signaling pathways were involved in the suppression of proliferation,and increased chemosensitivity upon DDIT4 downregulation.Conclusion:DDIT4 promotes GC proliferation and tumorigenesis,providing new insights into the role of DDIT4 in the tumorigenesis of human GC.

Negative regulation of caspase 3-cleaved PAK2 activity by protein phosphatase 1

The p21-activated kinase 2 (PAK2) is activated by binding of small G proteins, Cdc42 and Rac, or through proteolytic cleavage by caspases or caspase-like proteases. Activation by both small G protein and caspase requires autophosphorylation at Thr-402 of PAK2. Although activation of PAK2 has been investigated for nearly a decade, the mechanism of PAK2 downregulation is unclear. In this study, we have applied the kinetic theory of substrate reaction during modification of enzyme activity to study the regulation mechanism of PAK2 activity by the catalytic subunit of protein phosphatase 1 (PP1α). On the basis of the kinetic equation of the substrate reaction during the reversible phosphorylation of PAK2, all microscopic kinetic constants for the free enzyme and enzyme-substrate(s) complexes have been determined. The results indicate that (1) PP1α can act directly on phosphorylated Thr-402 in the acti-vation loop of PAK2 and down-regulate its kinase activity; (2) binding of the exogenous protein/peptide substrates at the active site of PAK2 decreases both the rates of PAK2 autoactivation and inactivation. The present method provides a novel approach for studying reversible phosphorylation reactions. The advantage of this method is not only its usefulness in study of substrate effects on enzyme modifica-tion but also its convenience in study of modification reaction directly involved in regulation of enzyme activity. This initial study should provide a foundation for future structural and mechanistic work of protein kinases and phosphatases.

Effect of Yanggan Jiedu Sanjie formula on human hepatocellular carcinoma Bel-7402 cells

OBJECTIVE: To observe the effect of Yanggan Jiedu Sanjie(YGJDSJ) formula on human hepatocellular carcinoma Bel-7402 cells.METHODS: Bel-7402 cells were treated with YGJDSJ. Cell proliferation was detected by cell counting kit-8 assay. Cell apoptosis was identified by Hoechst 33258 staining and flow cytometric analysis. Cell cycle distribution was quantified by flow cytometric analysis. Caspase activities were measured by commercial kit. Cell senescence was detected by senescence-associated β-galactosidase(SA-β-gal)staining. Protein expression and phosphorylation were identified by Western blot. Protein expression was knocked-down by siRNA.RESULTS: YGJDSJ inhibited proliferation of Bel-7402 cells in a dose-and time-dependent manner.YGJDSJ induced apoptosis and activated caspase-3, 8, and 9 in Bel-7402 cells. YGJDSJ-induced apoptosis was completely abrogated by a pan caspase inhibitor, Z-VAD-FMK. YGJDSJ also induced cell senescence, up-regulated cyclin-dependent kinase inhibitor 1 a(CDKN1 a) and CDKN2 a expression and down-regulated retinoblastoma protein(RB) phosphorylation in Bel-7402 cells. Specific knockdown of CDKN1 a and CDKN2 a significantly reduced YGJDSJ-induce cell senescence in Bel-7402 cells.CONCLUSION: YGJDSJ inhibited cell proliferation,induced caspase-dependent apoptosis and CDKN1 a/CDKN2 a-RB signalling mediated cell senescence in Bel-7402 cells. Our findings suggest that YGJDSJ might be potential for hepatocellular carcinoma treatment.

Activation of mammalian target of rapamycin contributes to pain nociception induced in rats by BmK I, a sodium channel-specific modulator

The mammalian target of rapamycin （mTOR） pathway is essential for maintenance of the sensitivity of certain adult sensory neurons. Here, we investigated whether the mTOR cascade is involved in scorpion envenomation-induced pain hypersensitivity in rats. The results showed that intraplantar injection of a neurotoxin from Buthus martensii Karsch, BmK I （10 pg）, induced the activation of mTOR, as well as its downstream molecules p70 ribosomal S6 protein kinase （p70 S6K） and eukaryotic initiation factor 4E-binding protein 1 （4E-BP1）, in lumbar 5-6 dorsal root ganglia neurons on both sides in rats. The activation peaked at 2 h and recovered 1 day after injection. Compared with the control group, the ratios of p-mTOR/p-p70 S6K/p-4E- BP1 in three types of neurons changed significantly. The cell typology of p-mTOR/p-p70 S6K/p-4E-BP1 immuno-reactive neurons also changed. Intrathecal administration of deforolimus, a specific inhibitor of mTOR, attenuated BmK I-induced pain responses （spontaneous flinching, paroxysmal pain-like behavior, and mechanical hypersensitivity）. Together, these results imply that the mTOR signaling pathway is mobilized by and contributes to experimental scorpion sting-induced pain.

Investigation and experimental validation of curcumin-related mechanisms against hepatocellular carcinoma based on network pharmacology

Objective:To determine the potential molecular mechanisms underlying the therapeutic effect of curcumin on hepatocellular carcinoma(HCC)by network pharmacology and experimental in vitro validation.Methods:The predictive targets of curcumin or HCC were collected from several databases.the identified overlapping targets were crossed with Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)analyses using the Database for Annotation,Visualization,and Integrated Discovery(DAVID)platform.Two of the candidate pathways were selected to conduct an experimental verification.The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide tetrazolium(MTT)assay was used to determine the effect of curcumin on the viability of Hep G2 and LO2 cells.The apoptosis and autophagy of Hep G2 cells were respectively detected by flow cytometry and transmission electron microscopy.Besides,western blot and real-time polymerase chain reaction(PCR)were employed to verify the p53 apoptotic pathway and adenosine 5’-monophosphate(AMP)-activated protein kinase(AMPK)autophagy pathway.Hep G2 cells were pretreated with pifithrin-α(PFT-α)and GSK690693 for further investigation.Results:The 167 pathways analyzed by KEGG included apoptosis,autophagy,p53,and AMPK pathways.The GO enrichment analysis demonstrated that curcumin was involved in cellular response to drug,regulation of apoptotic pathway,and so on.The in vitro experiments also confirmed that curcumin can inhibit the growth of Hep G2 cells by promoting the apoptosis of p53 pathway and autophagy through the AMPK pathway.Furthermore,the protein and messenger RNA(m RNA)of the two pathways were downregulated in the inhibitor-pretreated group compared with the experimental group.The damage-regulated autophagy modulator(DRAM)in the PFT-α-pretreated group was downregulated,and p62 in the GSK690693-pretreated group was upregulated.Conclusions:Curcumin can treat HCC through the p53 apoptotic pathway and the AMPK/Unc-51-like kinase 1(ULK1)autophagy pathway,in which the mutual transformation of autophagy and apoptosis may occur through DRAM and p62.