Ubiquitin-like 4A alleviates the progression of intracerebral hemorrhage by regulating oxidative stress and mitochondrial damage

Abstract

Acupuncture has obvious therapeutic effect on intracerebral hemorrhage (ICH). miR-34a-5p regulated by acupuncture was found to attenuate neurological deficits in ICH. However, the underlying mechanisms are unclear. Ubiquitin-like 4A (UBL4A) has not been studied in ICH. SD rats were injected with autologous blood to induce ICH and treated with Baihui-penetrating-Qubin acupuncture. Acupuncture resulted in an increase in forelimb placing test scores, and a decrease in corner test scores and brain water content of ICH rats. Histopathological examination showed that acupuncture inhibited ICH-induced inflammation, decreased damaged neurons and increased UBL4A expression. UBL4A overexpression increased cell viability, inhibited apoptosis, reduced reactive oxygen species (ROS) level and increased manganese superoxide dismutase (MnSOD) activity, mitochondrial membrane potential and mtDNA level in rat embryonic primary cortical neurons. miR-34a-5p knockdown increased UBL4A expression, apoptosis rate and ROS level in hemin-treated neurons. Dual luciferase assays showed that miR-34a-5p bound to UBL4A. Apoptotic cells and ROS level were increased in hemin-treated neurons with UBL4A and miR-34a-5p knockdown. We firstly demonstrate the inhibitory effect of UBL4A on neuronal apoptosis, and the regulation relationship between UBL4A and miR-34a-5p. This study provides a new candidate target for ICH treatment and more basis for elucidating the molecular mechanism of acupuncture. In the future, we will conduct a deeper exploration of the effects of UBL4A on ICH.

Introduction

Intracerebral hemorrhage (ICH) is a primary non-traumatic bleed in the brain parenchyma, accounting for 10–20% of all strokes [1]. The mortality rate of ICH ranges from 35% to 52%, and more than 50% of patients die within 2 d after ICH [2]. ICH is characterized by poor prognosis, neurological impairment and high mortality. Therefore, it is important to elucidate the pathological mechanisms of ICH to improve the patient survival.

Recent studies have shown that acupuncture has a significant therapeutic effect on ICH [3]. Acupuncture inhibits neuronal cell death [4], inflammation and ferroptosis in ICH rats [5]. A previous study by our group found that Baihui (DU20)-penetrating-Qubin (GB7) acupuncture effectively reduced brain edema and neuronal damage in ICH rats, and promoted nerve regeneration and neurological recovery [6]. Acupuncture at Baihui and Qubin acupoints improves the recovery rate of ICH by regulating the balance between mitochondrial autophagy and apoptosis [7]. miR-34a-5p is a member of the well-known family of tumor suppressor miRNAs. Acupuncture at Baihui and Qubin acupoints regulates the M1/M2 polarization balance of microglia and improves neurological deficits in ICH rats by regulating the miR-34a-5p pathway [8]. miR-34a-5p alleviates Abeta-induced neurotoxicity by inhibiting neuronal apoptosis and oxidative stress [9]. It also promotes inflammatory responses and apoptosis in rat trigeminal ganglion cells [10]. LINC00665 promotes lipopolysaccharide-induced apoptosis and inflammation in PC12 neuronal cells by targeting miR-34a-5p [11]. The above studies confirmed the neuroprotective effect of miR-34a-5p. However, the molecular mechanisms underlying this effect remain to be elucidated.

Ubiquitin-like protein 4A (UBL4A) is involved in the regulation of biological processes. The loss of UBL4A prevents inflammatory diseases by regulating NF-κB signaling in macrophages and dendritic cells [12]. UBL4A knockout mice could resist collagen-induced arthritis [13]. UBL4A exerts anti-tumor effects on autophagy-related proliferation and metastasis in pancreatic ductal adenocarcinoma [14]. Nuclear UBL4A forms complexes with other factors to mediate DNA damage signaling and cell death [15]. UBL4A deficiency accelerates colorectal tumorigenesis [16]. Under nutrient deprivation conditions, the absence of UBL4A leads to mitochondrial fragmentation and reactive oxygen species (ROS) accumulation and triggers caspase 9-dependent apoptosis by impairing the mitochondrial fusion process [17]. We found that UBL4A may be a potential target gene of miR-34a-5p through bioinformatics analysis. However, the function of UBL4A in ICH has not been reported and the regulation relationship between UBL4A and miR-34a-5p is unclear in ICH.

In conclusion, whether UBL4A mediates miR-34a-5p pathway induced by acupuncture to play a role in ICH was investigated in this study.

Materials and Methods

ICH model

All animal experiments were performed in accordance with the ARRIVE guidelines and received approval from the Experimental Animal Ethics Subcommittee of Beijing University of Chinese Medicine (NO. BUCM-4-202111702-4072). Healthy two-month-old male Sprague-Dawley (SD) rats were maintained under specific pathogen-free (SPF) conditions with controlled temperature (22 ± 1°C) and humidity (55–65%) and with a 12 h light/dark cycle. Food and water were freely available to the animals. A total of 72 animals were used for this experiment. These rats randomly received different treatments. Autologous blood injection was used to induce ICH in rats [18]. Anesthesia was induced in 3% isoflurane and maintained in 2% isoflurane. Autologous blood (50 µl) was collected from rat tail vein. A midline sagittal incision (~1 cm in length) was made to expose the skull. The stereotaxic coordinate was used to drill a small hole on the skull (3.5 mm right and 0.2 mm anterior to bregma with anterior fontanel as the center). A microinfusion syringe was inserted into the hole (the right striatum, 5.5 mm depth) and 50 µl of autologous blood without anticoagulant treatment was injected into rat at a rate of 10 µl/min. Then, the syringe was placed in the brain for 5–10 min and removed. The cranial hole was filled with bone wax and then sutured. After surgery, all rats received a subcutaneous injection of Meloxicam (1 mg/kg). It was reported that acupuncture at Baihui and Qubin points can promote functional recovery after cerebral hemorrhage by increasing the expression of neurotrophic factors [6]. A good clinical effect of acupuncture at Baihui point and Qubin point was observed in 68 patients with cerebral hemorrhage [19]. The rats were treated with Baihui (DU20)-penetrating-Qubin (GB7) acupuncture 30 min daily for 3 consecutive d 12 h after modeling. The size of acupuncture needle is 0.35 × 13 mm. During acupuncture, the animals showed no obvious pain behavior. Acupuncture at Qubin point (GB7) applied unilateral (affected side). After 3 d, the rats were euthanized with carbon dioxide and the tissues around the hematoma were collected for subsequent testing. Rats in the control group received the same surgical operation but no injection or treatment. Rats in the acupuncture group received the same surgical operation and acupuncture treatment but no injection.

Cell culture and treatment

The primary cortical neurons were isolated from embryos of rat brains according to the previous method [20] and cultured in B-27^TM Plus Neuronal Culture System media (Gibco, Carlsbad, CA, USA). The isolated primary cortical neurons were divided into four groups: i) lentiviral vector encoding UBL4A targeted shRNA (LV-shUBL4A), ii) lentiviral vector encoding negative control shRNA (LV-shNC), iii) lentiviral overexpression vector of UBL4A (LV-UBL4A) and iv) negative control of lentiviral overexpression vector (LV-vector) were synthesized by GENERAL BIOL Co. (Anhui, China). The shRNA sequence targeting Ubl4a was AGCTCAACCTAGTTGTTAAGC. At 48 h after transfection with lentivirus vectors, cells were treated with 50 µM hemin for 24 h to simulate the pathological environment of ICH in vitro.

Behavioral tests

For the forelimb placement test, the rat’s torso was held while the forelimbs were allowed to move freely. The rat’s tentacles were placed in rapid contact with the edge of the table. One point was recorded when the rat’s forelimb was reflexively extended towards the table, after ICH the rat’s forelimb was difficult to place on the other side of the injury. Each rat’s two forelimbs were tested 10 times each. For the corner test, two pieces of wood were fixed at a 30-degree angle. Rats may turn left or right after entering the angle of entrapment. Normal rats were equally likely to turn left or right, however, rats with brain hemorrhage were more likely to turn to the injured side. The experiment was repeated 10~15 times for each rat with a repetition interval of ≥30 s, and the number of left or right turns was counted. All experiments were performed in a blinded manner.

Brain water content

The severity of brain edema was assessed using the dry/wet weight method after 3 d of continuous acupuncture treatment. Whole brain of rats was removed. Each brain tissue was quickly weighed and the average of three repeats was used as the wet weight. The brain tissue was then placed in a desiccator for 24 h at 105°C for dehydration. The tissue was weighed to obtain the dry weight. Brain water content = [(wet weight − dry weight) / (wet weight)] × 100%. The brain weight was measured using a calibrated electronic analytical balance (FA1004, Lichen Technology, Shanghai, China) at room temperature with humidity less than 65%.

CCK-8

All experimental operations were performed according to the instructions of the Cell Counting Kit-8 (Keygen, Nanjing, China). Cells to be examined were inoculated into 96-well plates with 5 × 10³ cells per well. Then, 10 µl of CCK-8 reagent was added to each well. The whole process needs to be protected from light. The 96-well plates were then incubated at 37°C with 5% CO₂ for 2 h. The optical density was read on a microplate reader (Biotek, Winooski, VT, USA) at 450 nm.

Hematoxylin-eosin (HE) staining

HE staining was used to determine striatal hematoma. Brain tissues were embedded in paraffin and made into 5 µm-thick sections. The sections were incubated with xylene twice for 15 min each and immersed in 100% ethanol two times for 5 min each, followed by immersion in graded ethyl alcohols (95%, 85% and 75%) for 2 min each. Paraffin sections were stained with hematoxylin solution (Solarbio, Beijing, China) for 5 min, followed by eosin solution (Sangon, Shanghai, China) for 3 min. Finally, the sections were dehydrated with 100% ethanol, made transparent with xylene, and mounted with neutral balsam. The staining was observed under a light microscope (Olympus, Tokyo, Japan) and photographed. The results of HE staining were evaluated by a researcher who do not know the experimental grouping. Hematoma was defined by the presence of blood and/or inflammatory cells.

Fluoro-Jade B (FJB) staining

The FJB staining Kit (Merckmillipore, Burlington, MA, USA) was utilized to detect damaged neurons. Briefly, brain tissue sections were immersed in FJB staining solution for 20 min. The sections were dried at 50°C for approximately 5 min. Subsequently, the sections were soaked in xylene for 1 min and then covered with resin. Sections were observed under a microscope (Olympus) and photographed.

Immunofluorescence staining

Co-localization of UBL4A and NeuN in tissues were detected by double immunofluorescence staining. Tissue sections were fixed with 4% paraformaldehyde for 15 min and then blocked with 1% bovine serum albumin (BSA) for 15 min at room temperature. Tissue sections were blocked with UBL4A (1:100; 14253-1-AP; Proteintech, Wuhan, China) and Neuronal Nucleus antibody (NeuN; 1:300; ab104224; Abcam, Shanghai, China) overnight at 4°C, followed by FITC-labeled goat anti-rabbit IgG (1:200; ab6717; Abcam) and Cy3-labeled goat anti-mouse IgG (1:200; A-21424; Invitrogen, Carlsbad, CA, USA) were applied in a dark box for 90 min. After rinsing with PBS, 4’,6-diamidino-2-phenylindole (DAPI; Aladdin, Shanghai, China) was used as a counterstain. Confocal analysis was conducted under a fluorescence microscope (Olympus).

TUNEL staining

Cells (5 × 10⁴ cells) were treated with 0.1% Triton X-100 (Beyotime, Shanghai, China) in PBS for 5 min. TUNEL staining was performed according to the instructions of the In Situ Cell Death Detection Kit (Roche, Basel, Switzerland). Nuclei were stained with DAPI (D106471-5mg; Aladdin) and the slides were sealed with fluorescence quencher. Staining was observed under a fluorescence microscope (Olympus) and photographed.

Measurement of ROS, manganese superoxide dismutase (MnSOD) levels and mitochondrial membrane potential

A dichlorodihydrofluorescein diacetate (DCFH-DA) detection Kit (Beyotime) was used to evaluate ROS level in cells. Cells were seeded in 6-well plates at a concentration of 1 × 10⁶ cells per ml and 10 µM DCFH-DA was added to each well for 20 min. The reaction was performed at 37°C for 20 min. Cells were then resuspended in 500 µl PBS, and analyzed immediately by flow cytometer (Aceabio, San Diego, CA, USA). For SOD activity assay, cell supernatants were collected and the BCA Kit was used to determine the protein concentration. The total SOD and CuZn-SOD activities in the supernatant were measured separately according to the instructions of the MnSOD activity assay Kit (Njjcbio, Nanjing, China) with the xanthine oxidase method (hydroxylamine method). The MnSOD activity was obtained by subtracting the CuZn-SOD activity from the total SOD activity. Collection of 1 × 10⁷ cells was conducted for MnSOD analysis. The total SOD activity in the sample is calculated based on distilled water as a control and the calculation formula is “The activity of SOD enzyme = [(control group OD value-determination group OD value) /control group OD value] / 50% × (total volume of reaction solution / sample volume) / protein concentration of sample”. The calculation formula is consistent with the calculation formula of total SOD activity. Mitochondrial membrane potential of cells was measured using the Mitochondrial Membrane Potential Assay Kit (Leagene, Beijing, China). Cells (5 × 10⁵ cells) were collected and resuspended with PBS. Cells were incubated with 0.5 ml of JC-1 dyeing working solution at 37°C for 20 min in the dark, and then analyzed with a fluorescent microplate reader (Tecan, Männedorf, Switzerland).

Dual luciferase assay

Dual-luciferase activity was detected using a Dual-Luciferase Reporter Assay Kit (Keygen). The reporter vectors containing the wild-type or mutant Ubl4a 3′-UTR were transfected into cells using Lipofectamine 2000 (Invitrogen), and cells were transfected with miR-34a-5p mimics or negative control mimics (NC mimics). The empty vector was considered a negative control. FLOT-2 is a target of miR-34a-5p and suppressed by it [21]. Here, Flot-2 3′-UTR was used as a positive control. Cells were incubated in 12-well plates with 100 µl firefly luciferase and 100 µl renilla luciferase working solution at a concentration of 1 × 10⁴ cells per ml. Luciferase activity was detected with a microplate reader (Biotek). The fluorescence intensity of firefly was normalized by the renilla luciferase intensity and compared with the normalized intensity of empty vector condition.

Extraction of mitochondrial DNA (mtDNA)

Mitochondrial DNA Isolation Kit (K280-50, Biovision, Milpitas, CA, USA) was used to isolate mtDNA. In brief, cells were collected by centrifugation at 600 g for 5 min at 4°C. After washing with pre-cooled PBS, cells were incubated with Cytosol Extraction Buffer on ice for 10 min and homogenized in an ice-cold dounce tissue grinder. The homogenate was transferred to a 1.5 ml microcentrifuge tube and centrifuged at 1,200 g for 10 min at 4°C. The supernatant was transferred to a new centrifuge tube for a centrifugation at 10,000 g for 30 min. After removing the supernatant, repeat the centrifugation again. The pellet is isolated mitochondria. Mitochondria pellet was lysed in mitochondria lysis buffer and kept on ice for 10 min. Enzyme mix was added to the lysis solution followed by incubation at 50°C in a water bath for 60 min until the solution became clear. Finally, absolute ethanol was added to the mixture and incubated at −20°C for 10 min and followed by a centrifugation at room temperature for 5 min at 10,000 g. The pellet is mitochondrial DNA.

Real-time PCR

The total RNA from cells was extracted with TRIpure reagent (Bioteke, Beijing, China) and reverse transcribed into cDNA using BeyoRT II M-MLV Reverse transcriptase (Beyotime). SYBR Premix Ex TaqTM (Solarbio) based on real-time PCR (Bioneer, Daejeon, Korea) was carried out to analyze the relative expression levels of mRNAs. Reverse transcription of miR-34a-5p was conducted using a miRNA First Strand cDNA Synthesis kit (Sangon). Relative quantification of mitochondrial DNA level and the mRNA expression levels of target genes was calculated by the 2^−ΔΔCt method. The sequences of real-time PCR primers are the following: forward primer for Ubl4a, 5′-GCTGAATGTCCCTGTGC-3′; reverse primer for Ubl4a, 5′-ACCTGTCATAATCCCTCTGT-3′; forward primer for mtDNA, 5′-GTTAATGTAGCTTATAA-3′, reverse primer for mtDNA, 5′-TTGAATCCATCTAAGCATT-3′.

Western blot analysis

Proteins were extracted using RIPA lysate (Beyotime) and protein concentrations were measured using a BCA protein quantification Kit (Beyotime). Protein extracts from each sample were separated by SDS-PAGE and the separated proteins were transferred to PVDF membranes (Thermofisher, Waltham, MA, USA). The membranes were incubated with UBL4A antibody (1:1,000; 14253-1-AP; Proteintech); β-actin antibody (1:20,000; 66009-1-Ig; Proteintech) overnight at 4°C. After washing 3 times with TBST, goat anti-rabbit IgG (1:10,000; SA00001-2; Proteintech) and goat anti-mouse IgG (1:10,000; SA00001-1, Proteintech) were incubated for 1 h at 37°C. Western blots were analyzed using ECL luminescent liquid (7 Sea biotech, Shanghai, China).

Statistical analysis

All experimental data were presented as means ± SD. Data analysis was performed using GraphPad Prism 8.0. Forelimb placement test and corner turn test scoring data was analyzed statistically using the Kruskal-Wallis non-parametric test, followed by Duun’s test for multiple comparisons. Unpaired t test was used to analyze the difference between two groups for data with normal distribution and homogeneity of variance. Ordinary one-way ANOVA was used to analyze multiple groups of data that sacrificed homogeneity of variances and normal distribution and then Turkey test was used to post-hoc test. P-values <0.05 were considered statistically significant.

Results

UBL4A was highly expressed in brain tissue of rats after acupuncture

Rats received Baihui-penetrating-Qubin acupuncture at 12 h after autologous blood injection (Fig. 1a). All rats scored 100 in the forelimb placement test before ICH. ICH rats had a decrease in forelimb placement scores, an increase in cornering test scores and brain tissue water content (P<0.01). The three parameters were brought back towards normal levels with acupuncture (Figs. 1b and c). The results of western blot and real-time PCR demonstrated that the expression of UBL4A decreased in the brain tissue of ICH rats, and the expression of UBL4A increased after acupuncture (Fig. 1d) (P<0.01). Compared with the ICH group, the mRNA level of Ubl4a in the ICH+Acupuncture group was increased (Fig. 1d) (P<0.05). HE staining results showed that severe hemorrhage was detected in the brain striatum of ICH rats and the inflammatory cell infiltration was increased. Acupuncture treatment effectively alleviated the inflammation caused by ICH (Fig. 1e). FJB staining was used to detect neuronal degeneration after ICH, and acupuncture treatment reduced the number of FJB-positive cells triggered by ICH in brain tissue (Fig. 1f). Immunofluorescence further verified that the expression of UBL4A was reduced in ICH group and increased in ICH+Acupuncture group (Fig. 1g). In addition, it showed that acupuncture promoted the accumulation of UBL4A in the perinuclear area, which was different to the Control group.

Fig. 1.

(a) Schematic diagram showing the experimental procedure of the ICH rat. (b) Forelimb placing test and corner turn test of rats. (c) Brain water content of rats. (d) Real-time PCR and western blot were used to verify the expression of UBL4A in brain tissue. (e) Typical hematoxylin-eosin (HE) staining images presenting the striatal hematomas in rats. Scale bar: 500 µm (left) and 100 µm (right). (f) Fluoro-Jade B (FJB) staining was performed to detect neuronal cell death in rats. Scale bar: 50 µm. (g) Co-localization of ubiquitin-like protein 4A (UBL4A) and NeuN was detected by immunofluorescence staining. Scale bar: 50 µm. ICH, intracerebral hemorrhage. *P<0.05, **P<0.01, ns: no significance.

UBL4A reduced hemin-induced apoptosis of rat neurons

Hemin was used to mimic a hemorrhagic stimulation in vitro (Fig. 2a). The results in Fig. 2b showed that the expression of UBL4A was decreased in hemin-treated rat neurons (P<0.05). Overexpression and knockdown of UBL4A were successfully established by lentivirus in rat neurons (P<0.01) (Fig. 2b). Non-targeting shRNA (shNC) and empty vector were used as controls. Hemin treatment reduced the cellular activity of neurons (P<0.01). Compared with Hemin group, UBL4A knockdown reduced cell viability (P<0.01), while UBL4A overexpression increased cell viability (P<0.05) (Fig. 2c). TUNEL staining indicated that UBL4A knockdown increased the number of apoptotic cells with hemin treatment. Overexpression of UBL4A reduced the number of apoptotic cells (Fig. 2d). It has been reported that the loss of UBL4A induced mitochondrial fragmentation and ROS accumulation, triggering mitochondrial pathway-induced apoptosis [17]. We speculated that UBL4A might affect the apoptosis of neurons by regulating oxidative stress and mitochondrial damage.

Fig. 2.

(a) The diagram depicts the flow chart of the cell experiment. (b) Real-time PCR and western blot were used to verify the expression level of ubiquitin-like protein 4A (UBL4A) in neurons. (c) CCK-8 assay was performed to determine cell viability. (d) TUNEL staining was used to detect cell apoptosis. Scale bar: 50 µm. LV-shNC, negative control shRNA; LV-shUBL4A, shUBL4A knockdown; LV-vector, empty vector; LV-UBL4A, UBL4A overexpression. *P<0.05, **P<0.01.

UBL4A inhibited hemin-induced oxidative stress and mitochondrial damage in rat neurons

Upregulation of ROS level (P<0.01) (Fig. 3a) and downregulation of MnSOD activity (P<0.01) (Fig. 3b) was observed in neurons following hemin treatment. Compared with group treated with hemin, knockdown of UBL4A resulted in increased level of ROS and reduced activity of MnSOD (P<0.01). In the contrast, overexpression of UBL4A reduced ROS level and enhanced MnSOD activity. Mitochondrial membrane potential (Δψm) and mtDNA were used to evaluate mitochondrial damage. Compared with the control group, hemin decreased the mitochondrial membrane potential and mtDNA level. UBL4A inhibition also decreased the mitochondrial membrane potential and mtDNA level compared with Hemin+LV-shNC group (P<0.01) (Figs. 3c and d). The opposite results were observed in rat neurons with UBL4A overexpression.

Fig. 3.

(a) The level of reactive oxygen species (ROS) in cells was determined by flow cytometry using a dichlorodihydrofluorescein diacetate (DCFH-DA) based kit (cell number: ×10³). (b) Manganese superoxide dismutase (MnSOD) activity was measured with MnSOD assay kit. (c) Detection of mitochondrial membrane potential by JC-1 staining. (d) The relative mitochondrial DNA (mtDNA) level was detected by real-time PCR. LV-shNC, negative control shRNA; LV-shUBL4A, shUBL4A knockdown; LV-vector, empty vector; LV-UBL4A, UBL4A overexpression. *P<0.05, **P<0.01.

Reduced expression of miR-34a-5p alleviated hemin-induced neuronal damage in rats

The miR-34a-5p expression was elevated in hemin-treated neurons (P<0.01). Knockdown of miR-34a-5p led to a decrease in miR-34a-5p level (P<0.01) (Fig. 4a). miR-34a-5p knockdown increased the expression of UBL4A in hemin-treated neurons (P<0.01) (Fig. 4b). TUNEL staining illustrated that hemin increased the apoptotic neurons, whereas miR-34a-5p knockdown reduced apoptotic neurons (Fig. 4c). ROS level showed the same trend, with miR-34a-5p knockdown reducing the hemin-induced increase in ROS level (P<0.01) (Fig. 4d).

Fig. 4.

(a) Real-time PCR to verify the expression of miR-34a-5p in neurons. (b) Real-time PCR and western blot were used to verify the expression level of ubiquitin-like protein 4A (UBL4A) in neurons with miR-34a-5p knockdown. (c) TUNEL staining was used to detect cell apoptosis. Scale bar: 50 µm. (d) The level of reactive oxygen species (ROS) in cells was determined by flow cytometry using a dichlorodihydrofluorescein diacetate (DCFH-DA) based kit (cell number: ×10³). LV-shNC, negative control shRNA; LV-miR-34a-sponge, miR-34a-5p knockdown. **P<0.01.

UBL4A directly bound to miR-34a-5p

FLOT-2 has been found to be inhibited by miR-34a-5p [21], so we chose it as a positive control for the dual-luciferase experiment, and the empty vector without any inserted fragments was used as a negative control. As shown in Fig. 5a, the relative firefly luciferase activity was significantly reduced when miR-34a-5p and wild-type Ubl4a 3′-UTR were co-expressed (P<0.01). However, results in the miR-34a-5p and mutant Ubl4a 3′-UTR group shows no significant changes. Furthermore, the result of Ubl4a 3′-UTR group was similar with Flot-2 3′-UTR group. Therefore, UBL4A is a target of miR-34a-5p and can be suppressed by it.

Fig. 5.

(a) Predicted miR-34a-5p target sequences in 3′-UTR of Ubl4a (Left). Dual-luciferase reporter assay was conducted to verify whether ubiquitin-like protein 4A (UBL4A) was the target of miR-34a-5p (Right). NC mimics, negative control mimics; WT, wild type; MUT, mutant type. (b) Western blot was used to verify the expression level of UBL4A in neurons. (c) TUNEL staining was used to detect cell apoptosis. Scale bar: 50 µm. (d) The level of reactive oxygen species (ROS) in cells was determined by flow cytometry using a dichlorodihydrofluorescein diacetate (DCFH-DA) based kit (cell number: ×10³). LV-shNC, negative control shRNA; LV-miR-34a-sponge, miR-34a-5p knockdown. LV-shUBL4A, shUBL4A knockdown. *P<0.05, **P<0.01.

UBL4A, as a downstream target of miR-34a-5p, reduced hemin-induced neuronal damage in rats

UBL4A and miR-34a-5p was knocked down in neurons using the lentivirus (Fig. 5b). Simultaneous knockdown of miR-34a-5p and UBL4A in hemin-treated neurons resulted in an increase in apoptotic neurons (Fig. 5c) and ROS level (P<0.01) (Fig. 5d). The group knocking down miR-34a-5p alone showed opposite results, demonstrating that knockdown of UBL4A reversed the effect of knockdown of miR-34a-5p. Therefore, miR-34a-5p causes damage to neurons by inhibiting the expression of UBL4A under heme treatment.

Discussion

ICH induces a strong oxidative stress response and inflammatory processes [22]. ICH is caused by the rupture of a blood vessel, which may be followed by the expansion of a hematoma, leading to secondary injury induced by factors released from damaged tissue and extravasated blood (inducing inflammation) and lysis of erythrocytes, and cerebral oedema [23]. Interventions are needed to limit the adverse effects of oxidative stress and neuro-inflammation on brain function and to improve outcomes after ICH. It is important to understand the causative factors and pathogenesis of ICH, as is its treatment. In this study, we demonstrated the role of UBL4A in ICH and revealed the regulatory relationship between UBL4A and miR-34a-5p. However, this study used only one embryonic rat brain as the source of primary cortical neurons and failed to elucidate the differences between individuals or species. In addition, the current results have not been validated clinically, and the exploration of the molecular mechanism is only described in vitro.

micro-RNA is the most well-studied in the diagnosis and treatment of ICH [24]. miRNA mimics and antagonists can inhibit the progression of ICH in vivo and in vitro, such as reducing peripheral edema and hematoma size, reducing inflammation [25], and promoting neuronal survival [26]. It proves that miRNA has the potential to treat clinical ICH. In our study, we found that knockdown of miR-34a-5p significantly inhibited the heme-induced increase in ROS levels and cell apoptosis. Combined with our previous study [8], these findings once again demonstrate the importance of miR-34a-5p in ICH.

Dysregulated miRNAs and their potential targets in ICH are commonly associated with pathways such as neuroinflammation and cell death [27]. ICH induces brain damage through increased oxidative stress, and it significantly reduced SOD level in the ischemic cerebral cortex [28]. Herein, we find that UBL4A may alleviate ICH by reducing neuronal oxidative stress and mitochondrial damage. Inhibition of miR-34a-5p inhibited H₂O₂-induced apoptosis and oxidative damage in human lens epithelial cells [29]. Knockdown of miR-34a-5p inhibits oxidative stress damage in macrophages [30]. When miR-34a-5p was silenced, the carcinogenic-induced oxidative stress and inflammation of liver cancer cells were almost eliminated [31]. These studies revealed that miR-34a-5p play an important role in oxidative stress. In vitro experiments found that UBL4A and miR-34a-5p had opposite effects on heme-treated neurons. The dual luciferase assay validated the interaction between miR-34a-5p and UBL4A, and found that miR-34a-5p inhibited the expression of UBL4A. Knockdown of UBL4A reversed the effects of miR-34a-5p knockdown. In conclusion, miR-34a-5p may function through UBL4A. At present, there are few research reports on the function of UBL4A in diseases. In the future, we will also devote ourselves to exploring its potential functions and mechanisms.

Interestingly, our results also found that UBL4A was mainly located in the cytoplasm in the control group and accumulated in the perinuclear area after acupuncture treatment. In eukaryotic cells, approximately 30% of newly synthesized proteins misfold during early biogenesis, and these defective proteins may form toxic aggregates. Therefore, efficient disposal of these proteins is very important. In eukaryotes, their quality control starts from their biosynthetic site, the endoplasmic reticulum [32]. Endoplasmic reticulum-associated degradation (ERAD) is one of the mechanisms responsible for clearing misfolded proteins in the endoplasmic reticulum [33]. It has been reported that UBL4A can form protein complexes with other proteins and improve the efficiency of ERAD [34]. Based on the HPA database (http://www.proteinatlas.org) and related literatures [34, 35], UBL4A protein can be localized in the cytoplasm and nucleus. Under normal circumstances, UBL4A is mainly located in the cytoplasm [15]. But under some special circumstances, UBL4A can be enriched in the nucleus [34] or recruited to ribosomes [35]. Both endoplasmic reticulum and ribosomes were mainly distributed in the perinuclear area. Therefore, we speculate that acupuncture may affect the above functions of UBL4A.

In conclusion, we revealed that UBL4A overexpression alleviated ICH-related neurological deficits. Inhibition of miR-34a-5p alleviated hemin-induced neuronal damage. UBL4A is regulated by miR-34a-5p. This study is the first to verify the effect of UBL4A in ICH, and the regulatory relationship between UBL4A and miR-34a-5p. This provides more basis for elucidating the molecular mechanism of acupuncture treatment of ICH, provides new candidate targets for the treatment of ICH, and is of great significance for elucidating the function of UBL4A.

Conflict of Interest

The authors declare no conflict of interest.

Author Contributions

Dan Li, Le Wang and Peng Bai: conceived and designed the experiments. Dan Li, Le Wang, Shufeng Shi and Xiaofeng Deng: conducted the experiments. Yunong Li and Xuehan Zeng: analyzed the data. Shulin Li: wrote the article. All authors edited and reviewed the manuscript.

Ethical Statement

All animal experiments were performed in accordance with the ARRIVE guidelines and received approval from the Experimental Animal Ethics Subcommittee of Beijing University of Chinese Medicine (NO. BUCM-4-202111702-4072).

Funding

This work was supported by a grant from the National Natural Science Foundation of China (No. 82174514).