Object To explore the unfolded protein response (UPR) in the hippocampus of rats undergoing heroin relapse and the mechanisms underlying the acupuncture-mediated inhibition of brain damage caused by heroin relapse.
Methods 60 Sprague-Dawley rats (30 females and 30 males) were randomly divided into four groups: Control group, Heroin group, Heroin+acupuncture group, and Heroin+methadone group (n=15 each). In the latter three groups, a model of heroin addiction was established by successive increments of intramuscular heroin injections for 8 days, according to the exposure (addiction)→detoxification method. A UPR RT2 Profiler PCR array was used to screen for differentially expressed genes in the hippocampus. Apoptosis was detected by terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) staining. The protein expression levels of the following three differentially expressed genes were detected by Western blot to validate the results of the PCR array: heat shock protein (HSP)70, HSP105, and valosin-containing protein (Vcp).
Results The UPR RT2 Profiler PCR Array detection results indicated that acupuncture increased the expression levels of the molecular chaperones HSP70, HSP105, and Vcp. The degree of neuronal apoptosis in the hippocampus of rats in the Heroin+acupuncture and Heroin+methadone groups was significantly reduced compared with the untreated Heroin group (p<0.01). Protein expression of HSP70, HSP105, and Vcp in the Heroin+acupuncture and Heroin+methadone groups was significantly higher than the Heroin group (p<0.01).
Conclusions The positive effects of acupuncture on brain damage caused by heroin may be closely related to up-regulation of HSP70, HSP105, and Vcp, and reduced apoptosis.
- BASIC SCIENCES
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Drug abuse is currently one of the most serious public health and social problems worldwide. It is a global issue that has become a threat to human health, social stability, and economic development. The number of substance abusers is increasing each year in China and addicts are becoming younger.1 ,2
Acupuncture has been practised in China for thousands of years. Although there remains some controversy over its incorporation into orthodox Western medicine, it has become one of the most widely used alternative medicines in the world.3 In 1996, the WHO published a list of 64 medical problems considered amenable to acupuncture treatment that included drug abuse. Furthermore, a consensus panel convened by the National Institutes of Health (NIH) in 1997 concluded, albeit cautiously, that acupuncture might be efficacious in the treatment of drug addiction.4
Mental and behavioural disorders (including smoking, drinking, and heroin and cocaine use) are considered to belong to the ‘disease spectrum’ of traditional acupuncture.5 In addition, two Chinese randomised controlled trials have indicated a positive effect of acupuncture in heroin replase,6 ,7 and a meta-analysis has shown that acupuncture can be effective for the treatment of symptoms of opiate withdrawal when combined with opioid receptor agonists.8
Experimental studies have shown that significant ultrastructural pathological changes occur in the neurons of the cerebral cortex, hypothalamus, hippocampus, and pituitary gland of heroin-addicted rats and rhesus monkeys.9 ,10 The chronic effects of addictive substances can cause long-term changes in the tissues of the nervous system, including neuronal apoptosis and atrophy.11 ,12
Heroin has a neurotoxic effect and induces neuronal apoptosis in the hippocampus. Recent studies have demonstrated the role of acupuncture in promoting nerve cell repair and conferring cerebral protection.13 ,14 Our previous studies showed that acupuncture has an anti-apoptotic effect on the neurons of rats undergoing heroin relapse and that it protects brain tissues from damage.9 ,15 ,16 However, the potential biological mechanisms underlying the positive effect of acupuncture on heroin-induced neuronal damage remain unclear and in need of further exploration.
Recently, increasing evidence has emerged to support the role of the unfolded protein response (UPR) in maintaining endoplasmic reticulum homeostasis and preventing apoptosis.17 This mechanism may be the aetiological basis for a variety of diseases. We hypothesised that the protective effects of acupuncture on neuronal damage in the brain caused by addictive substances occurs due to modulation of the UPR. The aim of this study was to use a UPR PCR array to explore the mechanisms of action underlying the putative effects of acupuncture on neuronal damage in a rat model of heroin relapse.
All animal care and experimental procedures were approved by the Animal Care and Use Committee of Anhui University of Chinese Medicine (reference no. #AUCM-2014-0806) and were conducted in accordance with local guidelines for animal welfare consistent with the National Research Council's ‘Guide for the Care and Use of Laboratory Animals’. A total of 60 Sprague-Dawley rats (30 females and 30 males, 4 months old, clean grade, weighing 180–200 g) were provided by the Experimental Animal Center of Anhui Province (licence no. SCXR (Anhui) 2011–002) and maintained at 24°C and 40% relative humidity under a 12 h light/dark cycle. Rats were allowed free access to standard laboratory chow and water.
A rat model of heroin addiction was established by intramuscular (i.m.) injection of increasing amounts of heroin (85% purity, provided by the General Office of Drug Control of Anhui Province) for 8 consecutive days in a total of 45 rats. On the first day, rats received 0.8 mg/kg i.m. heroin. Beginning on the second day, the heroin dose was increased by 0.2 mg/kg per day and reached a peak of 2.2 mg/kg on the eighth day. Between the fourth and eighth days, the daily dose was administered in two divided doses at 07:00 and 19:00. After peak exposure, i.m. heroin injections were stopped for a total of 5 days to allow natural withdrawal and detoxification. According to the exposure (addiction)→detoxification method, the cycle was repeated in three stages (exposure→detoxification→exposure→detoxification→exposure→detoxification) to establish a model of heroin relapse.18 ,19 A Control group (n=15) was included that received saline via i.m. injection. The volume and frequency of administration were the same as for the Heroin group during the exposure phase of each cycle.
Following successful modelling of heroin relapse, the 45 rats were randomly subdivided into three groups (n=15 each): the Heroin+acupuncture group that was treated with acupuncture during the detoxification phase; the Heroin group that was restrained in the same way as the acupuncture-treated group but did not receive any treatment; and the Heroin+methadone group that received methadone intragastrically during the detoxification phase. In the Heroin+acupuncture group, 0.25×25 mm stainless steel needles (Suzhou Medical Supplies Co Ltd, Suzhou, China) were inserted to a depth of 12 mm at GV20 (Baihui, located at the medial aspect of the parietal bones) and GV14 (Dazhui, located between the seventh cervical and first thoracic vertebrae, in the midline of the back) and retained for 30 min. The rats remained in an awake and quiet state during treatment, which was administered during each detoxification phase daily at 07:00 for 5 continuous days (repeated three times during the experiment). Rats in the Heroin+methadone group received a tapering dose of methadone (Tianjin Central Pharm Co Ltd, China) intragastrically for 5 consecutive days during each detoxification phase. The dose was gradually reduced by 0.1 mg per day from 0.4 to 0 mg over the course of 5 days. Rats in the control group were restrained in the same way but did not receive any treatment. All rats were euthanased on the 39th day of the experiment under terminal anaesthesia using an intraperitoneal injection of 3 mL/kg 10% chloral hydrate.
Necropsy and tissue sampling
Under terminal anaesthesia using chloral hydrate (10%), the rats were transcardially perfused with 0.9% sodium chloride and 4% paraformaldehyde (PFA) and their brains were dissected out of the cranial cavity. The left hippocampus was isolated and either snap-frozen and stored at −80°C pending RNA extraction (n=3 per group) or protein extraction (n=6 per group), or fixed in 4% PFA overnight at 4°C and then embedded in paraffin pending terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) staining (n=6 per group).
RT2 profiler PCR array
To quantify gene expression associated with the UPR, we used the UPR RT2 Profiler PCR Array (SABiosciences, Qiagen, USA). RNA isolation, DNase treatment, and RNA clean-up were performed according to the manufacturer's protocol. The isolated RNA was reverse transcribed into cDNA using SuperScript II reverse transcriptase (Life Technologies, USA) according to the manufacturer's protocol. Quantitative PCR (qPCR) was performed using RT2 SYBR Green qPCR Master Mix (Invitrogen, USA) and an ABI7900 instrument (Applied Biosystems, USA). The corresponding software was used to calculate the Ct value of each gene in the PCR array and the ΔΔCt method was used to compare gene expression between groups. The analysis was completed by the Shanghai Kang Chen Bio-tech Company (Shanghai, China). The screening condition was up-regulation or down-regulation of genes by >2.0-fold. Genes of interest that exhibited differential regulation were then validated by measuring protein expression (see below).
Serial sections (4 μm thick) of paraffin-embedded left hippocampi were cut and the presence of apoptotic cells was detected by TUNEL staining using a commercial kit (catalogue no. 11684817910, Roche, USA) according to the manufacturer's instructions. Numbers of TUNEL-positive cells were counted in four randomly selected fields under a microscope (400×) and four sections per animal were used.
Total protein was extracted from snap-frozen hippocampal tissues using a commercial protein extraction kit (catalogue no. P0028, Beyotime, Jiangsu, China) and denatured. The protein concentration was determined using the BCA (bicinchoninic acid) protein assay kit (catalogue no. P0012S, Beyotime). Equal amounts of proteins were processed for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting using the following primary antibodies: anti-β-Actin as an internal reference (catalogue no. R-22, sc-130657, 1:3000, Santa Cruz, USA), anti-heat shock protein (HSP)70 (catalogue no. BS2741,1:800, Bioworld, USA), anti-HSP105 (catalogue no. BS1176,1:800, Bioworld, USA), and anti-valosin-containing protein (Vcp; catalogue no. 10736-1-AP, 1:1000, Proteintech, USA). These targets were chosen following screening using the UPR RT2 ProfilerPCR Array (see above and below).
Data are presented as mean±SD. The statistical analysis was performed with Student’s t test and one-way analysis of variance (ANOVA) using the Statistical Package for the Social Sciences (SPSS) V.17.0 (SPSS Inc, Chicago, Illinois, USA). The test of least significant difference (LSD) was used post hoc to analyse multiple pairwise comparisons. A value of p<0.05 was considered statistically significant.
UPR RT2 profiler PCR array detection
Table 1 shows the results of the UPR RT2 Profiler PCR test presented by study group (compared by Student's t test). Of the 84 genes included in the array, compared with the Control group, the Heroin group exhibited six down-regulated genes (that met the screening criteria of a fold-change ≥2). These genes included the neurotrophic factor Manf, the protein folding factors Hspa4 and Sil1, the protein disulphide isomerase Srebf1, the HSP Hsph1, and Vcp, which is related to the endoplasmic reticulum-associated protein degradation pathway, ubiquitination and HSPs.
Compared with the Heroin only group, 26 genes were up-regulated in the Heroin+acupuncture group (table 2). These genes included: the unfolded protein binding-associated factors Calr, Cct4, Cct7, Hspa2 and Sec63; the endoplasmic reticulum degradation pathway-associated proteins Herpud2, Nploc4, Nucb1, Os9, Sels and Vcp; the protein folding-associated factors Ppib, Sil1, Tcp1 and Hsp90b1; the HSP-related proteins Hspa4, Hspa5 and Hsph1; and other genes associated with endoplasmic reticulum protein folding quality control, ubiquitination, and apoptosis.
Compared with the Heroin group, the Heroin+methadone group exhibited two genes with differential expression, namely the protein folding-associated factor Ppic, which was down-regulated 2.17-fold, and Vcp, which was upregulated 3.4-fold. There were no significant differences in gene expression between the Heroin+acupuncture and Control groups.
Two genes were differentially expressed in the Heroin+acupuncture group relative to the Heroin+methadone group, namely the sterol regulatory element endoplasmic reticulum ring binding protein transcription factor Mbtps1, which was up-regulated 7.51-fold (p=0.023), and the cellular structural integrity associated protein Actb, which was up-regulated 4.8-fold (p=0.029). Mbtps1 was down-regulated 9.28-fold in the Heroin+methadone group relative to the Control group, which constituted the only significant difference comparing controls and methadone-treated heroin exposed rats.
HSP70, HSP105, and Vcp protein expression
Further analysis was performed on three genes of interest (HSP70, HSP105, and Vcp) that were down-regulated in Heroin versus Control groups and up-regulated in Heroin+acupuncture±Heroin+methadone versus Heroin groups. Figure 1 illustrates the results from the Western blotting for HSP70, HSP105, and Vcp protein expression in the hippocampus. Compared with the Control group, HSP70, HSP105, and Vcp protein levels in the Heroin group were significantly reduced (p<0.001, p=0.001, and p=0.002, respectively; figure 1B–D). Compared with the Heroin group, the protein expression of HSP70, HSP105, and Vcp was significantly greater in the Heroin+acupuncture group (p=0.004, p<0.001, and p=0.005, respectively) as well as the Heroin+methadone group (p=0.033, p=0.002, and p=0.036, respectively).
Neuronal apoptosis in the hippocampus
Figure 2 shows the results of the TUNEL staining of the hippocampus in each group. Neuronal apoptosis was rarely observed in the hippocampi of the Control group. Accordingly, the cell nuclei were mostly elliptical or circular and stained light blue (figure 2A). Compared with the Control group, neuronal apoptosis in the Heroin group was significantly increased and apoptotic bodies were observed, which had presumably formed after neuron fragmentation (figure 2B). Consequently, apoptotic cell counts were higher in Heroin versus Control groups (p<0.001; figure 2E). Conversely, lower levels of neuronal apoptosis in the hippocampus were observed in the Heroin+acupuncture group and Heroin+methadone group (figure 2C, D, respectively). Compared with the Heroin group, the total number of positive cells in the Heroin+acupuncture group and Heroin+methadone group was significantly decreased (p<0.001 and p<0.001, respectively; figure 2E), although the apoptotic cell counts still remained significantly higher than the Control group (p=0.012 and p<0.001, respectively). The reduction in the number of TUNEL positive cells in the Heroin+acupuncture group tended to be greater compared to the Heroin+methadone group (p=0.079).
Heroin relapse causes chronic and persistent damage to brain tissue in rats, which can result in a wide range of ultrastructural pathological lesions in multiple parts of the brain including the hippocampus, nucleus accumbens, and ventral tegmental area. Neuronal apoptosis is the main mode of cell death in the brain caused by heroin relapse. In addition, glial cells also demonstrate a series of ultrastructural changes including hyperplasia and degeneration of astrocytes.20 Acupuncture has been shown to regulate and maintain the biochemical balance of the central nervous system (CNS).21
In central nerve injury caused by heroin toxicity, acupuncture may protect the CNS from damage via an anti-apoptotic mechanism22 ,23 and/or repair CNS damage via an increase in the expression of neurotrophic factors. The most recent data on the effect of acupuncture on neurotrophins shows that it can prevent neuronal cell death and activate the signal pathway of brain derived neurotrophic factor (BDNF) to promote the survival and synaptic plasticity of neurons. This effect of acupuncture has been shown in many animal models.24
To further explore the mechanism by which acupuncture modulates brain damage caused by heroin use, we utilised a UPR PCR functional array to analyse the unfolded protein changes in the brains of rats undergoing heroin relapse and to examine the effect of the acupuncture intervention. UPR PCR functional arrays have rarely been used to explore brain damage caused by heroin and the potential positive effects of acupuncture on this process.
The UPR PCR array revealed differential expression in genes associated with protein folding, endoplasmic reticulum-associated protein degradation, neurotrophic factors, ubiquitination, and HSPs in rats undergoing heroin relapse. These findings suggest that brain damage caused by heroin relapse is characterised by a variety of different pathological processes that trigger a cascade of multifaceted physiological and biochemical events. Protein folding and endoplasmic reticulum-associated protein degradation are not conducive to anti-apoptotic processes and thereby increase the appearance of apoptotic cells in the hippocampus of rats undergoing heroin relapse.
In the present study, rats experiencing heroin relapse that received acupuncture treatment exhibited up-regulation of many genes, including those related to unfolded protein binding, the endoplasmic reticulum degradation pathway, HSPs, and ubiquitination. These proteins facilitate intracellular protein folding and are conducive to improving cell growth, development, differentiation, gene transcription, and other functions. Acupuncture also reduced the number of apoptotic cells in the hippocampus of these rats, which is consistent with our previous findings.14 ,25
Methadone is an internationally recognised drug that is widely used to treat opioid substance addiction. When observing and comparing the effects of methadone and acupuncture on brain damage in heroin relapse rats, we found that methadone use had a small effect on the differential expression of genes. Methadone down-regulated Ppic, which helps maintain basic cell viability, and up-regulated Vcp, which has an anti-apoptotic effect. When comparing the two treatments assessed herein, methadone had an enhancing effect on refolding, but its effect on membrane-binding transcription factor peptidase and β-actin appeared smaller than that produced by acupuncture.
HSP70, HSP105, and Vcp are chaperonins. HSP70 is the main chaperonin, and its function is to bind to nascent, unfolded, misfolded or focused proteins, causing them to dissociate and thereby reducing the generation of insoluble aggregates, which accelerates correct peptide chain folding and facilitates the degradation and removal of denatured proteins.26
This study found that expression levels of HSP70, HSP105, and Vcp in the hippocampus of untreated rats undergoing heroin relapse were lower than those in the healthy control group and acupuncture/methadone-treated heroin-exposed groups; this suggests that the neurons of rats experiencing heroin relapse have a reduced ability to interact with non-folding and/or denatured proteins and protect themselves against damage. The expression levels of HSP70, HSP105, and Vcp were higher in the control group and both treated heroin-exposed groups, suggesting that acupuncture-mediated regulation of HSP70, HSP105, and Vcp is beneficial for the repair or removal of variant proteins and nascent proteins. This potentially ensures that cells have an appropriate structural status and avoid damage from heroin/stressors. Collectively our results support the potential role of acupuncture for neuroprotection in heroin relapse and are consistent with previous studies on the up-regulation of HSP molecular chaperone genes that reduce apoptosis.27 ,28
Although we did not validate the other genes that were differentially expressed in the acupuncture-treated group, some other candidates are worthy of further discussion. The chaperonin containing tailless complex polypeptide 1 (CCT) is an important molecular chaperone that exists in the cytoplasm of eukaryotic cells.29 Its main function is to specifically facilitate the folding of intracellular nascent actin, tubulin and cyclin E.30 After acupuncture, the up-regulation of Cct4 and Cct7 may be the first line of defence against protein misfolding and accumulation. These proteins may be combined with intracellular proteins (while maintaining their spatial conformation) to prevent protein aggregation, assist in the degradation of hamartin in various abnormal protein folding diseases, and reduce cell damage.
TCP1 is mainly involved in the structure of the cytoskeleton.31 HSP90 is a steroid hormone binding protein that mainly exists in the form of dimers. It promotes activation of and stabilises the configuration of target proteins, and participates in signal transduction, cell cycle regulation, protein degradation and transportation in cells.32 A potential increase in TCP1 and Hsp90b1 following acupuncture might maintain the stability and activity of the protein and thereby promote cell growth.
In summary, we used the UPR PCR functional array to screen for genes that are differentially expressed following acupuncture and may play an important role in the protection against brain damage caused by heroin use. After rats undergoing heroin relapse received treatment with acupuncture or methadone, protein expression of the molecular chaperones HSP70, HSP105, and Vcp increased, which would be expected to improve the endoplasmic reticulum folding capacity. Acupuncture and methadone also mitigated the degree of neuronal apoptosis in the brains of rats undergoing heroin relapse. These changes may represent one of the mechanisms by which acupuncture at GV20 and GV14 modulates neuronal damage in the brains of rats undergoing heroin relapse. Whether increased expression of HSP70, HSP105, and Vcp can be considered a molecular target of acupuncture remains unknown and will require further research including in-depth exploration of upstream and downstream regulatory mechanisms.
Contributors XS designed the study. YZ performed most of the experiments, analysed the data and wrote the paper. XC, RZ, RX, SW, SY and JC performed some of the experiments and revised the paper.
Funding This research was funded by the National Natural Science Foundation of China (grant no. 81173325)
Competing interests None declared.
Provenance and peer review Not commissioned; externally peer reviewed.
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