Introduction

Glioblastoma multiforme (GBM), the most common and aggressive form of primary brain tumor in humans, causes an incidence of 3–8 cases per 100,000 population1. The histological classification of gliomas from grade I to IV in the 2016 WHO classification has been updated by the 2021 WHO CNS5 to include molecular biomarkers for different tumor types2,3. In addition, WHO CNS5 classifies gliomas into four different families: adult-type diffuse gliomas, pediatric-type diffuse low-grade gliomas, pediatric-type diffuse high-grade gliomas; and circumscribed astrocytic gliomas4. Clinically, common glioma treatment options include: chemotherapy, radiotherapy, surgical excision, and electric field therapy (TTF)5. However, accurate excision is difficult due to the invasive growth of these tumors. New therapeutic strategies such as gene therapy, immunotherapy, exosome therapy and oncolytic virus therapy face challenges including low efficiency of blood-brain barrier (BBB) drug delivery and difficulty targeting tumor hypoxic microenvironment5,6.

Engineered nanomaterials (ENMs) have been widely utilized in the treatment of cancer5,7,8. Receptor engineering mediated uptake of ENMs presents a potential method of targeted drug delivery through the BBB and can be employed for the targeted killing of cancer cells9,10. Metals and metal oxide nanoparticles, due to their small size, can also be applied in brain cancer treatment11. Among these ENMs, copper oxide nanoparticles (CuO NPs) offer the advantage of being cost-effective compared with gold nanoparticles (Au NPs) and silver nanoparticles (Ag NPs), and the released copper ions exhibit a strong killing effect on cancer cells. Recently, chemokinetic therapy (CDT) based on ENMs has been extensively researched12, which is the key to the action of CDT in the tumor microenvironment to produce hydroxyl radical (·OH) and accelerate the apoptosis of tumor cells13. CuO NPs are considered typical nanoscale enzymes capable of catalytically generating ·OH14. Jiang et al. synthesized CuO-decorated carbon nanoplatforms, which can release Cu2+and induce tumor cell apoptosis by generating hydroxyl radicals through Haber-Weiss and Fenton-like reactions15. Moreover, copper is involved in various cellular processes including mitochondrial respiration, antioxidant defense, REDOX signaling, kinase signaling, autophagy, and protein quality control16. Dysregulation of copper storage can lead to oxidative stress and cytotoxicity. Moreover, copper forms polymers by directly binding to the fatty acylation components of the tricarboxylic acid cycle, causing cytotoxicity and eventually cell death17. Therefore, the regulation of cellular copper content is particularly relevance to cancer therapy, as copper (Cu) has a higher effect on the growth and metastasis of cancer cells.

Thus, the application of CuO NPs in glioma therapy may contribute to addressing the dilemma of breaking the blood-brain barrier to administer drugs and regulate the hypoxic tumor microenvironment. However, few data are available on the treatment of glioma with CuO NPs, and fundamental research data are necessary before further consideration of the clinical safety of CuO NPs. In this study, we exploited and characterized the physicochemical properties of CuO NPs and investigated the therapeutic response of CuO NPs treatment to human glioma cell lines in vitro. Furthermore, the in vivo therapeutic effect of CuO NPs on glioma model rats was studied. Combined with the results of the study, the potential mechanism of CuO NPs anti-tumor was discussed and clarified.

Materials and methods

Preparation of cuo nps suspension

CuO NPs were purchased from Sigma-Aldrich (USA) and is used without further purification. Preparation of CuO NPs suspension: The CuO NPs were suspended in a sterilized dispersion (normal saline or cell culture solution) configured as a 2 mg/mL suspension. The prepared CuO NPs suspension was stored at 4℃ for future use. Before each use, the well-mixed suspension of CuO NPs was placed in an ice bath and continuously ultrasonic treatment for 1 h to obtain a uniformly scattered CuO NPs dispersion.

Characterization of cuo nps

The purchased-CuO NPs were observed by transmission electron microscope (TEM) and X-ray diffraction (XRD). Specifically, TEM (JEM-2010 FEF/JEM-2800, Japan) was used to observe the morphology and size of CuO NPs, and Image J (V1.8.0.112) was used to calculate the size of CuO NPS from the obtained images18. XRD spectroscopy (Ultima IV, Rigaku Corporation, Japan) was used to determine the crystal structure of CuO NPs in the 2θ range of 30–70°, using Jade 6.5 software for crystal identification.

Cell experiment

U87MG human glioma cell line (CL-0238, Procell) was cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco; Thermo Fisher Scientific, Inc.) at 37 °C with 5% CO2. The treatment of ENMs was incubated with 20 µg/mL CuO NPs for 1 h and 12 h.

Animal experiment

The 6-week-old rats (SPF male Wistar) were purchased from Beijing Vitonglihua Experimental Animal Technology Co., LTD., China. The rats were reared in SPF environmental conditions with a temperature of 20–26℃, humidity of 40–70%, light and darkness for 12 h each. After one week of adaptive feeding (100–120 g at the start of assays), animal exposure experiments were conducted, and the rats were randomly divided into blank control group (C), model group (M), and CuO NPs treatment group (E).

Modelling of glioma rats was performed as previously reported (Text S1)19. Rats were injected with CuO NPs (10 µg/g body weight) via tail vein, while control and model groups were injected with placebo (PBS)20. After the end of the exposure period, behavioral tests of rats were conducted. Then, the rats were anesthetized with 2% pentobarbital sodium (40 mg/kg), blood was collected from abdominal aorta, and glioma and para-cancerous hippocampus tissue were rapidly isolated on ice. Part of the hippocampus was fixed with 4% paraformaldehyde and 2.5% glutaraldehyde for pathological detection, immunohistochemical detection and ultrastructural detection, respectively. The remaining hippocampal tissue was stored in liquid nitrogen for later use.

Behavioral tests in rats

Marble burying test (MBT), Open field test (OFT) and Habituation/dishabituation olfactory test (H/DOT) were used to test the behavioral effects of CuO NPs treatment on glioma rats. Methodological details are given in Text S2, Text S3, and Text S4 in Supporting Information (SI).

Morris water maze test

The Morris Water Maze consists of a circular, opaque plastic pool 110 cm in diameter and 50 cm in height (divided into four quadrants) with a 10 cm diameter platform located 1.5 cm underwater (30 cm in depth)21. The test training phase was conducted continuously for 3 days, 4 times a day. The rat was thrown into the water facing the wall of the pool. The incubation period was recorded from the time the rats entered the water to the time they found the underwater concealed platform and stood on it. The incubation period was expressed in seconds (s). After the rats found the platform, they were allowed to stand on the platform for 10 s. The tests were carried out on days 1, 3 and 7, three times a day, with each trial in a different quadrant.

Histopathological examination

The pathological changes of rat hippocampus were observed by Hematoxylin and eosin (H&E) staining. H&E sections were observed and imprinted in bright areas using laser scanning confocal microscopy (LSM880 with Airyscan, ZEISS, Germany).

Western blot (WB) analysis

To begin with, ~ 100 mg hippocampus tissue was homogenized and treated with radioimmunoprecipitation assay (RIPA) lysis buffer (No: W062-1, Nanjing Institute of Bioengineering, China) and phenylmethanesulfonyl fluoride (PMSF, No: W044-1-1, Nanjing Institute of Bioengineering, China), 1000 g centrifuge for 5 min, supernatant was collected. Total protein quantitative assay kit (BCA, No: A045-3-2, Nanjing Jiancheng Bioengineering Institute, China) was used for protein quantification. The extracted samples were separated by sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) and imprinted on the membrane. Subsequently, the membrane was blocked with skimmed milk powder for 1 h and incubated at 4℃ overnight with primary antibody as shown in Table 1. The membranes were then rinsed with Tris-buffered saline/Tween (TBST) and incubated with secondary antibodies coupled with horseradish peroxidase. Finally, TBST again rinsed after scanning the film.

Table 1 Information on primary and secondary antibodies used in Western blot analysis.
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Eenzyme-linked immunosorbent assay (ELISA)

The levels of interleukin-1β (IL-1β, No: SEKM-0002), interleukin-6 (IL-6, No: SEKM-0007), tumor necrosis factor α (TNF-α, No: SEKM-0034), melatonin (MT, No: H256) and oxytocin (OT, No: H176) were measured in cells and hippocampal tissues using ELISA kits. ELISA kits for IL-1, IL-6 and TNF-α were purchased from Solarbio Biotechnology Co., LTD., Beijing, China. MT and OT ELISA test kits purchased from Nanjing Jiancheng Institute of Bioengineering, China. All tests were carried out in strict accordance with the manufacturer’s instructions.

Immunofluorescence

As with his sections, different groups of hippocampus and prefrontal cortex tissues were fixed, embedded, and sectioned. The sections were dewaxed and dehydrated, and antigen recovery was performed at 95 °C for 10 min. Subsequently, the paraffin sections were sealed at room temperature with 5% BSA for 1 h. For immunofluorescence, FitC-labeled fluorescent primary antibodies GFAP (Affinity, USA, 1:200) and IBA-1 (Merck, China, 1:200) were incubated overnight at 4℃. The next day, the rabbit IgG (H + L) Alexa Fluor 488 coupled with secondary antibody (CST, USA, 1:500) was washed with PBS and incubated for 1 h in the dark. After the DAPI was maintained, the sections were observed and photographed under a laser scanning confocal microscope (LSM880 with Airyscan, ZEISS, Germany).

Quantitative real-time PCR (qrt-PCR)

Total RNA extraction from hippocampus tissue cells and U87MG human glioma cells of each group was performed according to TRIzol reagent manual (Invitrogen). The total RNA obtained was treated with DNase I (Sigma Aldrich), and then quantified using the first strand cDNA synthesis kit (No: N118, Nanjing Jiancheng Institute of Bioengineering, China) for reverse transcription to generate cDNA. SuperRealPreMix Plus Kit (FP205, SYBR Green, TIANGEN China) was used for real-time qRT-PCR assay of 100 ng cDNA samples. qRT-PCR was performed using the IQ5 Multicolor qRT-PCR assay system (Bio-Rad, Hercules, USA) in strict accordance with the manufacturer’s instructions. The PCR primers used are listed in Table S1.

Measurement of neurotransmitters and oxidative stress

Neurotransmitters γ-aminobutyric acid (γ-GABA, No: H168-1-2), 5-Hydroxytryptamine (5-HT, No: H104-1-2), Acetylcholine (Ach, No: A105-1-1); and oxidative stress species Reactive oxygen species (ROS, No: E004-1-1), total superoxide dismutase (SOD, No: A001-3-2), and catalase (CAT, No: A007-2-1) was determined using a assay kit purchased from Jiancheng Bioengineering Institute, Nanjing, China. All procedures were carried out in strict accordance with the manufacturer’s instructions.

TEM analysis

Hippocampus tissue and cells collected from rats were fixed in 2.5% glutaraldehyde (Beijing Solarbio Technology Co., LTD.) at 4℃. The samples were then thoroughly rinsed with a 0.1 M phosphate buffer (pH 7.4) and fixed in 1% osmic acid (SSBT) at 4℃. The samples were thoroughly rinsed with phosphoric acid buffers, then sequentially dehydrated with gradient concentrations (30, 50, 70, 80, 85, 90, and 100%) of ethanol and embedded in epoxy resin. After the slices were sliced 50 nm by an ultra-thin micro slicer, the slices were dyed with uranium lead double (2% uranium acetate saturated water solution, lead citrate) at room temperature for 15 min and dried overnight at room temperature. Finally, hippocampal tissue was observed using TEM (FEI Talos F200x USA).

Statistical analysis

All tests were performed in three to six separate trials, with the number of repetitions detailed in the study. For behavioral experiments in rats, eight separate tests were performed. All data collected are presented as mean ± variance. One-way analysis of variance (ANOVA) was performed using SPSS 22.0 to assess inter-group differences. The symbols*, **, and *** represent statistical differences of p < 0.05, p < 0.01, and p < 0.001, respectively.

Results

Characterization of cuo nps

The characterization results of CuO NPs were shown in Fig. 1. TEM observation of CuO NPs showed that CuO NPs showed spherical morphology (Fig. 1a), with a mean particle size of 20.73 ± 8.69 nm (Fig. 1b). As shown in Fig. 1c, XRD analysis of the crystal structure of CuO NPs showed that the corresponding characteristic peak was ascribed to CuO (JCPDS 80-1268).

Fig. 1
Fig. 1The alternative text for this image may have been generated using AI.
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Characterization of CuO NPs. (a) TEM topography, (b) statistical distribution of CuO NPs particle size in TEM, and (c) XRD pattern of CuO NPs.

Effects of cuo nps on inflammation, oxidative stress, and pathology in rat hippocampus

Interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor α (TNF-α) are significantly upregulated in glioblastoma22. The treatment of glioma requires the regulation of hypoxic microenvironment in tumor area. Herein, we tested the effects of CuO NPs treatment on the activity of inflammatory cytokines, oxidase, and pathology in the hippocampus of glioma rats (Fig. 2). The results showed that the levels of inflammatory cytokines IL-1, IL-6 and TNF-α were dramatically increased in glioma rats compared to the control group (Fig. 2a). After treatment with CuO NPs, a significant decrease in inflammatory cytokines was observed (p < 0.001). In addition, CuO NPs treatment significantly increased the activities of catalase (CAT) and antioxidant oxidase (SOD) in the hippocampus of glioma model rats (p < 0.001), and the excess ROS produced by glioma modeling was significantly eliminated (Fig. 2b). HE staining of the rat hippocampus further revealed that the overall structure of the hippocampus of the model (M) rat was abnormal, with closely arranged but irregular cells, marked contraction and deep staining, obvious edema in the surrounding edema, and obvious inflammatory cell infiltration in the tissue. CuO NPs treatment eliminated edema and inflammatory cell infiltration (Fig. 2c). TEM observations of the rat hippocampus showed the same results (Fig. S1).

Fig. 2
Fig. 2The alternative text for this image may have been generated using AI.
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Inflammatory response, oxidative stress level, and pathological changes in the hippocampus before and after CuO NPs treatment. (a) The changes in the levels of inflammatory cytokines IL-1, IL-6 and TNF-α, (b) the levels of CAT, SOD and ROS, and (c) HE staining of the hippocampus. The number of parallel samples used is n = 6.

The effect of CuO NPs treatment on IBA-1 (Ionized calcium binding adapter molecule 1) in the hippocampus and prefrontal cortex of rats was further observed by immunofluorescence. The results showed that CuO NPs reduced the level of IBA-1 in the hippocampus and prefrontal cortex of rats (Fig. 3 and Fig. S2).

Fig. 3
Fig. 3The alternative text for this image may have been generated using AI.
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Immunofluorescence observation of Hippocampal tissue after CuO NPs treatment.

Cuo nps reduce the levels of glioma biomarkers in hippocampus tissues

Western Blot (WB) and real-time quantitative PCR (qRT-PCR) were used to confirm the expression of glioma-related proteins and genes in the hippocampus of model rats treated with CuO NPs (Fig. 4).

Fig. 4
Fig. 4The alternative text for this image may have been generated using AI.
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Effect of CuO NPs on glioma rats. (a) Representative Western Blot results, (b) expression of EphA2, YKL-40, MMP-9 and PGC-1α relative to GADPH, and (c) relative expression of PTEN, EphA2, YKL-40 and MMP-2 genes. The number of parallel samples is n = 6.

EphA2 is overexpressed in about 60% of GBM, and is also highly expressed in astrocytoma and significantly enhanced with the increase of pathological grade23. Its overexpression is directly related to poor prognosis of patients and inversely proportional to patient survival rate. YKL-40 is also believed to be significantly related to the occurrence of glioma, and its content is inversely proportional to the prognosis of patients with survival24. MMP-2 and MMP-9 are significantly correlated with the occurrence of glioma, and are considered as potential biomarkers for detection of glioma25.PTEN is a tumor suppressor gene. In the treatment of GBM, the abnormal activation of AKT signaling pathway due to the deletion or mutation of PTEN makes cells resistant to tyrosine kinase inhibitors, which affects the therapeutic effect26. Thus, EphA2, YKL-40, MMP-2, MMP-9 and PTEN were selected as glioma-related proteins and genes. Glioma modeling resulted in increased contents of EphA2, YKL-40 and MMP-9 proteins in the model rats, and the three proteins in the glioma rats were down-regulated after CuO NPs treatment, and the down-regulated effects were more significant with the extension of the treatment cycle (Fig. 4a and b; Table 2). The qRT-PCR tests (Fig. 4c) showed that the expressions of EphA2, MMP-2 and MMP-9 in the hippocampus of model rats were significantly down-regulated after CuO NPs treatment (p < 0.001), while the content of PTEN was significantly up-regulated (p < 0.05).

Table 2 Statistical differences in Western blot analysis of rat brain tissue. Note: the * symbol outside the parentheses, inside () and [] indicates the difference between E-7 d and M group, E-1 d and E-3 d, respectively.
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Cuo nps improved spatial recognition and memory in rats

The neuroprotective effect of CuO NPs treatment on glioma model rats was studied (Fig. 5). Melatonin (MLT) regulates biological rhythm and has anti-tumor function27. Oxytocin (OT) coordinates a range of social behaviors in animals, such as mother-infant bonding, social cognition, and mate bonding28. Herein, we found the effect of CuO NPs treatment on MLT and OT (Fig. 5a and b). The results showed that glioma modeling resulted in decreased levels of MLT and OT in the rats, while CuO NPs treatment significantly increased the levels of MLT (p < 0.005) and OT (p< 0.001) in the hippocampus. Neurotransmitters have been identified as potential targets for glioblastoma, and neurotransmitters such as γ-aminobutyric acid (γ-GABA), 5-Hydroxytryptamine (5-HT), and acetylcholine (Ach) have been identified as key players in GBM behavior29. The detection of CuO NPs treated model rats showed that the content of γ-Gaba was up-regulated, while the content of 5-HT and Ach was down-regulated (Fig. 5c), these changes were significantly different (p < 0.001).

Fig. 5
Fig. 5The alternative text for this image may have been generated using AI.
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Effect of CuO NPs treatment on spatial recognition and memory ability in rats. (a) Changes of Melatonin (MLT) levels in rats hippocampus before and after treatment, (b) changes in hippocampus Oxytocin (OT), (c) changes in neurotransmitters γ-GABA, 5-HT, and Ach, (d) Marble burying test (MBT) the number of marble beads buried by rats, (e) Open field test (OFT) the moving distance of rats and (f) the stay time in the central area, (g) Habituation/dishabituation olfactory test (H/DOT) experiment of rats on a particular taste recognition, (H) Morris water maze experiment rats in the time duration of the platform in quadrant, (I) the escape latency in Morris water maze test, and (j) path tracing of rats in the Morris Water maze test. The number of parallel rats used in each experiment ranged from 6 to 8.

Next, four behavioral tests including MBT, OFT, H/DOT and Morris Water maze were used to explore the effect of CuO NPs treatment on spatial recognition and memory ability of glioma rats after treatment30,31. Bead burial is a living habit of rodents. The bead burial behavior of glioma rats was significantly inhibited after modeling, and the inhibition of bead burial behavior caused by glioma was alleviated by CuO NPs treatment (Fig. 5d). In OFT studies, model rats spent longer time in the center of the field and moved longer distances (Fig. 5e and f), which may be due to glioma-induced hyperexcitability in rats, which was significantly alleviated by treatment with CuO NPs (p < 0.001). The new and old odor discrimination tests showed that the influence of modeling on the odor discrimination ability of rats was not significant (p > 0.05), and CuO NPs did not cause significant changes (Fig. 5g). Morris water maze experiment showed that CuO NPS-treated model rats moved longer distances in the platform quadrant than untreated model rats (Fig. 5h) and shorter escape incubation period (Fig. 5i). In addition, CuO NPs treatment increased the number of cross-platform model rats (Fig. 5j). The shorter the incubation period and the more cross-platform times, the better the memory of the rats.

Therapeutic effect of cuo nps on human brain glioma cells

Finally, the therapeutic effect of CuO NPs on human glioma cells was verified by U87MG human glioma cell line. Targeted activation of PGC-1α can improve the response of glioma patients to therapy32. Therefore, PGC-1α was co-selected to verify the therapeutic effect of CuO NPs on U87MG human glioma cells. As shown in WB results (Fig. 6a and b), CuO NPs down-regulated the expression of EphA2, YKL-40 and MMP-9, and increased the expression of PGC-1α in human glioma cells, and the differences were significant (p < 0.001). Meanwhile, the expression of PGC-1α was significantly up-regulated with the extension of treatment time (p < 0.005). The qRT-PCR tests (Fig. 6c) showed that after CuO NPs treatment, the expressions of EphA2, YKL-40 and MMP-2 in human glioma cells were significantly down-regulated (p < 0.001), while the up-regulated expression of PTEN was not different (p > 0.05).

Fig. 6
Fig. 6The alternative text for this image may have been generated using AI.
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Effect of CuO NPs therapy on human glioma cells. (a) Representative Western Blot results, (b) EphA2, YKL-40 and MMP-9 protein expression relative to GADPH, and (c) relative expression of PTEN, EphA2, YKL-40 and MMP-2 genes. The number of parallel samples is n = 3.

Discussion

Glioma is a common tumor in adults, but clinical diagnosis and treatment of glioma is still limited to prolonging the survival time of patients22. Engineering nanomaterials (ENMs), especially metals and metal oxide nanomaterials, have small particle sizes and can release metal ions slowly in application engineering33,34,35. The development and progress of nanotechnology provide new opportunities and challenges for the treatment of glioma36. CuO nanoparticles (NPs) have lower economic cost than traditional Ag NPs and Au NPs, and release Cu2+ in application, which is also biotoxin to cancer cells. These properties make CuO NPs offer greater potential in glioma treatment. However, few studies have systematically evaluated the therapeutic effect of CuO NPs on glioma. Herein, we used CuO NPs with a particle size of 20.73 ± 8.69 nm to test its therapeutic effect on glioma rats and U87MG human glioma cell line.

The treatment of CuO NPs ameliorated oxidative stress, inflammatory response and tissue edema in the hippocampal tissues of glioma rats. Treatment with CuO NPs inhibited the expression of IL-1, IL-6, and TNF-α in hippocampal tissue of glioma rats and alleviated integral cell edema. Meanwhile, CuO NPs treatment promoted the levels of oxide scavenging enzymes as well as hydrogen peroxide scavenging enzymes in hippocampal tissues of glioma rats and down-regulated ROS levels. The possible explanation is that Cu2+ released by CuO NPs will catalyze the in situ decomposition of endogenous H2O2to produce ·OH, and then the rats will spontaneously induce the secretion of CAT and SOD enzymes in response to the production of ·OH, which helps to alleviate the hypoxic microenvironment around glioma15,37. Hypoxia is a widespread trait in 90% of solid tumors, and this characteristic is closely related to tumor proliferation, differentiation, angiogenesis, energy metabolism, drug resistance and poor prognosis of patients38. CuO NPs can inhibit the inflammatory response and regulate the hypoxic microenvironment in the hippocampus of glioma rats.

Treatment with CuO NPs restricted activation of the EphA2/YKL-40/MMP-9 signaling pathway in rats thereby constraining glioma development and progression. The expressions of EphA2, YKL-40 and MMP-9 in the hippocampus of rats treated with CuO NPs were down-regulated (Fig. 4). These results indicate that CuO NPs can inhibit the breakthrough of the histological barrier of glioma, prevent the formation of tumor blood vessels and benefit the prognosis of glioma rats. EphA2 is involved in tumor angiogenesis39. YKL-40 is associated with glioma occurrence and prognostic survival. MMP plays a key role in glioma invasion, invasion and metastasis by degrading various protein components in extracellular matrix (ECM) and destroying the histological barrier that prevents tumor cell invasion40. Among them, the expressions of MMP-9 are mainly related to the invasiveness and angiogenesis of glioma41. These results indicate that CuO NPs is a potential treatment method for glioma rats by inhibiting the tissue barrier breakthrough of glioma cells in the hippocampus of glioma rats, inhibiting the formation of tumor blood vessels through restricted EphA2/YKL-40/MMP-9 signaling pathway, thus limiting the occurrence and development of tumors. The effects of CuO NPs on the U87MG human glioma cell line were also tested42, and the results showed that CuO NPs also had a tumor-suppressive effect on human glioma cells through inhibition of the EphA2/YKL-40/MMP-9 signaling pathway. Moreover, after CuO NPs treatment, the expression of PTEN in the U87MG human glioma cell was significantly up-regulated. By antagonizing the activity of tyrosine kinase and other phosphorylases, PTEN can trigger a cascade reaction, affect cell proliferation and metabolism, and inhibit the occurrence and development of tumors26. This suggests that PTEN is a potential target of CuO NPs in the treatment of human glioma32.

Treatment with CuO NPs improved spatial recognition and memory of glioma rats. The neuroprotective investigation of CuO NPs in glioma rats (Fig. 5) showed that melatonin and oxytocin levels were upregulated in hippocampal tissue of model rats after CuO NPs treatment, which contributed to the restoration of biological rhythms and social relationships in glioma rats28,29. γ-GABA levels were upregulated, which helped to inhibit excessive neural signaling and reduce excitability in glioma rats43. The levels of 5-HT and Ach, as important neurotransmitters affecting glioma behavior, were significantly down-regulated after CuO NPs treatment29. Glioma-secreted pro-inflammatory factors, such as IL-6, IL-1β, and TNF-α, play a crucial role in the functional structure of the brain that directly contributes to psychiatric disorders44. Treatment with CuO NPs significantly suppressed the expression of the relevant pro-inflammatory factors in the hippocampus of the model rats, which likewise ameliorated the psychiatric disorders of the rats. Behavioral investigations revealed that glioma modeling leads to the production of excitation and anxiety in rats, and treatment with CuO NPs for 7 d significantly alleviated g hyperexcitability and anxiety in model rats. In addition, Morris water maze as well as H/DOT showed that CuO NPs treatment improved spatial recognition and memory in glioma rats. The results suggest that CuO NPs can play a neuroprotective role in alleviating excitation and anxiety.

Overall, treatment with CuO NPs can catalyze the decomposition of H2O2 in situ to produce ·OH, which in turn induces the expression of CAT and SOD and improves the hypoxic microenvironment of tumors. Meanwhile, the release of CuO NPs could inhibit the expression of pro-inflammatory factors such as IL-6, IL-1β, and TNF-α, suppressing the inflammatory response while possibly psychiatric disorders in rats. Inhibition of the EphA2/YKL-40/MMP-9 signaling pathway is considered to be an important pathway for CuO NPs to restrict the deterioration of gliomas.

It should be noted that there are still shortcomings and challenges in this work. Firstly, the uptake and biotoxin effects of CuO NPs in rats (even at lower supplemental doses) need to be investigated, as it is still considered a potentially toxic heavy metal oxide nanoparticle45. The cellular uptake of nanoparticles is thought to be mediated through the endocytosis pathway. The results of Joshi et al. suggested that the intracellular uptake of CuO NPs and the subsequent release of toxic intracellular copper ions are unlikely to be responsible for the toxicity of the CuO NPs, but rather are a result of the release of extracellular copper ions from the particles and the subsequent uptake of copper ions by copper transporters (e.g., Ctr1)46. The main toxicity pathway of CuO NPs is to increase the production of reactive oxygen species (ROS)47. Regarding our results, treatment with CuO NPs significantly inhibited ROS overaccumulation, inflammatory response and tissue edema in hippocampal tissues of glioma model rats, as well as improved spatial cognition and memory in model rats, with no detrimental toxic effects detected for the time being. In addition, whether CuO NPs can break through the blood-brain barrier and reach the lesion is the key to the treatment of glioma15. Therefore, it is necessary to conduct biological modification and modification of CuO NPs to improve its biocompatibility, mitigate biotoxicity, and achieve targeted delivery to glioma occurring regions48. For example, the results of Dobrucka et al. showed that biosynthesized Au-CuO NPs were more effective in killing glioma cells49.

Conclusion

In this study, the possibility of engineering nanomaterial CuO NPs in the treatment of brain glioma was tested by in vivo experiments in rats and in vitro experiments of human glioma cell lines. With a tiny size, CuO NPs has the potential to breach the blood-brain barrier. CuO NPs treatment can reduce the inflammatory response of glioma rats, promote the production of ·OH free radicals in the local microenvironment of cancerous tissue, and eventually lead to the increase of CAT and SOD enzyme content. In addition, CuO NPs treatment can clear ROS in the hippocampus of glioma rats. CuO NPs can promote the release of neurotransmitters by inhibiting the expression of glioma indicator genes and proteins, thus improving the spatial recognition and memory ability of glioma model rats. Further, through verification of human glioma cell lines, CuO NPs exposure also inhibited the expression of glioma indicator genes and proteins. In conclusion, CuO NPs is a new potential glioma treatment drug, and this work provides data support for the application of ENMs in the treatment of glioma. Of course, more modification and modification strategies of CuO NPs are still needed to improve their biocompatibility and achieve more efficient target-oriented transport.