Cannabidiol Attenuates Methamphetamine-Induced Cardiac Inammatory Response and Necrosis through The PKA/CREB Signaling Pathway

Methamphetamine (MA) abuse is a major global public health problem, with cardiovascular issues becoming an increasingly recognized complication. Cannabidiol (CBD) has gained recent attention, due to its various pharmacological properties. However, whether CBD has therapeutic effects on MA-induced cardiotoxicity remains unknown. In the present study, we investigated whether CBD has a protective or therapeutic effect on MA-induced cardiac damage in rats via the protein kinase A (PKA)/cyclic adenosine monophosphate response element-binding (CREB) signaling pathway. Thirty rats were randomly divided into ve groups. The rats were administered MA by intraperitoneal injection (IP) once a day for 4 weeks, with CBD (40 or 80 mg/kg, IP) treatment 1 h prior to the MA injections. Body and heart weights were measured, and morphological changes were determined using hematoxylin & eosin and Masson’s trichrome staining. The serum levels of interleukin-6 (IL-6) and IL-10 were detected using enzyme linked immunosorbent assay (ELISA) kits. The protein expression levels of PKA, phospho-PKA (p-PKA), CREB, phospho-CREB (p-CREB) and cardiac troponin I (cTnI) in the myocardium were detected by western blot analysis. Results showed that the heart-to-body weight ratio increased signicantly following MA administration but decreased with CBD treatment. Chronic administration of MA resulted in a cardiac inammatory response and progressive development of brosis, while CBD treatment attenuated these lesions in a dose-dependent manner. MA administration increased IL-6 but decreased IL-10 levels, which were reversed by CBD pretreatment. Moreover, MA signicantly increased the cTnI level, but this was decreased by CBD treatment at 80 mg/kg. The protein expression levels of PKA, p-PKA, CREB, and p-CREB increased following MA administration, but signicantly decreased with CBD treatment. Overall, these results indicate that chronic MA administration leads to cardiotoxicity, including cardiac inammatory response, brosis, and myocardial necrosis, but these effects can be attenuated by CBD pretreatment. Our research suggests a potential application of CBD for MA-induced cardiotoxicity, which may attenuate inammatory response and necrosis through the PKA/CREB signaling pathway.


Introduction
Methamphetamine (MA) is a highly addictive class of synthetic stimulant and one of the most popular drugs of abuse worldwide(World Drug Report 2020). The number of young MA users has increased dramatically in recent years, resulting in criminal detention and medical resource shortages. Thus, MA abuse has become of increasing concern and is a major global public health problem (Kohno et al., 2020, Spivak et al., 2020. The molecular structure of MA consists of a methyl group added to the base structure of amphetamine (Fig. 1A). MA is highly lipophilic, allowing it to readily cross the blood-brain barrier (Homer et al., 2008), where it can affect the central nervous system by increasing catecholamine  Razavi et al., 2020). However, MA is a sympathomimetic amine substance, and can have a series of sides effects on multiple organs, such as the brain, heart, kidney, and spleen, following overdose and chronic administration (Rusyniak, 2011, Gurel, 2016, Wu et al., 2016, Isoardi et al., 2020. Of note, cardiovascular injury caused by MA use has gained recent attention.
Hypertensive heart disease, pulmonary hypertension, delayed cardiomyopathy, and cardiogenic shock are frequently found in MA abusers, with sudden unexpected death also reported (Nishida et al., 2003, Segawa et al., 2019, Dalal et al., 2020, Hendrickson and Strauss, 2020. A retrospective study on autopsy found that cardiovascular disease is the second leading cause of death in MA abusers, after accidental drug toxicity (Darke et al., 2017). Furthermore, 68% of MA poisoning-related deaths are reported to show changes in cardiovascular pathology (Akhgari et al., 2017). Cardiovascular complications from MA are increasingly recognized and include vasoconstriction, hypertension, arrhythmia, aortic dissection, acute coronary syndromes, pulmonary arterial hypertension, atherosclerotic coronary artery disease, cardiomyopathy, and heart failure (Paratz et  In ammation is an important risk factor for cardiovascular disease (Sethwala et al., 2021), and also plays a key role in MA-induced cardiac injury. Endomyocardial biopsies from the left ventricles of patients with MA-associated cardiomyopathy show increases in myocyte damage and markers of in ammation and brosis (Schurer et al., 2017). Myocarditis and endocarditis are also found in MA abusers during postmortem examination (Darke et al., 2017). The pathological development of cardiac in ammation can lead to arteriosclerosis, coronary heart disease, heart structure remodeling, and dilated cardiomyopathy (Gao et al., 2015, Kevil et al., 2019, Reddy et al., 2020. However, the molecular mechanism of myocardial in ammation induced by MA exposure has not yet been fully elucidated.
Reducing mitochondrial membrane potential and promoting ROS expression induced by MA may potentiate the in ammatory process (Potula et al., 2010). Toll-like receptor 4 (TLR4) plays an important role in immune and in ammatory responses by activating nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), which further regulates the expression of cytokines. The TLR4/NF-κB signaling pathway is involved in the pathology of MA-induced in ammation in BV2 cells, increasing the level of proin ammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β Although autopsy, clinical, and animal studies provide compelling evidence of cardiovascular dysfunction, the Food and Drug Administration has not approved any speci c treatments or drugs for cardiac complications caused by MA. Cannabidiol (CBD) (Fig. 1B) is a non-psychoactive component of Cannabis sativa (Mechoulam and Hanus, 2002), which is considered safe and well tolerated in high doses and chronic use (Bergamaschi et al., 2011, Shayesteh et al., 2019. In recent years, CBD has been applied in the treatment of various diseases, such as epilepsy, schizophrenia, addiction, anxiety, and neonatal ischemic encephalopathy ( In the present study, we explored the potential protective effects of CBD and involvement of the PKA/CREB signaling pathway in MA-induced cardiac in ammation. We evaluated the therapeutic effects of CBD on cardiac in ammation induced by MA and the expression of key factors of the PKA/CREB signal pathway in the myocardium of rats, which may provide a novel strategy for the treatment of cardiotoxicity induced by MA abuse.

Reagents and chemicals
We legally obtained MA (purity above 98%) from the Yunnan Provincial Public Security Department. The MA was dissolved in saline and administered at a dose of 10 mg/kg via intraperitoneal injection (IP). The Rats were housed together (6 rats/cage) and provided with free access to food and water under standard conditions (room temperature of 22 ± 1 °C, humidity of 50%-60%, and 12-h:12-h light: dark cycle). All rats were acclimatized to the new environment for 3 days prior to the start of the experiments. The rats were randomly divided into ve experimental groups (n = 6/group): i.e., solvent control group (saline solution containing 5% DMSO + 5% Tween-80, 10 ml/kg, IP), CBD 80 group (CBD, 80 mg/kg, IP), MA group (MA, 10 mg/kg, IP), CBD 40 (40 mg/kg) + MA group, and CBD 80 (80 mg/kg) + MA group. The rats received MA or CBD once a day (at 09:00 am) for 4 weeks, with the MA (10 mg/kg) injections given 1 h after CBD treatment (40 or 80 mg/kg, IP), and weighed weekly for dose adjustment. All rats were sacri ced by narcotic overdose 24 h after the last injection. Cardiac blood (for serum) and hearts were collected. Some hearts were xed in 4% paraformaldehyde solution, and some hearts were stored at −80 °C until further analysis. The experimental protocols are shown in Fig. 1C.

Histopathological examination
The hearts were xed in 4% paraformaldehyde solution, dehydrated, embedded in para n wax, and sliced into 5-μm thick sections using a microtome (RM2235, Leica, Germany). The sections were then stained with hematoxylin & eosin (H&E) to assess in ammatory and necrotic changes in tissue and with Masson's trichrome to assess brotic changes. The heart tissue sections were examined using a digital pathological section scanning system (KF-PRO-005-EX, KFBIO, Ningbo, China)(Song et al., 2020).

Enzyme linked immunosorbent assay (ELISA)
The cardiac blood serum levels of pro-in ammatory cytokine IL-6 and anti-in ammatory cytokine IL-10 were measured using rat ELISA kits (Mlbio, Shanghai, China) in accordance with the manufacturer's protocols. Absorbance was recorded at 450 nm.

Statistical analysis
All data are expressed as mean ± standard deviation (SD). Statistical analyses were performed with SPSS v21.0 (IBM SPSS, Chicago, USA), and graphs were constructed using GraphPad Prism v6.0 (GraphPad Software, USA). One-way analysis of variance (ANOVA) was used to analyze experimental data, followed by Tukey's post-hoc tests for comparing individual groups. Paired t-test was applied to evaluate body weight between pre-and post-drug administration. Signi cance was de ned at P < 0.05.

Results
Effects of CBD on MA-induced body and heart weight changes in rats Body weight was measured before drug injection and at 1 to 4 weeks after drug administration. Body weight showed a time-dependent increase and was signi cantly higher after 4 weeks of administration than before administration in each group (P < 0.01, Fig. 2A). Weight gain was lower in the MA group than in the control group but showed no signi cant difference between the two groups after 4 weeks of administration. Compared with the MA group, CBD had no effect on body weight at either dose (40 or 80 mg/kg) ( Fig. 2A). The hearts were weighed immediately after dissection. As shown in Fig. 2B, although heart weight increased slightly in the MA group compared to the control group, this increase was not statistically signi cant. Interestingly, however, 1-h pretreatment with 80 mg/kg of CBD reduced MAinduced cardiomegaly compared to the MA group (P < 0.01). The heart-to-body weight ratio increased signi cantly in the MA group compared to the control group (P < 0.01, Fig. 2C), whereas CBD (80 mg/kg) pretreatment for 1 h reduced the ratio compared to the MA group (P < 0.01, Fig. 2C). These results suggest that chronic exposure to MA for 4 weeks can induce cardiomegaly in rats, to some extent, which can be inhibited by administration of CBD (80 mg/kg).

CBD attenuates MA-induced cardiac in ammatory response
The H&E-stained heart tissues in the MA group showed myocardial ber disorder, mild interstitial oedema, mild myocyte hypertrophy, vacuolization and karyolysis, as well as distinct mononuclear in ammatory in ltration in interstitial spaces or around blood vessels, and focal lesions with necrosis. As shown in Fig.   3C, focal mononuclear in ammatory in ltration was observed with myocardial necrosis and brosis. The myocardial structure of the control and CBD 80 groups was mostly normal (Fig. 3A and 3B). However, pretreatment with 40 or 80 mg/kg CBD for 1 h before MA administration resulted in gradual and effective suppression of cardiac lesions. As shown in Fig. 3D and 3E, only slight mononuclear in ammatory in ltration in the interstitial spaces or around blood vessels was observed, mainly manifested as myocardial cell degeneration. Myocardial brosis is a progressive pathological process of chronic in ammation. Here, Masson's trichrome staining was used to evaluate brosis in the myocardium, resulting in blue-brown-stained nuclei, red-stained cytoplasm and myo ber, and blue-stained collagen bers. As shown in Fig. 3H, more blue-stained collagen brils in the perivascular and interstitial spaces were observed in the MA group than in the control group. Compared with the MA group, 40 mg/kg CBD pretreatment reduced the extent of brosis, although perivascular brosis remained (Fig. 3I), whereas there was almost no brosis in the 80 mg/kg CBD group (Fig. 3J). To further assess MA-induced in ammation and effects of CBD on in ammation, we evaluated the cardiac blood serum levels of proin ammatory cytokine IL-6 and anti-in ammatory cytokine IL-10 via ELISA. As shown in Fig. 4A, the level of IL-6 increased signi cantly in the MA group compared to that in the control group. However, IL-6 expression decreased dose-dependently following CBD pretreatment. In addition, MA signi cantly reduced the expression of IL-10 compared to that in the control group, whereas CBD pretreatment increased the level of IL-10 in a dose-dependent manner (Fig. 4B). Thus, chronic administration of MA in rats can cause a cardiac in ammatory response and progressive development of brosis, while CBD treatment shows protective effects against this pathology.

CBD attenuates MA-induced myocardial necrosis in left ventricle
To assess the extent of myocardial damage caused by MA exposure, we evaluated the left ventricle level of cTnI, an important biomarker of myocardial necrosis, via western blot analysis. As shown in Fig. 4C, MA signi cantly increased the expression of cTnI compared to that in the control group (P < 0.01), whereas pretreatment with 80 mg/kg CBD attenuated the level of cTnI compared to that in the MA group (P < 0.05), although no effect was observed under 40 mg/kg CBD.
CBD down-regulates PKA, p-PKA, CREB, and p-CREB expression in left ventricle following MA-inducedcardiotoxicity As shown in Fig. 5A and 5B, the expression levels of PKA (P < 0.01) and p-PKA (P < 0.05) were signi cantly higher in the MA group compared to the control group. However, pretreatment with 40 mg/kg or 80 mg/kg CBD reduced the expression levels of PKA and p-PKA compared to that in the MA group, with 80 mg/kg CBD more effective (PKA: P < 0.01; p-PKA: P < 0.001). As shown in Fig. 5C and 5D, the expression levels of CREB (P < 0.01) and p-CREB (P < 0.05) were signi cantly higher in the MA group compared to that in the control group. However, pretreatment with 40 mg/kg or 80 mg/kg CBD reduced the expression levels of CREB and p-CREB compared to that in the MA group. Interestingly, compared with 40 mg/kg CBD, 80 mg/kg CBD treatment did not signi cantly reduce the level of CREB, but was more effective for p-CREB (P < 0.01).

Discussion
Previous studies have indicated that body mass index and heart weight are signi cantly higher in MA abusers than in non-MA users (Abdullah et al., 2020). In addition, compared with deaths caused by multiple drug toxicities, those from MA toxicity show heavier hearts (Darke et al., 2018). In the current study, we demonstrated that rat body weight increased signi cantly after 4 weeks of MA administration (Fig. 2A). These results are similar to previous ndings showing an increasing trend in body weight in mice receiving MA (2 mg/kg, IP, 10 days), followed by withdrawal (7 days)(Garcia-Carmona et al., 2018), but differ from other research showing no signi cant differences in body weight in mice receiving MA (0-6 mg/kg) via subcutaneous injection 5 days a week for 4 weeks(Abdullah et al., 2020). Heart weight was higher in the MA group than in the control group, but not signi cantly. Relative heart weight, calculated as the heart-to-body weight ratio, increased signi cantly in the MA group compared to the control group (Fig. 2C), consistent with that found in mice (i.e., increased 4-5 mg/week for 8 weeks at 35 mg/kg MA and 2 mg/week after 20 weeks at 40 mg/kg) (Marcinko et al., 2019). In contrast, other research has reported no signi cant differences in relative heart weight in rats after an acute dose of 50 mg/kg MA or in mice after 10 days of exposure to 2 mg/kg MA (Islam et al., 2009, Garcia-Carmona et al., 2018). These discrepancies could be explained by the different concentrations and treatment times of MA administration, animal species tested, or speci c experimental protocols used. The current study is also the rst to report on the protective effects of CBD (80 mg/kg) against MA-induced cardiomegaly in rats.
Various autopsy reports have revealed that cardiomyopathy, coronary artery stenosis, valvular heart disease, and in ammatory heart disease are involved in many MA-toxicity deaths, with myocyte hypertrophy, myocarditis, endocarditis, pericarditis, perivascular and interstitial brosis, ber necrosis, collagen deposition, and subendocardial myocardial infarction found via microscopic Notably, in ammatory response, brosis, and necrosis of myocardial tissue were con rmed by H&E staining, ELISA analysis, Masson's trichrome staining, and western blotting. Compared to the MA group, however, CBD pretreatment alleviated these lesions to a certain extent.
Long-term MA administration in ApoE −/− mice can lead to a signi cant increase in the levels of plasma Creactive protein, in ammatory cytokines (ICAM-1, VCAM-1, TNF-α), and neuropeptide Y in the aortic root and myocardial tissue, which promote in ammation and atherosclerosis (Gao et al., 2015). Furthermore, acute exposure to MA in mice (30 mg/kg for 6 h) results in a signi cant increase in serum IL-6, TNF-α, and IL-10, with a further increase under MA exposure and water-restraint stress (Tomita et al., 2011). Both the Nfkbiz gene (a regulator of NF-κB) and the Nr4a1 gene (a transcription factor) are up-regulated by NF-κB signaling activation, which is associated with in ammatory response (Yamamoto et al., 2004, Shinone et al., 2010. Similarly, mRNA expression of Nfkbiz and Nr4a1 in the heart and TNF-α, IL-1β, and IL-6 levels in serum are signi cantly induced in mice after MA exposure (30 mg/kg), with further increases in TNF-α and IL-6 when the mice are restrained after MA administration (Shinone et al., 2010). These studies indicate that in ammation plays a key role in myocardial damage induced by MA, which can be aggravated by additional environmental stimuli. In the present study, IL-6 increased and IL-10 decreased after MA administration, but these changes were reversed by CBD (40 or 80 mg/kg), suggesting that CBD may have an anti-in ammatory protective effect on myocardial damage induced by MA (Fig. 4A and 4B).
Previous research has indicated that CBD treatment (2 µg/µl) can inhibit the increase in IL-1β mRNA expression in the prefrontal cortex of rats following MA exposure (Karimi-Haghighi et al., 2020). Furthermore, CBD treatment (1.5 mg/kg, IP, 10 weeks) can signi cantly decrease pro-in ammatory cytokine IL-23, its receptor, CXCL-9, and CXCL-11 in mice with spinal cord injury, but not IL-6 or INF-γ (Li et al., 2018). In rats with myocardial ischemic reperfusion injury, CBD (5 mg/kg, IP, 7 days) can reduce infract size, myocardial in ammation, and serum IL-6 (Durst et al., 2007). Myocarditis, focal and diffuse myocardial brosis, and myocardial dysfunction are reported in patients with pheochromocytoma, indicating that catecholamine toxicity may lead to myocarditis and myocardial brosis (Ferreira et al., 2016). This is supported by our study, whereby MA induced a cardiac in ammatory response and myocardial brosis, but these effects were attenuated by CBD in a dose-dependent manner (Fig. 3 and   Fig. 4). Thus, CBD exhibited considerable preventive and therapeutic effects against cardiac damage induced by MA exposure, which may be mediated by a reduced in ammatory response.
To evaluate the extent of myocardial necrosis caused by MA, we detected cTnI levels in the left ventricle using western blot analysis, as shown in Fig. 4C. Creatine kinase myocardial band (CK-MB) is a key biomarker of myocardial infarction. Autopsy studies have shown high cTnI expression in fatal MA abusers, with CK-MB levels also increased in cardiac and peripheral blood (Zhu et al., 2007) and pericardial and cerebrospinal uids of MA abusers (Wang et al., 2011). Elevated levels of cTnI and CK-MB are indicative of increased myocardial necrosis, as found in our study following MA administration.
However, we also found that CBD (80 mg/kg) pretreatment decreased cTnI levels compared to the MA group, indicating that high-dose CBD may have a protective effect on cardiac damage. Similar ndings have been reported in rabbits with acute myocardial infraction, with CBD administration (100 µg/kg) signi cantly decreasing plasma levels of cTnI and reducing ischemic injury in the myocardium (Feng et al., 2015).
The distribution of MA in the major organs of MA-sensitized rats is reported to be higher in the brain and heart than in the kidney, blood, and abdominal muscle, and delayed e ux of MA in the heart may be associated with cardiac toxicity (Nakagawa et al., 2003). The brain corticotrophin releasing factor system, which is associated with cardiac sympathetic control, is activated by chronic MA administration and withdrawal in mice, which further activates the sympathetic pathways in the heart with increased levels of phospho-tyrosine hydroxylase (p-TH) and p-heat shock protein 27 (p-HSP 27), which may be the mechanism of cardiovascular risk related to MA abuse(Garcia-Carmona et al., 2018). Although various pathological mechanisms have been investigated, the mechanism underlying myocardial injury caused by MA remains unclear. Our data showed that the PKA/CREB signaling pathway was activated and p-PKA and p-CREB increased in rats under chronic MA administration. These ndings suggest that the PKA/CREB pathway participated in MA-induced myocardial in ammation and myocardial pathology. Increased cellular cAMP promotes the dissociation of PKA, the catalytic subunit of which migrates to the nucleus and phosphorylates CREB at a single phospho-acceptor site (ser 133), with p-CREB then promoting further transcription (Mayr and Montminy, 2001). PKA is the key kinase for CREB phosphorylation (Meyer et al., 2000), and CREB plays an important role in drug addiction (Zhou and Zhu, 2006). PKA, p-PKA, CREB, and p-CREB are highly expressed in different brain regions of MA-induced conditioned place preference (CPP) rats and in SH-SY5Y cells, but can be inhibited by gastrodin (Yang et al., 2020b). The cAMP/PKA/CREB pathway is also involved in the apoptosis of cortical neurons induced by MA, but can be regulated by the neuroprotective effects of gastrodin . In this study, the expression levels of PKA, p-PKA, CREB, and p-CREB decreased following CBD pretreatment, indicating that CBD may attenuate myocardial in ammation and cardiac pathology by mediating the PKA/CREB signaling pathway. Similarly, CBD has shown potential therapeutic effects on MA-induced CPP in rats via the PI3K/AKT/GSK-3β/CREB signaling pathway (Yang et al., 2020a).
To the best of our knowledge, this study is the rst to report on the protective effects of CBD on cardiac pathology elicited by chronic MA exposure in rats, with inhibition of cardiomegaly and reversal of histopathology, in ammatory response, and necrosis. Results showed that CBD at 40 and 80 mg/kg had a protective effect on MA-induced cardiac damage, although the effect was stronger at the higher concentration (80 mg/kg). These ndings are similar with previous study showing that CBD at 80 mg/kg, but not 40 mg/kg, can reduce motivation of self-administered MA and drug-seeking behavior after extinction (Hay et al., 2018). CBD may exhibit cardioprotective effects by modulating the expression of crucial components of the cAMP/PKA/CREB signaling pathway. Our study highlights the potential clinical application of CBD in MA-induced cardiac pathology.
Our study demonstrated that chronic MA administration induced cardiomegaly and cardiac pathology in rats, with a notable increase in in ammatory response and myocardial necrosis. Interestingly, CBD pretreatment signi cantly and dose-dependently reduced the in ammatory response and myocardial necrosis via regulation of the PKA/CREB signaling pathway. These results indicate that CBD may have potential clinical application for the treatment of MA-induced cardiotoxicity. However, the speci c molecular mechanism of MA-induced cardiotoxicity and the protective effects of CBD need to be further investigated.

Declarations Author Contributions
The authors declare that all data were generated in-house and that no paper mill was used. S.H. and L.L. were responsible for overall direction of the project. Q.N. performed experiments and drafted the manuscript. W.D. and B.S. performed daily injections of methamphetamine and cannabidiol. G.Y. and H.Y. performed experiments. R.Z. and Y.P. collated data. Y.Y. performed data analysis. All authors read and approved the nal manuscript. Availability of data and materials Data and gures are available upon request.

Competing Interests
The authors have no relevant nancial or non-nancial interests to disclose.

Ethics approval
All animal studies were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals of KMU, and all experimental procedures were approved by the Ethics Committee on Animal Care and Use from KMU (Approval code: kmmu2020403).

Consent to Participate
Not applicable.

Consent to Publish
Not applicable.    Effects of CBD on MA-induced cytokine and cTnI expression. Rats were pretreated with CBD (40 or 80 mg/kg, IP) for 1 h before administration of MA (10 mg/kg, IP), once a day for 4 weeks. Serum levels of IL-6 (A) and IL-10 (B) were detected by ELISA. (C) cTnI expression in left ventricle was measured by western blot analysis. Data are mean ± SD, * * P < 0.01, * * * P < 0.001 vs. control group; # P < 0.005, ## P < 0.01, ### P < 0.001 vs. MA group.

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