In vitro parasiticidal effects of PIK-75 inhibition of Nrf2 against Echinococcus granulosus protoscoleces

In vitro parasiticidal effects of PIK-75 inhibition of Nrf2 against Echinococcus granulosus protoscoleces | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article In vitro parasiticidal effects of PIK-75 inhibition of Nrf2 against Echinococcus granulosus protoscoleces Guangyao Tang, Ziyu Liu, Longjun Wang, Sheng Liu, Bin Yang, Hailong Lv This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3013557/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Introduction The aims of this study were to investigate the role of the nuclear factor E2-related factor 2 (Nrf2) signaling pathway in Echinococcus granulosus protoscoleces, and to examine the effects of PIK-75 inhibition on Nrf2 activity. Methods Nrf2 protein expression and localization in protoscoleces were examined via confocal immunofluorescence microscopy. Reactive oxygen species (ROS detection kit) was used to detect ROS level in protoscoleces. The effects of PIK-75 on activity of heme oxygenase1(HO-1), NAD(P)H quinone oxidoreductase 1 (NQO1), glutathione peroxidase (GSH-Px) and thioredoxin peroxidase (TPx) were characterized using ELISA. Evidence of parasite damage and death was observed by scanning electron microscopy(SEM). Western bolting was used to detect the expression level of Nrf2 protein. In addition, caspase-3 activity was detected using an assay kit. Results The study found that Nrf2 is primarily localized in the protoscoleces cytoplasm, and PIK-75 treatment could increased ROS level( P <0.05) in the early time, and reduced NQO-1, HO-1, GSH-Px and TPx ( P <0.05) activity in protoscoleces. SEM showed that PIK-75-treated protoscoleces presented damage in the protoscoleces region. Western-blot showed that the Nrf2 protein expression had decreased significantly. Caspase-3 activity clearly increased in protoscoleces treated for 24 and 48 h with PIK-75 compared with that in controls ( P <0.05). Conclusion The present investigation demonstrated that PIK-75 had an inhibitory effect on the Nrf2 signaling pathway. We also provide evidence that PIK-75 may serve as a potential therapeutic agent for the treatment of protoscoleces. The use of PIK-75 as a treatment for protoscoleces, however, requires further research. Echinococcus granulosus protoscoleces Nrf2 signal pathway PIK-75 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Introduction Cystic echinococcosis (CE) is a parasitic infestation caused by the larval stage of the cestode Echinococcus granulosus , which occurs most commonly in the liver and lungs ( 1 ). It is a major health problem worldwide, especially in pastoral communities ( 2 ). Traditionally, the most effective method of treating hydatid cysts is surgery, but surgery presents risks, including anaphylaxis and protoscoleces dissemination due to cystic fluid spillage, cannot be excluded ( 3 ). Consequently, percutaneous aspiration irrigation and respiration (PAIR) has been introduced to the clinic as a safe, minimally invasive, therapy that presents a potential alternative to surgery ( 4 , 5 ). In recent years, various local chemotherapy drugs have emerged either clinically or at the experimental stage ( 6 ). However, adverse side effects have been observed with both scolicidal agents, which may cause bile duct function abnormalities ( 7 ), and H 2 O 2 , which has not gained wide application owing to potential complications and low efficacy ( 8 ). The use of high concentrations of hypertonic saline solution has been found to be extremely effective for short periods against scolices of hydatid cysts. However, disadvantages, including intracranial bleeding, acute hypernatremia, convulsions, necrosis, and myelinolysis, have been reported ( 9 , 10 ). Irrigation using formaldehyde solution has been discontinued owing to the development of sclerosing cholangitis and acute pancreatitis ( 11 , 12 ). Therefore, there is a clinical need for safe and effective scolicidal solutions. The nuclear factor E2-related factor 2 (Nrf2)/Kelch-like ECH-associated protein 1 (KEAP1) signaling pathway is the primary pathway responsible for cell defense against oxidative stress and maintaining intracellular redox equilibrium at physiological levels ( 13 ). It is well established that Nrf2 is the key regulator of antioxidant expression via antioxidant response elements (AREs) located in gene regulatory regions ( 14 , 15 ). Nrf2 plays a key role in reducing electrophiles and reactive oxygen species (ROS) by promoting the transcription of antioxidant and detoxifying enzymes such as heme oxygenase 1 (HO-1). This exerts a strong anti-oxidant and anti-apoptotic effect favoring cancer cell growth and resistance to therapy, thus decreasing DNA mutations and damage and preventing genomic instability in normal cells ( 16 ). Several studies have shown that parasitic helminths possess defensive tactics against host-generated oxygen radicals, including both enzymatic and non-enzymatic antioxidant systems such as superoxide dismutase (SOD), glutathione (GSH), NAD(P)H quinone oxidoreductase 1 (NQO-1), glutathione peroxidase (GSH-Px), and thioredoxin peroxidase (TPx)( 17 ). These enzymes play an important role in E. granulosus defenses against oxidative damage ( 18 ). Evidence has accumulated indicating that Nrf2, a promising therapeutic target for anti-infection mechanisms, promotes apoptotic cell death and tissue damage in host cells infected by microorganisms such as bacteria, viruses, and parasites. Thus, inhibiting Nrf2 would be an efficient strategy in anti-parasitic disease therapy ( 19 ). PIK-75 has been demonstrated to be a potent inhibitor of Nrf2 in human pancreatic cancer cells by inducing its proteasomal degradation ( 20 ). Hence, the present study investigated the potential efficacy of PIK-75 in vitro against E. granulosus protoscoleces. Materials and methods Protoscoleces culture. Protoscoleces of E. granulosus were collected aseptically from liver hydatid cysts of infected sheep slaughtered in an abattoir located in the northeast of Xinjiang uygur autonomous region, China. Briefly, protoscoleces were allowed to settle in a 50mL Falcon tube, were washed several times in phosphate-buffered saline [PBS, pH 7.2] and placed into culture medium (RPMI 1640 containing 100 U/mL of penicillin and 100 mg/mL of streptomycin) supplemented with 10% fetal bovine serum. Cultures were incubated at 37℃ in a 5% CO2 atmosphere, with medium changes every 3–5 days. Drug treatment of E.granulosus protoscoleces. Treatment of protoscoleces was initiated within 5 days of in vitro culture. The viability of protoscoleces was assessed by 0.1% eosin staining exclusion assay every day. When the percentage of viable protoscoleces in the sediment was 95% or more, they were considered to be distributed to 6-well plates (~ 2500 to 3500 per well). PIK-75 was dissolved in dimethylsulfoxide (DMSO) and stored at -20˚C. we used the following final concentration of the drugs: 0.4 µM, 0.8 µM, 1.2 µM, 1.6 µM and 2.0 µM, respectively. The corresponding dilution of DMSO served as control. Treatments were carried out at 37℃, 5% CO2. This experiment was repeated three times under identical experimental condition. Confocal immunofluorescence. Samples were washed in PBS and fixed in 4% paraformaldehyde in 0.1 M PBS (pH 7.4) overnight at 4℃, and then embedded in paraffin. The embedding process was carried out in the tissue processor which comprised one immersion in 75%, 85%, 95% ethanol (4 hours each), three immersion in absolute ethanol (60 mins each), two immersion in xylol (50 mins each) and two immersion in liquid paraffin at 62℃ (120 mins each). Sections of 4 µm thickness were obtained with a microtome (HM 325 Microm) and placed on adhesive-coated glass slides to avoid their detachment during immunofluorescence processing. Sections were deparaffinized in two changes of xylene and dehydrated in descending concentrations of ethanol (100%, 95%, 80%, and 70%) until water. Antigen retrieval was performed with 0.01 M Citric Acid (pH 6.0) at 130℃, 700W (8 ~ 10 mins). After blocking with 3% H2O2 for 10 mins at room temperature, they were incubated with each primary antibody (Santa Cruz Biotechnology, Inc.) at 4℃ overnight. After washing in PBS, the sections were incubated with FITC-conjugated secondary antibodies at room temperature in the dark for 1 h. The tissues were stained with PI in the dark for 3 mins, and then blocked with 50% glycerinum. PBS was used as a negative control. The specimens were visualized and photographed with a scanning confocal microscope (LSM 510 META, Carl Zeiss). Detection of reactive oxygen species generation After the treatment of protoscoleces with PIK-75, protoscoleces were washed with PBS, stay natural precipitation, then collecting the protoscoleces, and add the DCFH-DA probe, the initial work concentration was 10 µM, in 37°C incubation for 1 hour and then use the Bio-Rad fluorescence enzyme standard instrument to observe readings. ELISA Kit study. Protoscoleces were cultured in 6-well plates. All protoscoleces were pretreated with each indicated agent for the indicated time. Protoscoleces were washed twice in PBS and lysed on ice for 20 mins in lysis buffer. Samples were centrifuged at 12000 g for 10 min at 4℃. Supernatants were recovered and frozen at − 80℃ until assay. The levels of HO-1and NQO-1 were quantified using ELISA kit (Wuhan USCN Business Co., Ltd). Assays were performed according to the manufacturer’s instructions. The absorbance (450 nm) for each sample was analyzed using microplate reader (Bio-RAD, USA). The levels of GSH-Px and TPx were quantified using ELISA kit Jiancheng(Nanjing, China). Assays were performed according to the manufacturer’s instructions. Using super micro UV VIS spectrophotometer to detect OD value (Thermo, USA). SEM analysis. Protoscoleces were washed three times in PBS pH 7.2, and placed into 4% glutaraldehyde for at least 24 h at 4℃. For SEM analysis, the settled specimens were dehydrated by sequential incubations in increasing concentrations of ethanol (50%, 70%, 80%, 90% and 100%), and were finally immersed in hexamethyl-disilazane and air-dried under a fume hood. The dehydrated protoscoleces were sputter-coated with gold and observed under a LEO1430VP scanning electron microscope operating at 25 kV. Western Bloting Analysis Protoscoleces were collected after PIK-75 treatments and washed with ice-cold PBS. The protoscoleces were lysed with RIPA lyse buffer containing PMSF (1:100) for 10 min on ice, after that we used Cell Ultrasonic Crusher to smash protoscoleces, and then centrifuged at 12000g for 10min. A microplate reader was used to determine the protein concentrations. The supernatant was designated as whole protoscoleces protein extract and kept at -80℃. Subsequently, the samples were boiled in Laemmli loading buffer. Each 40 mg aliquots of total protein extracts were separated on an 8%, 10%, or 12% SDS-polyacrylamide gel, followed by transfer of the proteins to a polyvinylidene fluoride (PVDF) membrane. Membranes were blocked with Tween 20 (TBST) supplemented with 5% nonfat dry milk for 1–2 hours, followed by incubation with primary antibodies at 4℃ for 12–24 hours. Membranes were incubated with the goat anti-rabbit IgG antibody at temperature for 2–3 hours, after the the membranes had been washed three times with PBST. The bands were visualized using an ECL detection kit and quantified using Image Lab software, after being washed three times with PBST. Caspase-3 activity assay. Protoscoleces were treated as indicated and caspase-3 activity was measured from cell lysates with Caspase-3 Assay kit (APPLYGEN, China) according to the manufacturer's protocol. Luminescence was measured using microplate reader. Relative luminescence units were determined by calculating luminescence values from samples as a percentage of values from control samples. The experiments were considered in three independent experiments performed in duplicate. Statistical Analysis. All data are shown as the arithmetic mean ± S.E.M. The results were subjected to paired t-tests and one-way ANOVA with Least significant difference (LSD) test in order to determine the statistical significant differences from the control using SPSS 17.0 software. P values less than 0.05 were considered significant. Results Localization of Nrf2 in E. granulosus protoscoleces. The localization profile of Nrf2 in protoscoleces was demonstrated using immunofluorescence. Immunocytochemical staining showed that under control conditions, Nrf2 was localized in the cytoplasm of the cells of the protoscoleces. (Figs. 1, 2). After 24 h incubation with both low and high concentrations of PIK-75, Nrf2 levels in protoscoleces decreased relative to control (Fig. 2). The generation of reactive oxygen species We also measured intracellular ROS by using a DCFH-DA-based assay after protoscoleces were treated with PIK-75 for 2d and 5d. As shown in Figure 3, incubation of protoscoleces with PIK-75 induced a continuous time- and concentration-dependent increase in ROS production, for the complete 5d, when compared to the control protoscoleces, as observed with the microplate reader (P<0.05). Effects of PIK-75 on the expression of Antioxidant enzymes in protoscoleces. Protoscoleces were pre-treated for 2 or 5 days with 0.8 μM or 1.6 μM PIK-75, and HO-1, NQO-1, GSH-Px and TPx expression was measured using an ELISA kit to examine whether PIK-75 modulates HO-1, NQO-1, GSH-Px and TPx expression in cultured protoscoleces (Figs.4,5,6,7). All protoscoleces treated with PIK-75 showed a time- and dose-dependent decrease in HO-1, NQO-1, GSH-Px and TPx expression (P<0.05). PIK-75 suppress the expression of Nrf2 protein in vitro We investigated the effect of PIK-75 treatment on the expression of Nrf2 in protoscoleces by using western bolt technology. As shown in Figure 8, the level of Nrf2 protein was decreased by PIK-75 in a concentration- dependent manner. The 1.6μM PIK-75 treatment caused a significant decrease in the expression of PIK-75 at 5d when compare to 1000 μM control group. Effects of PIK-75 on E. granulosus protoscoleces in vitro. Following the isolation of protoscoleces from hydatid cysts, the viability of protoscoleces was assessed using the eosin exclusion assay. The survival of E. granulosus protoscoleces after exposure to DMSO or various concentrations of PIK-75 is shown in Figure 9,10. It was observed that protoscoleces cultured with 2.0 μM PIK-75 died at a considerably faster rate than protoscoleces cultured with the lower concentrations. After 5 days of exposure to 0.4 μM PIK-75, protoscoleces viability was approximately 81.3%, had started presenting evaginations (Figs. 9, 10b). The viability of the protoscoleces incubated with 0.8 μM PIK-75 for 2 days was 72.88%, and several slight pits were apparent in all protoscoleces (Figs. 9, 10c). After 5 days treatment with 0.8 μM PIK-75, survivability had been reduced to 53.2%, and the appearance of tegumental alterations and sucker region distortion was observed (Figs. 9, 10d). PIK-75 clearly showed decreased efficacy at a concentration of 1.6 μM, as 55.1% of protoscoleces were still viable after 2 days of treatment. Scanning electron microscopy (SEM) showed ultrastructural changes, including tegumental contraction with loss of hooks, and rostellar disorganization (Figs. 9, 10e). The rate of protoscoleces death increased over time. Only a small fraction (~22%) of protoscoleces was viable in cultures treated with 1.6 μM PIK-75 after 5 days. Furthermore, the complete destruction of the rostellum and the germinal layer was observed by SEM at this time point (Figs. 9, 10f). However, control protoscoleces that had been incubated in RPMI Medium 1640 with DMSO for 7 days presented ~95.4% viability, with no observed changes in morphology or ultrastructure throughout the experimental period (Figs. 9, 10a). PIK-75-induced apoptosis in protoscoleces. To study the impact of Nrf2 inhibition-induced apoptosis, protoscoleces were treated with different concentrations of PIK-75 for 24 or 48 h (Figure 11). Apoptosis was determined by assaying caspase-3 activation. All concentrations induced a significant increase of caspase-3 activity at both time points compared with controls. Discussion The aim of this study was to investigate the efficacy of PIK-75 against inhibition of the Nrf2/KEAP1 signaling pathway in E. granulosus protoscoleces. In vitro culture of protoscoleces was employed to demonstrate the parasiticidal effects of PIK-75. Nrf2-KEAP1-ARE is a pivotal signaling pathway in maintaining cellular oxidative equilibrium ( 21 ). Nrf2, referred to as the master regulator of antioxidant, detoxification, and cell defense gene expression, is bound in the cytoplasm to KEAP1. This study determined Nrf2 localization and expression in E. granulosus protoscoleces using confocal immunofluorescence microscopy, and found that although Nrf2 is widely distributed, it is primarily localized in the cytoplasm. As a phosphoinositide 3-kinase (PI3K) inhibitor, PIK-75 has demonstrated potent anti-cancer activity in several cancer cell lines ( 22 ) and in a cervical cancer xenograft model ( 23 ). Duong HQ et al. have demonstrated that Nrf2 protein levels and activity were reduced by PIK-75 in human pancreatic cancer cell lines ( 14 ). To some degree, the ability to inhibit Nrf2 via a PI3K/Akt pathway inhibitor was expected, because it had been reported that Nrf2 stability is regulated by the PI3K/Akt/glycogen synthase kinase (GSK) 3β pathway ( 14 ). Using confocal immunofluorescence microscopy, it was found that in vitro treatment of protoscoleces with increasing doses of PIK-75 resulted in decreasing immunoreactivity intensity compared to untreated controls. Furthermore, confocal microscopy did not reveal any consistent translocation of Nrf2 from the cytoplasm to the nucleus in PIK-75-treated protoscoleces. All this suggests that Nrf2 protein is primarily expressed in the cytoplasm, and its levels decrease during PIK-75-treatment in a dose-dependent manner. Generally, ROS overproduction is deleterious to cell structures and cell macromolecular constituent functions, leading to excessive expression of cytokines and inflammatory factors, which in turn results in acute inflammation, atheromatous plaques, sepsis, and tissue injury ( 24 ). To defend against these deleterious actions, cells contain multiple antioxidant enzymes expressed in various regions to scavenge ROS ( 25 ). According to a new study, the induction of HO-1 could protect cells from oxidative stress in acute hepatic injury model ( 26 ). In addition, HO-1 has been shown to exert a particularly strong anti-apoptotic effect, favoring cancer cell growth and therapeutic resistance. Ma Q has found that, in the development of chronic obstructive pulmonary disease (in particular, emphysema) and lung cancer, the adaptive activation of Nrf2 led to increased expression of NQO1, GSH-Px2, and GSH, all of which suppress CS (cigarette smoke)-induced ROS production and oxidative damage ( 27 ). Some experts have also demonstrated that GSTs were well represented in Fasciola hepatica ( 28 , 29 ). In the present study, PIK-75 has been shown to induce higher levels of intracellular ROS in the early time, and ELISA data showed that treating cultured protoscoleces with PIK-75 (0.8, and 1.6 µM) for either 2 or 5 days caused a significant decrease in both HO-1, NQO-1, GSH-Px and TPx production in a time- and dose-dependent manner. Taken together, PIK-75 has a clear inhibitory effect on antioxidant enzyme production in E. granulosus protoscoleces. In addition, we investigated PIK-75 treatment effects on protoscoleces viability and morphology in vitro. PIK-75 increased the mortality of protoscoleces in a dose-dependent manner, as protoscoleces cultured with 2.0 µM PIK-75 showed considerably higher mortality than protoscoleces cultured with other concentrations. SEM revealed many ultrastructural changes in PIK-75-treated protoscoleces, including numerous pits appearing at the tegument, disruption of hooks and microtriches of the scolex region, collapse of the sucker region, and protoscoleces contraction. In contrast, those parasites cultured in RPMI 1640 Medium with DMSO did not present significantly altered morphology or ultrastructure. These results demonstrate that PIK-75 negatively affects E. granulosus protoscoleces. Caspases, including the upstream “initiator” and downstream “effector” groups, belong to a family of cysteine proteases, which are involved in the cleavage of aspartic acid. Caspase-3, a member of the downstream “effector” group, has been identified as a key regulator of growth-promoting signals generated from dying cells. Moreover, Caspase-3 has been shown to be involved in the “execution” phase of cellular apoptosis ( 30 ). Other studies have demonstrated significant increases in not only cell apoptosis, but also in caspase-3 activity in drug-treated protoscoleces ( 31 ). In this study, caspase-3 activity in the PIK-75-treated group was significantly higher than in the control group at both 24 h and 48 h. Furthermore, protoscoleces were more influenced by high concentrations of PIK-75, rendering those cells more sensitive to apoptosis. These findings indicate that PIK-75 possibly mediates apoptosis of protoscoleces through the caspase-3 pathway. To conclude, this study has elucidated the Nrf2 distribution pattern in protoscoleces of E. granulosus , and observed decreased levels of Nrf2-mediated antioxidases in protoscoleces treated with PIK-75. This study has also demonstrated that the Nrf2 inhibitor PIK-75 exerts a satisfactory scolicidal effect, presenting eventual reduction of parasite viability that leads to death. These successful results will provide a theoretical basis for the development of novel treatments for hydatid disease in the future. Further studies for the comprehensive elucidation of the mechanisms of PIK-75-mediated Nrf2 downregulation in E. granulosus protoscoleces will be required in order to avoid any potential side effects of PIK-75. Declarations Acknowledgements We would like to thank Prof. Hailong Lv Department of Hepatobiliary Surgery, The First Affiliated Hospital, School of Medicine, Shihezi University for helpful suggestions, and Bin Yang at the Department of Cardiac Surgery, The Seventh People’s Hospital of Zhengzhou City for expert technical assistance. We are also indebted to the Department of Histology and Embryology, Medical College of Shihezi University for providing us the experimental platform. Ethical Statement The protocol of the study was reviewed and approved by the Ethics Committee of the Seventh People’s Hospital of Zhengzhou. Consent to participate Applicable. Consent for publication Applicable. Competing interests The authors declare that they have no competing interests. Authors’ contributions Conceived and designed the study and supervised the project: Guangyao Tang, Ziyu Liu, Hailong Lv and Bin Yang. Undertook the study and data analysis: Guangyao Tang and Longjun Wang. Contributed to analysis using various tools: Sheng Liu and Ziyu Liu. Wrote the paper: Guangyao Tang, Ziyu Liu, and Hailong Lv. Contributed to the interpretation of findings and drafting of the manuscript: Longjun Wang and Sheng Liu. All authors read and approved the final version of the manuscript. Funding This study was supported by grants from the National Natural Science Foundation of China (grant U1303121 and 81560334 to Hailong Lv, )and Technology Research Plan Joint Construction Project Foundation of Henan (grant LHGJ20220837 to Bin Yang). Data availability Not applicable. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3013557","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":207997595,"identity":"33a9d19b-b54b-4d55-9f18-b97daa3f9a9c","order_by":0,"name":"Guangyao Tang","email":"","orcid":"","institution":"The Seventh People’s Hospital of Zhengzhou City","correspondingAuthor":false,"prefix":"","firstName":"Guangyao","middleName":"","lastName":"Tang","suffix":""},{"id":207997596,"identity":"6389b1c7-d9a3-4e9d-a42b-da77064029bb","order_by":1,"name":"Ziyu Liu","email":"","orcid":"","institution":"Xinxiang Medical University","correspondingAuthor":false,"prefix":"","firstName":"Ziyu","middleName":"","lastName":"Liu","suffix":""},{"id":207997597,"identity":"ea93e832-f426-408a-bf7a-4e156b18bbb2","order_by":2,"name":"Longjun Wang","email":"","orcid":"","institution":"Qinghai University","correspondingAuthor":false,"prefix":"","firstName":"Longjun","middleName":"","lastName":"Wang","suffix":""},{"id":207997598,"identity":"a8f01c2b-60c9-45ff-ad3a-af91e009dcb8","order_by":3,"name":"Sheng Liu","email":"","orcid":"","institution":"Xinxiang Medical University","correspondingAuthor":false,"prefix":"","firstName":"Sheng","middleName":"","lastName":"Liu","suffix":""},{"id":207997599,"identity":"eb98ea4e-3966-4345-b5f7-61db31f38f2c","order_by":4,"name":"Bin Yang","email":"","orcid":"","institution":"The Seventh People’s Hospital of Zhengzhou City","correspondingAuthor":false,"prefix":"","firstName":"Bin","middleName":"","lastName":"Yang","suffix":""},{"id":207997600,"identity":"63464479-aa54-45e6-ac4e-d2ddb77b926b","order_by":5,"name":"Hailong Lv","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzUlEQVRIiWNgGAWjYBACfmb+h4//GNjIsbE3EKlFsr2H2YCnIM2Yn+cAkVoMzpxhE+D5cChx5owEYm2ZkXuMQcLgAOOGm4833mCosYkmqIVfIi/tgYHBHWaD22nFFgzH0nIbCNuSYG6QYPCMzeB2jpkEY8NhwloMbiSYSRwwOMxjcPMMsVrOnDGTbDA4LCE5g4dILZLtbcnGDAZpBvw8QL8kEOMXfmbmg48Z/tjUt7Ef3njjQ40NYS0ojpRIIEU5RAupOkbBKBgFo2BkAAA20UGgARMEkQAAAABJRU5ErkJggg==","orcid":"","institution":"Shihezi University","correspondingAuthor":true,"prefix":"","firstName":"Hailong","middleName":"","lastName":"Lv","suffix":""}],"badges":[],"createdAt":"2023-06-02 08:44:26","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3013557/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3013557/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":38401708,"identity":"f3e0cc17-f27b-4e21-bc8d-76b7a1edb668","added_by":"auto","created_at":"2023-06-12 14:19:54","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":209979,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLocalization and expression of the Nrf2 protien in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eE. granulosus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e protoseoleces.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-3013557/v1/468a2dcd24abb3e56acdd15f.png"},{"id":38403554,"identity":"806c203b-38b4-479f-846f-568a0cbaf626","added_by":"auto","created_at":"2023-06-12 14:35:54","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":512842,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of PIK-75 on localization and expression of the Nrf2 protien in protoseoleces\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA:Control protoseoleces; B:protoscoleces incubated with PIK-75 at concentrations of 0.8μM for 24h : protoscoleces incubated with PIK-75 at concentrations of 1.6 μM for 24h\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-3013557/v1/68810bc790fc907646222de0.png"},{"id":38401704,"identity":"b6706772-c113-4ef9-97b9-3169308b9da1","added_by":"auto","created_at":"2023-06-12 14:19:54","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":112677,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImpact of PIK-75 on ROS level in E. granulosus protoscoleces after 2 days and 5 days post-incubation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBar graph indicates:* Compared with the control group,P\u0026lt;0.05;# Intra-group comparison,P\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-3013557/v1/220e7e3ca3ba92d34a395302.png"},{"id":38403552,"identity":"b61400c0-8ed3-4ace-992f-9441295121f2","added_by":"auto","created_at":"2023-06-12 14:35:54","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":122682,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of PIK-75 on HO-1 activity in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eE. granulosus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e protoscoleces after 2d and 5d post-incubation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBar graph indicates:* Compared with the control group,P\u0026lt;0.05;# Intra-group comparison,P\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-3013557/v1/5734e77379747cfa9f7ce2a5.png"},{"id":38402448,"identity":"d4c2c564-7190-49bc-b173-f94d03a242af","added_by":"auto","created_at":"2023-06-12 14:27:54","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":112881,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of PIK-75 on NQO-1 activity in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eE. granulosus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e protoscoleces after 2d and 5d post-incubation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBar graph indicates:* Compared with the control group,P\u0026lt;0.05;# Intra-group comparison,P\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-3013557/v1/22e37ebf4dd0d1126266d11f.png"},{"id":38402446,"identity":"54d837c6-f69e-4124-be9e-c8c144a13d56","added_by":"auto","created_at":"2023-06-12 14:27:54","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":116683,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of PIK-75 on GSH-Px activity in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eE. granulosus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e protoscoleces after 2d and 5d post-incubation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBar graph indicates:* Compared with the control group,P\u0026lt;0.05;# Intra-group comparison,P\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-3013557/v1/64dc6d0d7875cc1c15177855.png"},{"id":38401706,"identity":"fffdb148-2fa6-4fc9-b515-c8ff7015f577","added_by":"auto","created_at":"2023-06-12 14:19:54","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":112833,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of PIK-75 on TPx activity in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eE. granulosus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e protoscoleces after 2d and 5d post-incubation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBar graph indicates:* Compared with the control group,P\u0026lt;0.05;# Intra-group comparison,P\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-3013557/v1/8e0d2449f639bfde49365f2f.png"},{"id":38403553,"identity":"13681ef5-f9a3-42d6-83f5-49ea22d75022","added_by":"auto","created_at":"2023-06-12 14:35:54","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":87375,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLevel of Nrf2 protein in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eE. granulosus \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eprotoscoleces incubated with PIK-75 (0.8 and 1.6 μmol/L) for 2 d and 5 d.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-3013557/v1/b86b6ec44c7f907772fdaa43.png"},{"id":38401713,"identity":"0c0bd03a-6ef4-4c24-9c6c-c217eab86e3a","added_by":"auto","created_at":"2023-06-12 14:19:55","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":133674,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLoss of viability of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eE. granulosus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eprotoscoleces incubated in vitro with PIK-75 at different concentrations.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure9.png","url":"https://assets-eu.researchsquare.com/files/rs-3013557/v1/170fad5cef3be221689e9df4.png"},{"id":38401714,"identity":"1cb3eb37-0672-49f9-964f-140fc7e5e3e3","added_by":"auto","created_at":"2023-06-12 14:19:55","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":677175,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSEM of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eE. granulosus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e protoscoleces cultured in vitro with PIK-75\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eScanning of \u003cem\u003eE. granulosus \u003c/em\u003e\u0026nbsp;protoscoleces incubated in vitro PIK-75 at either time point 5days(C,E) or 2 days(D,F). (A): Control group, invagination type; (B) : protoscoleces incubated with DMSO, evaginated type ; (C-F): altered protoscoleces incubated with PIK-75 in concentrations of 0.8μM (C, D) and 1.6μM (E, F).\u003c/p\u003e","description":"","filename":"Figure10.png","url":"https://assets-eu.researchsquare.com/files/rs-3013557/v1/4ccdcb252b650346145cda99.png"},{"id":38401712,"identity":"3b3474df-cc7c-4ebc-91d6-b7ea402d639d","added_by":"auto","created_at":"2023-06-12 14:19:55","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":103590,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eActivity of caspase-3 in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eE. granulosus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e protoscoleces incubated with PIK-75 for 24 h、48 h\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBar graph indicates:* Compared with the control group,P\u0026lt;0.05;# Intra-group comparison,P\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"Figure11.png","url":"https://assets-eu.researchsquare.com/files/rs-3013557/v1/c2899a1ba1327147eac435cb.png"},{"id":39089603,"identity":"4d7b1674-e1d0-4dc4-8f20-2e2c55ff8007","added_by":"auto","created_at":"2023-06-26 15:29:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3144070,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3013557/v1/d55d4918-6b89-4231-abd2-1e546b6ba2bf.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"In vitro parasiticidal effects of PIK-75 inhibition of Nrf2 against Echinococcus granulosus protoscoleces","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCystic echinococcosis (CE) is a parasitic infestation caused by the larval stage of the cestode \u003cem\u003eEchinococcus granulosus\u003c/em\u003e, which occurs most commonly in the liver and lungs (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). It is a major health problem worldwide, especially in pastoral communities (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTraditionally, the most effective method of treating hydatid cysts is surgery, but surgery presents risks, including anaphylaxis and protoscoleces dissemination due to cystic fluid spillage, cannot be excluded (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Consequently, percutaneous aspiration irrigation and respiration (PAIR) has been introduced to the clinic as a safe, minimally invasive, therapy that presents a potential alternative to surgery (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn recent years, various local chemotherapy drugs have emerged either clinically or at the experimental stage (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). However, adverse side effects have been observed with both scolicidal agents, which may cause bile duct function abnormalities (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e), and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, which has not gained wide application owing to potential complications and low efficacy (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). The use of high concentrations of hypertonic saline solution has been found to be extremely effective for short periods against scolices of hydatid cysts. However, disadvantages, including intracranial bleeding, acute hypernatremia, convulsions, necrosis, and myelinolysis, have been reported (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Irrigation using formaldehyde solution has been discontinued owing to the development of sclerosing cholangitis and acute pancreatitis (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Therefore, there is a clinical need for safe and effective scolicidal solutions.\u003c/p\u003e \u003cp\u003eThe nuclear factor E2-related factor 2 (Nrf2)/Kelch-like ECH-associated protein 1 (KEAP1) signaling pathway is the primary pathway responsible for cell defense against oxidative stress and maintaining intracellular redox equilibrium at physiological levels (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). It is well established that Nrf2 is the key regulator of antioxidant expression via antioxidant response elements (AREs) located in gene regulatory regions (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Nrf2 plays a key role in reducing electrophiles and reactive oxygen species (ROS) by promoting the transcription of antioxidant and detoxifying enzymes such as heme oxygenase 1 (HO-1). This exerts a strong anti-oxidant and anti-apoptotic effect favoring cancer cell growth and resistance to therapy, thus decreasing DNA mutations and damage and preventing genomic instability in normal cells (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). Several studies have shown that parasitic helminths possess defensive tactics against host-generated oxygen radicals, including both enzymatic and non-enzymatic antioxidant systems such as superoxide dismutase (SOD), glutathione (GSH), NAD(P)H quinone oxidoreductase 1 (NQO-1), glutathione peroxidase (GSH-Px), and thioredoxin peroxidase (TPx)(\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). These enzymes play an important role in \u003cem\u003eE. granulosus\u003c/em\u003e defenses against oxidative damage (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Evidence has accumulated indicating that Nrf2, a promising therapeutic target for anti-infection mechanisms, promotes apoptotic cell death and tissue damage in host cells infected by microorganisms such as bacteria, viruses, and parasites. Thus, inhibiting Nrf2 would be an efficient strategy in anti-parasitic disease therapy (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePIK-75 has been demonstrated to be a potent inhibitor of Nrf2 in human pancreatic cancer cells by inducing its proteasomal degradation (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Hence, the present study investigated the potential efficacy of PIK-75 in vitro against \u003cem\u003eE. granulosus\u003c/em\u003e protoscoleces.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e \u003cb\u003eProtoscoleces culture.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eProtoscoleces of \u003cem\u003eE. granulosus\u003c/em\u003e were collected aseptically from liver hydatid cysts of infected sheep slaughtered in an abattoir located in the northeast of Xinjiang uygur autonomous region, China. Briefly, protoscoleces were allowed to settle in a 50mL Falcon tube, were washed several times in phosphate-buffered saline [PBS, pH 7.2] and placed into culture medium (RPMI 1640 containing 100 U/mL of penicillin and 100 mg/mL of streptomycin) supplemented with 10% fetal bovine serum. Cultures were incubated at 37℃ in a 5% CO2 atmosphere, with medium changes every 3\u0026ndash;5 days.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDrug treatment of\u003c/b\u003e \u003cb\u003eE.granulosus\u003c/b\u003e \u003cb\u003eprotoscoleces.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTreatment of protoscoleces was initiated within 5 days of in vitro culture. The viability of protoscoleces was assessed by 0.1% eosin staining exclusion assay every day. When the percentage of viable protoscoleces in the sediment was 95% or more, they were considered to be distributed to 6-well plates (~\u0026thinsp;2500 to 3500 per well). PIK-75 was dissolved in dimethylsulfoxide (DMSO) and stored at -20˚C. we used the following final concentration of the drugs: 0.4 \u0026micro;M, 0.8 \u0026micro;M, 1.2 \u0026micro;M, 1.6 \u0026micro;M and 2.0 \u0026micro;M, respectively. The corresponding dilution of DMSO served as control. Treatments were carried out at 37℃, 5% CO2. This experiment was repeated three times under identical experimental condition.\u003c/p\u003e \u003cp\u003e \u003cb\u003eConfocal immunofluorescence.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eSamples were washed in PBS and fixed in 4% paraformaldehyde in 0.1 M PBS (pH 7.4) overnight at 4℃, and then embedded in paraffin. The embedding process was carried out in the tissue processor which comprised one immersion in 75%, 85%, 95% ethanol (4 hours each), three immersion in absolute ethanol (60 mins each), two immersion in xylol (50 mins each) and two immersion in liquid paraffin at 62℃ (120 mins each). Sections of 4 \u0026micro;m thickness were obtained with a microtome (HM 325 Microm) and placed on adhesive-coated glass slides to avoid their detachment during immunofluorescence processing. Sections were deparaffinized in two changes of xylene and dehydrated in descending concentrations of ethanol (100%, 95%, 80%, and 70%) until water. Antigen retrieval was performed with 0.01 M Citric Acid (pH 6.0) at 130℃, 700W (8\u0026thinsp;~\u0026thinsp;10 mins). After blocking with 3% H2O2 for 10 mins at room temperature, they were incubated with each primary antibody (Santa Cruz Biotechnology, Inc.) at 4℃ overnight. After washing in PBS, the sections were incubated with FITC-conjugated secondary antibodies at room temperature in the dark for 1 h. The tissues were stained with PI in the dark for 3 mins, and then blocked with 50% glycerinum. PBS was used as a negative control. The specimens were visualized and photographed with a scanning confocal microscope (LSM 510 META, Carl Zeiss).\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eDetection of reactive oxygen species generation\u003c/h2\u003e \u003cp\u003eAfter the treatment of protoscoleces with PIK-75, protoscoleces were washed with PBS, stay natural precipitation, then collecting the protoscoleces, and add the DCFH-DA probe, the initial work concentration was 10 \u0026micro;M, in 37\u0026deg;C incubation for 1 hour and then use the Bio-Rad fluorescence enzyme standard instrument to observe readings.\u003c/p\u003e \u003cp\u003e \u003cb\u003eELISA Kit study.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eProtoscoleces were cultured in 6-well plates. All protoscoleces were pretreated with each indicated agent for the indicated time. Protoscoleces were washed twice in PBS and lysed on ice for 20 mins in lysis buffer. Samples were centrifuged at 12000 g for 10 min at 4℃. Supernatants were recovered and frozen at \u0026minus;\u0026thinsp;80℃ until assay. The levels of HO-1and NQO-1 were quantified using ELISA kit (Wuhan USCN Business Co., Ltd). Assays were performed according to the manufacturer\u0026rsquo;s instructions. The absorbance (450 nm) for each sample was analyzed using microplate reader (Bio-RAD, USA). The levels of GSH-Px and TPx were quantified using ELISA kit Jiancheng(Nanjing, China). Assays were performed according to the manufacturer\u0026rsquo;s instructions. Using super micro UV VIS spectrophotometer to detect OD value (Thermo, USA).\u003c/p\u003e \u003cp\u003e \u003cb\u003eSEM analysis.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eProtoscoleces were washed three times in PBS pH 7.2, and placed into 4% glutaraldehyde for at least 24 h at 4℃. For SEM analysis, the settled specimens were dehydrated by sequential incubations in increasing concentrations of ethanol (50%, 70%, 80%, 90% and 100%), and were finally immersed in hexamethyl-disilazane and air-dried under a fume hood. The dehydrated protoscoleces were sputter-coated with gold and observed under a LEO1430VP scanning electron microscope operating at 25 kV.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eWestern Bloting Analysis\u003c/h2\u003e \u003cp\u003eProtoscoleces were collected after PIK-75 treatments and washed with ice-cold PBS. The protoscoleces were lysed with RIPA lyse buffer containing PMSF (1:100) for 10 min on ice, after that we used Cell Ultrasonic Crusher to smash protoscoleces, and then centrifuged at 12000g for 10min. A microplate reader was used to determine the protein concentrations. The supernatant was designated as whole protoscoleces protein extract and kept at -80℃. Subsequently, the samples were boiled in Laemmli loading buffer. Each 40 mg aliquots of total protein extracts were separated on an 8%, 10%, or 12% SDS-polyacrylamide gel, followed by transfer of the proteins to a polyvinylidene fluoride (PVDF) membrane. Membranes were blocked with Tween 20 (TBST) supplemented with 5% nonfat dry milk for 1\u0026ndash;2 hours, followed by incubation with primary antibodies at 4℃ for 12\u0026ndash;24 hours. Membranes were incubated with the goat anti-rabbit IgG antibody at temperature for 2\u0026ndash;3 hours, after the the membranes had been washed three times with PBST. The bands were visualized using an ECL detection kit and quantified using Image Lab software, after being washed three times with PBST.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCaspase-3 activity assay.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eProtoscoleces were treated as indicated and caspase-3 activity was measured from cell lysates with Caspase-3 Assay kit (APPLYGEN, China) according to the manufacturer's protocol. Luminescence was measured using microplate reader. Relative luminescence units were determined by calculating luminescence values from samples as a percentage of values from control samples. The experiments were considered in three independent experiments performed in duplicate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis.\u003c/h2\u003e \u003cp\u003eAll data are shown as the arithmetic mean\u0026thinsp;\u0026plusmn;\u0026thinsp;S.E.M. The results were subjected to paired t-tests and one-way ANOVA with Least significant difference (LSD) test in order to determine the statistical significant differences from the control using SPSS 17.0 software. P values less than 0.05 were considered significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eLocalization of Nrf2 in E. granulosus protoscoleces.\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe localization profile of Nrf2 in protoscoleces was demonstrated using immunofluorescence. Immunocytochemical staining showed that under control conditions, Nrf2 was localized in the cytoplasm of the cells of the protoscoleces.\u0026nbsp;(Figs. 1, 2). After 24 h incubation with both low and high concentrations of PIK-75, Nrf2 levels in protoscoleces\u0026nbsp;decreased relative to control\u0026nbsp;(Fig. 2).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe generation of reactive oxygen species\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe also measured intracellular ROS by using a DCFH-DA-based assay after protoscoleces were treated with PIK-75 for 2d and 5d. As shown in Figure 3, incubation of protoscoleces with PIK-75 induced a continuous time- and concentration-dependent increase in ROS production, for the complete 5d, when compared to the control protoscoleces, as observed with the microplate reader \u0026nbsp;(P\u0026lt;0.05).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffects of PIK-75 on the expression of Antioxidant enzymes in protoscoleces.\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eProtoscoleces were pre-treated for 2 or 5 days with 0.8 μM or 1.6 μM PIK-75, and HO-1, NQO-1, GSH-Px and TPx expression was measured using an ELISA kit to examine whether PIK-75 modulates HO-1, NQO-1, GSH-Px and TPx expression in cultured protoscoleces\u0026nbsp;(Figs.4,5,6,7). All protoscoleces treated with PIK-75 showed a time- and dose-dependent decrease in HO-1, NQO-1, GSH-Px and TPx expression (P\u0026lt;0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePIK-75\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003esuppress\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;the expression of Nrf2 protein in vitro\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe investigated the effect of\u0026nbsp;PIK-75\u0026nbsp;treatment on the expression of Nrf2 in protoscoleces by using western bolt technology. As shown in Figure 8, the level of Nrf2 protein was decreased by\u0026nbsp;PIK-75\u0026nbsp;in a concentration- dependent manner. The 1.6μM\u0026nbsp;PIK-75\u0026nbsp;treatment caused a significant decrease in the expression of\u0026nbsp;PIK-75\u0026nbsp;at 5d when compare to 1000 μM control group.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffects of PIK-75 on \u003cem\u003eE. granulosus\u003c/em\u003e protoscoleces in vitro.\u003c/strong\u003e \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFollowing the isolation of protoscoleces from hydatid cysts, the viability of protoscoleces was assessed using the eosin exclusion assay. The survival of \u003cem\u003eE. granulosus\u003c/em\u003e protoscoleces after exposure to DMSO or various concentrations of PIK-75 is shown in Figure\u0026nbsp;9,10. It was observed that protoscoleces cultured with 2.0 μM\u0026nbsp;PIK-75 died at a considerably faster rate than protoscoleces cultured with the lower concentrations. After 5 days of exposure to 0.4 μM PIK-75, protoscoleces viability was approximately 81.3%, had started presenting evaginations (Figs. 9, 10b). The viability of the protoscoleces incubated with 0.8 μM PIK-75 for 2 days was 72.88%, and several slight pits were apparent in all protoscoleces (Figs. 9, 10c). After 5 days treatment with 0.8 μM PIK-75, survivability had been reduced to 53.2%, and the appearance of tegumental alterations and sucker region distortion was observed (Figs. 9, 10d). PIK-75 clearly showed decreased efficacy at a concentration of 1.6 μM, as 55.1% of protoscoleces were still viable after 2 days of treatment. Scanning electron microscopy (SEM) showed ultrastructural changes, including tegumental contraction with loss of hooks, and rostellar disorganization (Figs. 9, 10e). The rate of protoscoleces death increased over time. Only a small fraction (~22%) of protoscoleces was viable in cultures treated with 1.6 μM PIK-75 after 5 days. Furthermore, the complete destruction of the rostellum and the germinal layer was observed by SEM at this time point (Figs. 9,\u0026nbsp;10f). However, control protoscoleces that had been incubated in RPMI Medium 1640 with DMSO for 7 days presented ~95.4% viability, with no observed changes in morphology or ultrastructure throughout the experimental period (Figs. 9, 10a).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePIK-75-induced apoptosis in protoscoleces.\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo study the impact of Nrf2 inhibition-induced apoptosis, protoscoleces were treated with different concentrations of PIK-75 for 24 or 48 h (Figure 11). Apoptosis was determined by assaying caspase-3 activation. All concentrations induced a significant increase of caspase-3 activity at both time points compared with controls.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe aim of this study was to investigate the efficacy of PIK-75 against inhibition of the Nrf2/KEAP1 signaling pathway in \u003cem\u003eE. granulosus\u003c/em\u003e protoscoleces. In vitro culture of protoscoleces was employed to demonstrate the parasiticidal effects of PIK-75.\u003c/p\u003e \u003cp\u003eNrf2-KEAP1-ARE is a pivotal signaling pathway in maintaining cellular oxidative equilibrium (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Nrf2, referred to as the master regulator of antioxidant, detoxification, and cell defense gene expression, is bound in the cytoplasm to KEAP1. This study determined Nrf2 localization and expression in \u003cem\u003eE. granulosus\u003c/em\u003e protoscoleces using confocal immunofluorescence microscopy, and found that although Nrf2 is widely distributed, it is primarily localized in the cytoplasm.\u003c/p\u003e \u003cp\u003eAs a phosphoinositide 3-kinase (PI3K) inhibitor, PIK-75 has demonstrated potent anti-cancer activity in several cancer cell lines (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e) and in a cervical cancer xenograft model (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). Duong HQ et al. have demonstrated that Nrf2 protein levels and activity were reduced by PIK-75 in human pancreatic cancer cell lines (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). To some degree, the ability to inhibit Nrf2 via a PI3K/Akt pathway inhibitor was expected, because it had been reported that Nrf2 stability is regulated by the PI3K/Akt/glycogen synthase kinase (GSK) 3β pathway (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Using confocal immunofluorescence microscopy, it was found that in vitro treatment of protoscoleces with increasing doses of PIK-75 resulted in decreasing immunoreactivity intensity compared to untreated controls. Furthermore, confocal microscopy did not reveal any consistent translocation of Nrf2 from the cytoplasm to the nucleus in PIK-75-treated protoscoleces. All this suggests that Nrf2 protein is primarily expressed in the cytoplasm, and its levels decrease during PIK-75-treatment in a dose-dependent manner.\u003c/p\u003e \u003cp\u003eGenerally, ROS overproduction is deleterious to cell structures and cell macromolecular constituent functions, leading to excessive expression of cytokines and inflammatory factors, which in turn results in acute inflammation, atheromatous plaques, sepsis, and tissue injury (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). To defend against these deleterious actions, cells contain multiple antioxidant enzymes expressed in various regions to scavenge ROS (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). According to a new study, the induction of HO-1 could protect cells from oxidative stress in acute hepatic injury model (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). In addition, HO-1 has been shown to exert a particularly strong anti-apoptotic effect, favoring cancer cell growth and therapeutic resistance. Ma Q has found that, in the development of chronic obstructive pulmonary disease (in particular, emphysema) and lung cancer, the adaptive activation of Nrf2 led to increased expression of NQO1, GSH-Px2, and GSH, all of which suppress CS (cigarette smoke)-induced ROS production and oxidative damage (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). Some experts have also demonstrated that GSTs were well represented in Fasciola hepatica (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). In the present study, PIK-75 has been shown to induce higher levels of intracellular ROS in the early time, and ELISA data showed that treating cultured protoscoleces with PIK-75 (0.8, and 1.6 \u0026micro;M) for either 2 or 5 days caused a significant decrease in both HO-1, NQO-1, GSH-Px and TPx production in a time- and dose-dependent manner. Taken together, PIK-75 has a clear inhibitory effect on antioxidant enzyme production in \u003cem\u003eE. granulosus\u003c/em\u003e protoscoleces.\u003c/p\u003e \u003cp\u003eIn addition, we investigated PIK-75 treatment effects on protoscoleces viability and morphology in vitro. PIK-75 increased the mortality of protoscoleces in a dose-dependent manner, as protoscoleces cultured with 2.0 \u0026micro;M PIK-75 showed considerably higher mortality than protoscoleces cultured with other concentrations. SEM revealed many ultrastructural changes in PIK-75-treated protoscoleces, including numerous pits appearing at the tegument, disruption of hooks and microtriches of the scolex region, collapse of the sucker region, and protoscoleces contraction. In contrast, those parasites cultured in RPMI 1640 Medium with DMSO did not present significantly altered morphology or ultrastructure. These results demonstrate that PIK-75 negatively affects \u003cem\u003eE. granulosus\u003c/em\u003e protoscoleces.\u003c/p\u003e \u003cp\u003eCaspases, including the upstream \u0026ldquo;initiator\u0026rdquo; and downstream \u0026ldquo;effector\u0026rdquo; groups, belong to a family of cysteine proteases, which are involved in the cleavage of aspartic acid. Caspase-3, a member of the downstream \u0026ldquo;effector\u0026rdquo; group, has been identified as a key regulator of growth-promoting signals generated from dying cells. Moreover, Caspase-3 has been shown to be involved in the \u0026ldquo;execution\u0026rdquo; phase of cellular apoptosis (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). Other studies have demonstrated significant increases in not only cell apoptosis, but also in caspase-3 activity in drug-treated protoscoleces (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). In this study, caspase-3 activity in the PIK-75-treated group was significantly higher than in the control group at both 24 h and 48 h. Furthermore, protoscoleces were more influenced by high concentrations of PIK-75, rendering those cells more sensitive to apoptosis. These findings indicate that PIK-75 possibly mediates apoptosis of protoscoleces through the caspase-3 pathway.\u003c/p\u003e \u003cp\u003eTo conclude, this study has elucidated the Nrf2 distribution pattern in protoscoleces of \u003cem\u003eE. granulosus\u003c/em\u003e, and observed decreased levels of Nrf2-mediated antioxidases in protoscoleces treated with PIK-75. This study has also demonstrated that the Nrf2 inhibitor PIK-75 exerts a satisfactory scolicidal effect, presenting eventual reduction of parasite viability that leads to death. These successful results will provide a theoretical basis for the development of novel treatments for hydatid disease in the future. Further studies for the comprehensive elucidation of the mechanisms of PIK-75-mediated Nrf2 downregulation in \u003cem\u003eE. granulosus protoscoleces\u003c/em\u003e will be required in order to avoid any potential side effects of PIK-75.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank Prof. Hailong Lv\u0026nbsp;Department of Hepatobiliary Surgery, The First Affiliated Hospital, School of Medicine, Shihezi University for helpful suggestions, and Bin Yang\u0026nbsp;at the\u0026nbsp;Department of Cardiac Surgery, The Seventh People’s Hospital of Zhengzhou City\u0026nbsp;for expert technical assistance.\u0026nbsp;We are also indebted to the Department of Histology and Embryology, Medical College of Shihezi University for providing us the experimental platform.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe protocol of the study was reviewed and approved by the Ethics Committee of the Seventh People’s Hospital of Zhengzhou.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eApplicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eApplicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors’ contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceived and designed the study and supervised the project: Guangyao Tang, Ziyu Liu, Hailong Lv and Bin Yang. Undertook the study and data analysis: Guangyao Tang and Longjun Wang. Contributed to analysis using various tools: Sheng Liu and Ziyu Liu. Wrote the paper: Guangyao Tang, Ziyu Liu, and Hailong Lv. Contributed to the interpretation of findings and drafting of the manuscript: Longjun Wang and Sheng Liu. All authors read and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by grants from the National Natural Science Foundation of China (grant U1303121 and 81560334 to Hailong Lv, )and Technology Research Plan Joint Construction Project Foundation of \u0026nbsp;Henan (grant LHGJ20220837 to Bin Yang).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003e\u003cspan\u003eBudke CM, Carabin H, Ndimubanzi PC, Nguyen H, Rainwater E, Dickey M, Bhattarai R, Zeziulin O, Qian MB .2013. A systematic review of the literature on cystic echinococcosis frequency worldwide and its associated clinical manifestations. 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Parasitol Res 109(2):453\u0026ndash;9. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s00436-011-2276-9\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Echinococcus granulosus protoscoleces, Nrf2 signal pathway, PIK-75","lastPublishedDoi":"10.21203/rs.3.rs-3013557/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3013557/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eIntroduction \u003c/strong\u003eThe aims of this study were to investigate the role of the nuclear factor E2-related factor 2 (Nrf2) signaling pathway in \u003cem\u003eEchinococcus granulosus\u003c/em\u003e protoscoleces, and to examine the effects of PIK-75 inhibition on Nrf2 activity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods \u003c/strong\u003eNrf2 protein expression and localization in protoscoleces were examined via confocal immunofluorescence microscopy. Reactive oxygen species (ROS detection kit) was used to detect ROS level in protoscoleces. The effects of PIK-75 on activity of heme oxygenase1(HO-1), NAD(P)H quinone oxidoreductase 1 (NQO1), glutathione peroxidase (GSH-Px) and thioredoxin peroxidase (TPx) were characterized using ELISA. Evidence of parasite damage and death was observed by scanning electron microscopy(SEM). Western bolting was used to detect the expression level of Nrf2 protein. In addition, caspase-3 activity was detected using an assay kit.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults \u003c/strong\u003eThe study found that Nrf2 is primarily localized in the protoscoleces cytoplasm, and PIK-75 treatment could increased ROS level(\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05) in the early time, and reduced NQO-1, HO-1, GSH-Px and TPx (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05) activity in protoscoleces. SEM showed that PIK-75-treated protoscoleces presented damage in the protoscoleces region. Western-blot showed that the Nrf2 protein expression had decreased significantly. Caspase-3 activity clearly increased in protoscoleces treated for 24 and 48 h with PIK-75 compared with that in controls (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion \u003c/strong\u003eThe present investigation demonstrated that PIK-75 had an inhibitory effect on the Nrf2 signaling pathway. We also provide evidence that PIK-75 may serve as a potential therapeutic agent for the treatment of protoscoleces. The use of PIK-75 as a treatment for protoscoleces, however, requires further research.\u003c/p\u003e","manuscriptTitle":"In vitro parasiticidal effects of PIK-75 inhibition of Nrf2 against Echinococcus granulosus protoscoleces","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-06-12 14:19:49","doi":"10.21203/rs.3.rs-3013557/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c62e3abe-6ef0-4a8b-878a-f6a796ef1ea3","owner":[],"postedDate":"June 12th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-06-26T15:29:29+00:00","versionOfRecord":[],"versionCreatedAt":"2023-06-12 14:19:49","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3013557","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3013557","identity":"rs-3013557","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}