The Effects of Chronic Cannabinoid Administration on the Haematological Parameters, Oxidative Stress, BNP, and the Gene Expression of TNF–A in a Rat Model of Pulmonary Arterial Hypertension

Chayil Gunpath^*
*Correspondence: Chayil Gunpath, Department of Human Physiology, School of Laboratory Medicine and Medical Science, University of KwaZulu-Natal, Durban, South Africa, Email:

Keywords

Pulmonary arterial hypertension • Cannabidiol • Oxidative stress • Brain natriuretic peptide • Tumour necrosis factor

Abbreviations

CBD: Cannabidiol; BNP: Brain Natriuretic Peptide; PAH: Pulmonary Arterial Hypertension; TNF: Tumour Necrosis Factor; MCT: Monocrotaline; T-AOC: Total Antioxidant Capacity

Introduction

Pulmonary arterial hypertension

Pulmonary Arterial Hypertension (PAH) is a rare, progressive, and often fatal disorder with multifactorial causes. PAH is a pathophysiological and haemodynamic condition that mainly affects the pulmonary arterioles and is characterised by progressive increases in pulmonary vascular resistance, as well as pulmonary artery pressure, which ultimately leads to inflammation of the pulmonary arteries as well as hypoxia, leading to the eventual failure of the right side of the heart leading to premature death [1]. It is known to affect all age groups but is becoming increasingly prevalent in elderly populations and occurring more often in females than males. The present estimates suggest that there is a 1% prevalence in the global population, which increases to up to 10% in people aged more than 65 years [2]. There is no racial or ethnic group that is known to have a higher frequency of PAH in patients [3]. Post COVID-19, there was a global increase seen in patients who had and currently have COVID-19. PAH is now seen as a risk factor for worsened course of COVID-19 and elevated mortality, which makes PAH even more significant now and in the foreseeable future [4]. Research is still ongoing to determine the pathogenesis of PAH, and to develop more effective treatments and therapeutic interventions. The current therapeutic option that is available is a drug called selexipag. It is currently the gold standard for treatment of PAH. It is a phosphodiesterase inhibitor, targeting the Prostacyclin (PGI2) pathway, that works by slowing the progression of PAH by causing vasodilation and may even reverse some damage done to the heart and lungs. The downside to selexipag is that it is not easily affordable or readily available and is prescribed only after PAH has progressed to an irreversible stage [5].

Cannabinoids

Cannabinoid administration has become an increasingly popular co-treatment for a variety of heart and lung diseases as it has vasodilatory properties and is known to decrease resting blood pressure [6]. Several studies suggest Cannabidiol (CBD) has great potential for therapeutic use as an agent with antiepileptic, antipsychotic, anxiolytic, analgesic, anti-inflammatory, and neuroprotective properties, and has promising therapeutic implications in vascular diseases especially in the treatment of pulmonary hypertension. CBD, which is the non-psychoactive component derived from the Cannabis plant, produces its biological effects through the interaction with cannabinoid receptors in the body, CB1 and CB2, of the endocannabinoid system. The endocannabinoid system is a widespread neuromodulatory system that is expressed throughout the body and is responsible for maintaining the balance of several homeostatic processes. Both the cannabinoid receptors regulate a variety of central and peripheral physiological functions that include neuronal development, energy metabolism as well as cardiovascular, respiratory, and even reproductive functions [7]. As cannabis is becoming largely legalised around the world due to its medicinal properties, there is now research opportunities to study its role in different physiological and pathological processes. There is reason to believe that CBD administration has the properties to influence expression of certain cytokines involved in inflammation and hypoxia that contribute to PAH development, and that it either works on its own, or in combination with available treatments such as selexipag (Figure 1).

Figure 1. Shows the molecular pathways and mechanisms of CBD administration within the cardiovascular system. CBD is involved in regulating several pathways, as well as regulating the Ca²⁺/K⁺ intake, protecting cardiomyocytes from inflammation and oxidative stress, promoting cellular survival, and decreasing immune proliferation. CBD also helps to protect mitochondria and regulates their biogenesis, which improves their cellular energy supply. In vascular vessels, CBD causes vasodilatation, lowering blood pressure, and in turn protects the heart. Arrows indicate changes in activity for each inflammatory cell/molecule/mechanism/inflammatory cell.

Oxidative stress

Oxidative Stress (OS) can be associated with both hyperoxia and hypoxia and is characterised by increase in Reactive Oxygen (ROS) and Nitrogen (RNS) species that is typically generated by an underlying disease process, or an imbalance between the production and accumulation of ROS in cells and tissues and the ability of a biological system to detoxify these reactive products [8]. Excessive accumulation of ROS can lead to cellular and tissue damage. In recent years, several antioxidants have been exploited for their supposed beneficial effects against oxidative stress, such as Vitamin E, flavonoids, and polyphenols, but with the recent emergence and surge of CBD research, there is a case for using CBD as a therapeutic option for OS as it contains both anti-inflammatory and antioxidant properties. The chemistry and pharmacology of CBD as well as cannabinoid receptors and other components of the endocannabinoid system with which it interacts have been extensively researched and in addition to this, clinical studies have contributed to our general understanding of the therapeutic potential of CBD for many diseases, including those associated with OS.

CBD has shown to affect the redox balance by modifying the level and activity of both oxidants and antioxidants. Unlike other antioxidants, CBD interrupts free radical chain reactions, capturing free radicals or transforming them into less deleterious forms. CBD reduces oxidative conditions by preventing the formation of superoxide radicals generated by xanthine oxidase and NADPH oxidase. CBD also reduces ROS production by chelating transitional metal ions involved in the Fenton reaction to form extremely reactive hydroxyl radicals. The antioxidant activity of CBD begins at the level of protein transcription by activating the redox-sensitive transcription factor, Nuclear Erythroid 2-related factor (Nrf2), which is responsible for the transcription of cytoprotective genes, including antioxidant genes.

Inflammation and OS play a vital role and are essential in the development and progression of pulmonary hypertension by increasing lipid peroxidation and reducing antioxidant defences. Reis, et al. also confirms this fact by finding increased levels of circulating cytokines, such as TNF-α in his study investigating the relation between CBD and OS [9]. One reason for the increased levels of plasma cytokines in patients with PAH may be related to oxidative stress present in these patients and the body’s efforts to regain vascular homeostasis through activation of the inflammatory cascade, which promotes excessive formation of ROS’s and RNA and/or decreased antioxidant defences.

Biomarkers in PAH

Search is still ongoing for a novel biomarker, a naturally occurring characteristic or gene by which PAH can be identified, that can be used to detect the early onset by a simple test. The only biomarkers so far recommended by the current guidelines for risk stratification are the B-type Natriuretic Peptide (BNP) and amino-terminal pro-Btype natriuretic peptide. Tumour Necrosis Factor (TNF-α) is a proinflammatory cytokine that is present in neurons and glia. Due to the inflammatory response of PAH, excessive production of this cytokine contributes significantly to the inflammatory process, and this could potentially be used as a biomarker for PAH. In a joint study by Lu, et al. they found that CBD significantly suppressed mRNA levels of TNF-α in mouse lungs that were hypoxic [10]. The same could be applied to the heart as existing clinical data has shown that circulating levels of TNF-α are elevated after heart failure and subsequently higher levels were associated with increased mortality. A comprehensive understanding of TNF-α signalling in the failing heart is still incomplete and existing published data identify both pathogenic and cardioprotective mechanisms of TNF-α in the affected heart. Targeting the TNF-α pathway in heart disease may show therapeutic benefits with anti-TNF-α therapy [11].

Brain natriuretic peptide

Natriuretic peptides are a family of hormones secreted primarily by the heart, kidneys, and brain that cause vasodilation and natriuresis. They include Brain Natriuretic Peptide (BNP), C-type natriuretic peptide and urodilatin. BNP is a product of the early-response gene NPPB and is mainly synthesised de novo and secreted by the ventricular myocardium in response to hormonal, mechanical or sympathetic stimulation. In PAH, volume overload, transmural pressure, hypoxia, or pro-inflammatory factors induce transcription of NPPB to produce 134-amino acid preproBNP. A signal peptide is then removed in the sarcoplasmic reticulum, leaving 108-amino acid proBNP. This is then cleaved on secretion into the bloodstream to produce two biomarkers of 32-amino acid BNP and 76-amino acid NT-proBNP. BNP then binds to the natriuretic peptide receptor-A, which is expressed primarily in the kidney, aorta, lung, and adipose tissue [12]. Receptor activation leads to an increase in the intracellular secondary messenger cyclic guanosine monophosphate resulting in vasodilation, natriuresis, aldosterone inhibition and lipolysis. It is well established in current research that BNP and NTproBNP correlate with several pulmonary haemodynamic metrics that are associated with survival.

In a study by Leuchte, et al. they tried to establish plasma BNP as a simple, non-invasive, and observer independent parameter for assessing disease severity in patients with primary pulmonary hypertension [13]. They demonstrated significant correlations between BNP levels and haemodynamic parameters obtained during right heart characterisation. Their results were in line with the hypothesis that natriuretic peptides are part of a physiological counterregulatory system in hypertension patients with progressive right heart failure. In this context, activation of the natriuretic peptide system will ameliorate pulmonary hypertension owing to the direct vasodilatory properties of BNP.

With regards to CBD and its effects on BNP in a pulmonary model, there is a gap in the literature and its implications are yet to be studied in detail. From existing knowledge, it could be possible that CBD in turn increases the level of BNP because of its antioxidant and inflammatory affects.

Aims and objectives

This study aims to determine the effects of CBD and selexipag on haematological factors, haemoglobin and platelets, in a monocrotaline-induced rat model of PAH, and the effect that CBD has on the gene expression of TNF-α in the cardiac tissue versus the blood. It also seeks to investigate whether co-administration with selexipag has any effects on the expression of this factor as well as on oxidative stress levels and BNP. It is therefore hypothesised that cannabinoids, and possibly in combination with selexipag, have a significant effect on the gene expression of TNF-α in the cardiac tissue of an MCT-induced rat model of PAH, as well as on the oxidative stress and BNP levels by potentially increasing T-AOC in the heart and increasing BNP levels in vivo.

Materials and Methods

Ethical clearance was granted from the Animal Research Ethics Committee (AREC) (AREC/014/019M), animal handling and competency training was done at the Biomedical Resource Unit (BRU) housed on the University of KwaZulu-Natal campus.

Animal groupings and housing

40 Healthy adult male Sprague-Dawley rats with bodyweight between 200 g and 220 g were obtained from the Biomedical Research Unit (BRU) at the University of Kwa-Zulu-Natal, Westville campus. Rats were randomly divided into 5 groups of 8 animals each: Control group, MCT control group, MCT-selexipag group, MCT-CBD group, and MCT-CBD+selexipag group. The rats were housed in cages based on the different groups mentioned above. Cages had enough space for rats to move around freely. Environmental enrichment was also provided. They were housed under standard laboratory conditions of 18-22°C room temperature, 50-70% humidity, with a 12-hour light/dark cycle. Standard rat diet and water was available ad libitum. Fresh water and food were given daily, and weights were measured twice weekly.

Rat model of monocrotaline-induced PAH

MCT, a pyrrolizidine alkaloid, selectively damages the pulmonary vascular endothelium leading to pathological vascular remodelling and hypertrophy of the pulmonary arteries, resulting in increased vascular resistance, and ultimately increased pulmonary arterial pressure. The pulmonary vascular effect is usually seen after 2-3 weeks of administration of this drug.

Drug preparation

Monocrotaline: Monocrotaline was prepared at a standard dosage of 60 mg/kg by mixing 500 mg of MCT with 6 ml of Dulbecco’s Phosphate-Buffered Solution (D-PBS). Thereafter, 2.45 ml of 0.1N HCL was added to dissolve the compound and 14.5 ml of 0.1N NaOH to neutralise the pH. Dosages per rat were calculated at 0.1 ml per 100 g (5 mg/100 μl) and was administered accordingly via intraperitoneal injection with the aid of 27 g needle. The pulmonary vascular effect is usually seen after 2/3 weeks after administration.

Selexipag: Selexipag was prepared at a standard dosage of 3 mg/ kg. 75 mg of selexipag was dissolved thoroughly in 100 ml deionized water to get a dosage of 0.75 mg/250 g and was administered to the stomach orally with the aid of a syringe in accordance with each rat’s individual weight.

CBD: ADCO CBD pain 200 (20 mg/1.5 ml) purified CBD oil, obtained from a local pharmacy, was calculated at a dosage of 0.2 ml/250 g, and again, administered to the stomach orally with the aid of a syringe in accordance with each rat’s individual weight.

Protocol

Day 0: At the start of the study, 8 rats were injected with saline for the control group and 32 rats were injected intraperitoneally with monocrotaline (60 mg/kg) with the aid of a 27 g needle.

Day 18: Cannabinoid preparation: Commercially available CBD (ADCO, South Africa) was administered and delivered to the stomach by oral gavage. For oral administration, in order to control for effects of stomach contents on absorption, the rats were fasted for 8 hours prior to drug administration. CBD and selexipag (10 mg/kg) were administered orally once every other day for 2 weeks. A study done by Hložek, et al. which aimed at profiling the pharmacokinetic and behavioural effects of CBD, THC, and their combination after different routes of administration on rats, reported that oral administration is the best administration route compared to pulmonary (smoking) and subcutaneous administration for medical usage of cannabinoids because of efficient absorption and long half-life span of the drugs [14]. In addition, they reported that oral administration has the following behavioural effects: Locomotor inhibition and no or less psychoactive effects compared to other administration routes.

Day 31: At the end of the experimental protocol, food was removed 12 hours before and all rat groups were sacrificed by euthanasia. The right lung, heart, kidney, and blood were harvested from all animals. Tissue samples were stored at -70°C in a bio freezer until required for analysis (Figure 2).

Figure 2. Physiological responses of MCT-induced PAH model.

Shortcomings during experimental protocol: It is important to note that during the course of the trial, due to the nature of the experimentation and the physiological effects of monocrotaline on the cardiopulmonary system, there was a total of 11 rats that had succumbed to pulmonary oedema. PAH was confirmed on all autopsies done on the animals. The trial was stopped one week shorter than the original end date to prevent any further loss while still maintaining a significant number of samples that would produce statistically accurate results. There were a total of 3 rats from the MCT control group, 1 rat from the MCT-selexipag group, 4 rats from the MCT-CBD group, and 3 rats from the MCT-CBD-selexipag group that were lost. Possibilities for prevention in the future could include lowering the MCT dose and/or increasing the selexipag/CBD dosages.

Haematological analysis

Two blood samples from each rat were drawn post euthanasia and collected in EDTA tubes. They were immediately put on ice and transported to the laboratory for analysis. A full haematological profile was done using a Beckman-Coulter haemolyzer, and the results were recorded.

One sample of blood from each rat was centrifuged at 10000 g for 10 minutes to separate the blood and plasma. Plasma samples were then extracted and transferred to polypropylene tubes and frozen at -70°C until time for BNP and Total Antioxidant Capacity (T-AOC) analysis.

Plasma BNP: Optical densities were determined with a microplate reader after using Elabscience’s rat BNP ELISA Kit (E-EL-R0126). Standard competitive-ELISA preparation was strictly followed according to the manufacture’s guidelines. All samples and standards were prepared in duplicate.

Plasma Total Antioxidant Capacity (T-AOC): Optical densities were determined with a microplate reader after using Elabscience’s total antioxidant capacity colorimetric assay (Ferric reducing ability of protein, FRAP, method) (E-BC-K225-M). The assay was strictly prepared according to the manufacture’s guidelines. All samples and standards were prepared in duplicate.

Heart tissue preparation

Whole heart samples were thoroughly washed with Phosphate Buffer Solution (PBS), dried, and weights were recorded. Hearts were homogenised at the ratio of the volume of homogenised medium (PBS) (2-6°C) (ml): The weight of the heart (g)=9:1. Heart homogenate was then centrifuged for 10 minutes at 10000 g at 4°C. Supernatant was taken out and transferred to EP tubes, then preserved on ice until detection.

Heart TNF-α: Optical densities were determined with a microplate reader after using Elabscience’s rat TNF-α ELISA Kit (E-EL-R2856). Standard sandwich-ELISA preparation was strictly followed according to the manufacture’s guidelines. All samples and standards were prepared in duplicate.

Heart Total Antioxidant Capacity (T-AOC): Optical densities were determined using Elabscience total antioxidant capacity colorimetric assay (Ferric reducing ability of protein, FRAP, method) (E-BC-K225- M). The assay was strictly prepared according to the manufacture’s guidelines. All samples and standards were prepared in duplicate.

Before determining the heart T-AOC, total protein concentration of each heart sample had to be determined first in order to calculate the resulting T-AOC. This was done using Elabscience’s bradford protein colorimetric assay (E-BC-K168-M). Samples were diluted for 15x, and the assay was carried out strictly according to the manufacturers guidelines with standards and samples prepared in duplicate.

Results

All graphs, standard curves, and statistical analysis were carried out using GraphPad prism 10 statistical software. BNP and TNF-α concentrations were extrapolated from their respective standard curves. Protein concentration and the resulting heart T-AOC levels were calculated from their respective simple linear standard curves. Plasma T-AOC levels were also calculated from a simple linear standard curve (Table 1 and Figure 3).

Haematological profiles

Table 1. Full haematological profile showing the averages between all groups.

Plasma BNP

Figure 3. Sigmoidal graph showing the BNP standard curve.

Welch’s unpaired T tests (Two-tailed) and ANOVA was carried out to find significance amongst and between groups. Welch’s corrections were chosen, as the number of samples between each group varied.

BNP levels decreased across the MCT control, MCT-selexipag, and MCT-CBD groups, with the MCT-CBD group seeing the most significant decrease (p<0.05), when compared to the control group. Interestingly, the combination group of CBD and selexipag showed a significant increase, and proved the most effective, compared to the other two treatment groups (p<0.05), with levels of BNP rising to just above ‘normal’ control group levels. Welch’s ANOVA showed significant difference amongst all groups (p<0.05) (Figures 4 and 5). 

Figure 4. The effect of CBD administration on BNP expression in plasma of MCT-induced PAH (*amount represents significance between groups).

Plasma Total Antioxidant Capacity (T-AOC)

Figure 5. Linear graph showing T-AOC standard curve.

The T-AOC standard curve that was generated has 5 points on the curve, instead of 8, as there were pipetting errors that occurred on the last 3 dilutions that rendered incorrect optical densities. These 3 points were disregarded to generate the linear graph shown. This had no impact on the calculated results as all the optical densities fell below the 5th point on the graph. The same standard curve was used for both calculations of the respective blood and heart T-AOC (Figure 6).

Figure 6. The effect of CBD administration on T-AOC expression in plasma of MCT-induced PAH (*amount represents significance between groups).

T-AOC levels dropped across all groups compared to the control group, with the largest decrease seen in the MCT-CBD group (p<0.05). Similarly, to the BNP levels, the most effective treatment can be seen in the combination treatment group of MCT and selexipag, with levels rising close to that of the control group levels and higher than that of the MCT control group. The combination treatment group showed significance when compared to the MCT+selexipag group (p<0.05), but not with the MCT+CBD group. Welch’s ANOVA showed significant difference amongst all groups (p<0.05).

Heart T-AOC

Before calculation of the heart T-AOC, total protein concentration had to first be determined. This was done using bradford’s protein colorimetric assay (Figures 7-9).

Figure 7. Linear graph showing bradford protein standard curve.

Figure 8. The effect of CBD administration on protein concentration in heart tissue of MCT-induced PAH (*represents significance between groups).

Figure 9. The effect of CBD administration on T-AOC in heart tissue of MCT-induced PAH (*represents significance between groups).

Welch’s ANOVA proved significance amongst all the groups (p<0.05). Unpaired Welch’s T-test’s also showed significant differences between the control group and the MCT control group, as well as between the control group and only two of the treatment groups; the MCT+selexipag group and the MCT+CBD group (p<0.05). The highest T-AOC concentration was picked up in the combination treatment group and was the only group that did not show significance when compared against the control group (Figures 10 and 11).

Heart TNF-α

Figure 10. Sigmoidal graph showing the TNαF -standard curve

Figure 11. The effect of CBD administration on TNF-α expression in heart tissue of MCT-induced PAH (*amount represents significance between groups).

The MCT control group recorded the lowest levels of TNF-α compared to any other group, whilst the MCT-CBD group recorded the highest levels. There was a significant difference between the control and MCT control group, as well as between the MCT control and MCTCBD group (p<0.05). There was, however, no significance between the other two treatment groups and the control group, or MCT control group. Welch’s ANOVA showed there was a significant difference amongst all groups (p<0.05).

It is also worth noting that the large standard deviations recorded across various groups could be attributed to the long storage times of the samples, possibly affecting the bioavailability and integrity of the samples. Another factor to take into consideration is the pipetting technique when dealing with the samples and the various ELISA kit components.

Discussion

From the results of the BNP levels in the blood, we can see that the combination of CBD and selexipag proved to be the most effective treatment compared to the individual treatments of CBD and selexipag, which did not prove to be effective as they showed no significance in increasing BNP levels. This suggests that there is some kind of synergistic interaction between CBD and selexipag that possibly takes place in the prostacyclin pathway, that drives the BNP levels back up to normal. It is clear to see that due to the combination of CBD and selexipag, that there was activation of the natriuretic peptide system. Further research will need to be done to investigate this phenomenon as it shows great promise as a therapeutic option for increasing BNP that would aid in lowering blood pressure, due to BNP’s direct vasodilatory properties, and potentially reversing some of the symptoms and damage caused by PAH.

Interestingly, there seems to be a direct correlation between the results of BNP levels compared to the T-AOC levels in the blood. The results closely mirror each other, where again, the combination group of CBD and selexipag showed to be the most effective, having the highest levels compared to the other two treatment groups. This supports the claim that once T-AOC levels increase, this subsequently decreases OS and thus increases BNP levels, which can be seen in the resulting T-AOC levels. Recent studies have demonstrated the relationship between intensification of oxidative stress and inflammation and the severity of ventricular dysfunction that could lead to heart failure. Leong, et al. showed that combining BNP with oxidative stress and inflammatory markers may increase the detection of ventricular systolic dysfunction and early-stage heart failure. This could prove particularly useful in monitoring PAH progression or used as a screening tool for PAH detection.

There were similar trends between T-AOC levels in the blood versus the heart tissue. As expected, both the control groups had the highest T-AOC levels. The co-treatment group also had the highest TAOC levels amongst the three treatment groups, owing to the possible synergistic nature, or potentiation, between CBD and selexipag. Possible explanations for this could be due to a variety of different factors, such as absorption, distribution, metabolism, or excretion. Heart T-AOC levels were higher amongst all groups when compared to the blood T-AOC. When looking at the MCT-CBD group specifically, T-AOC levels in the blood recorded the lowest levels, even lower than the MCT control group. This was not the case for heart T-AOC. Heart T-AOC levels, for the MCT-CBD group, more than doubled when compared to the blood. This could suggest that CBD plays a role in attenuating oxidative stress, as well as inflammation, in the myocardium as a result of PAH, strongly suggesting that it may have great therapeutic potential in the treatment of cardiovascular disorders. This can be supported by a study conducted by Rajesh, et al. where they investigated CBD’s influence on oxidative stress and cardiac dysfunction, finding similar results [15]. The increase in TAOC indicates that there is increased absorption of antioxidants resulting in an improved antioxidant defence status in vivo. This suggests that due to the nature of PAH’s excessive stress on the cardiovascular system, it could elicit this type of response. It is important to note that total antioxidant capacity was determined, and not any specific antioxidant independently, that any particular antioxidant could possibly alter the total status of antioxidant capacity of either the blood or heart tissue.

As investigated by Irwin, et al. they found that higher levels of TNF- α were associated with increased mortality [16]. This holds true as the highest mortality (4) was recorded in the group with the highest recorded TNF-α levels, the MCT-CBD group. From this finding we can assume that CBD does not positively influence heart TNF-α levels by decreasing it. CBD, in this case, could have possibly added to the inflammatory response in the heart progressing PAH, even with CBD’s known anti-inflammatory effects, which is why we see increased TNF- α levels [17]. CBD therefore could potentially be used as a screening method for TNF-α and PAH detection. Interestingly, the MCT control group had the lowest levels of TNF-α, where it was predicted to have the highest. Possible explanations for this may be due to the disease state at the time of sacrifice and sample collection, and due to the early conclusion of the experiment, adequate time was not given for CBD and selexipag treatments to run their full course and influence TNF-α expression to the levels that were expected. Another factor to take into consideration is how each animal responded to the drugs and treatment given. Any possible underlying illness could also play a role in influencing results.

Focus on the haematological profile was given specifically to platelet count and haemoglobin level, as these showed significant changes across all groups. Other haematological parameters remained insignificant across all the groups as individual counts varied quite considerably. Platelet counts showed that the MCT-CBD group contained the highest number of platelets in the blood. Pignatelli, et al. explains that higher circulating levels of TNF-α could account for the hyperfunction of platelets as TNF-α is able to activate platelets trough the stimulation of the arachidonic pathway [18]. With regards to CBD’s possible effect on platelets, Randall explained how CBD promotes platelet activation, indicating that they might also be thrombogenic [19]. These could be possible explanations to account for the high platelet levels. Similar to platelet count, haemoglobin levels were also highest in the MCT-CBD group. Atsma, et al. explained that haemoglobin levels were positively associated with hypertension, where a rise in haemoglobin would result in higher blood pressure [20]. This could be another explanation for the high TNF-α levels and backs up the claim that there were higher levels of inflammation in the heart as well as possible damage to the pulmonary arteries where CBD, and TNF-α, activated excessive platelet production to try and reverse damage. This can be backed up by the subsequent T-AOC and BNP levels observed. The downside to excessive platelet activation is that this may lead to the development of thrombocythemia, however no symptoms of this were observed during the trial and postmortem. Another significant downside is that excessive platelet could also in fact contribute to the development of PAH as investigated by Zanjani. CBD ultimately should not be considered as a treatment option, at least on its own, to treat PAH (SupplementaryTables).

Conclusion

From the results generated it is clear to see both the positive and negative effects that CBD can have on the expression on TNF-α, BNP, and oxidative stress. It seems CBD’s efficacy lies in where other drugs are taken in conjunction with it, in this case selexipag. There is promise with regards to CBD and selexipag being taken in combination to increase BNP levels, by activation of the natriuretic peptide system which will ameliorate pulmonary hypertension owing to the direct vasodilatory properties of BNP. Further research would therefore need to be conducted to investigate the synergistic relationship that CBD and selexipag possibly has. In contrast to that and in this case, CBD seems to increase TNF-α and possibly further drive PAH and inflammation which can be supported by the platelet count and haemoglobin levels, as well as mortality rate in that group. By possibly increasing/decreasing amounts of MCT and/or CBD, as well as further prolonging the experiment itself, we could potentially see a decrease in TNF-α levels. Research would need to be done to investigate why CBD contributed to a rise in TNF-α, and why the lowest levels were recorded in the MCT control group. Interestingly, T-AOC in the heart specifically in the MCT-CBD group was the highest, owing to CBD’s potential to attenuate oxidative stress and counteract the increased stress and inflammation the heart was under during PAH.

Synthesis

With the increased prevalence and mortality rate of pulmonary arterial hypertension, there is a case to be made with regards to finding a more effective, cheaper, and easily accessible treatment than the current gold standard treatment, selexipag. The ease on cannabis legislation globally for both recreational and medicinal use allows for more research to be conducted on a variety of diseases, particularly cardiovascular diseases, given CBD’s known immunomodulatory, anti-inflammatory, and analgesic properties.

As of recent times, there is substantial evidence that exists within the literature that shows CBD’s efficacy in treating and managing various symptoms of hypertensive conditions, however not enough with regards to PAH, and co-treatment with selexipag. This study therefore aimed to show how CBD, alone and in combination with selexipag, affected key biomarkers brain natriuretic peptide and TNF- α of PAH, as well as the oxidative stress and key haematologic factors, haemoglobin, and platelets, in a rat model of PAH. All these factors together helped paint a picture of PAH progression within the heart and vascular system and how the treatments affected the overall disease state and management of symptoms upon analysis of biological samples.

The main finding of the study showed that CBD’s efficacy lies not in being taken alone, but rather in combination with selexipag. The co-administration, in fact, proved more effective than either CBD and selexipag taken in isolation showing the potential for adjunctive therapy. CBD and selexipag together showed to increase BNP and blood T-AOC levels back to control levels. Leong, et al. explains how combining BNP with oxidative stress and inflammatory markers, such as TNF-α, may increase detection of ventricular dysfunction and early-stage heart failure in PAH which could be useful as a screening tool for PAH severity. Looking at haematological parameters such as platelet count and haemoglobin levels could also aid in PAH detection/disease state. CBD in isolation increased heart T-AOC and TNF-α levels back to control levels. CBD, in isolation, could ultimately be used as an effective screening method for TNF-α and PAH detection as higher levels indicate increased chance of mortality, as demonstrated in this study. Irwin, et al. demonstrated this in their study between TNF-α levels and mortality in PAH models.

These findings are particularly significant as it shows there is promise in CBD to decrease stress and increase antioxidant defence in the cardiovascular system during PAH and to possibly reverse any damage caused by means of vascular remodelling. Rajesh, et al. conducted a study investigating CBD’s influence on oxidative stress and cardiac dysfunction in a hypertensive model, finding similar results.

Limitations

A significant limitation of this study is the non-pressure measurement by right heart catheterization, an invasive procedure to establish an increased pressure in the right ventricle due to PAH.

Acknowledgements

I wish to acknowledge Dr. A. Nadar, of the University of Kwa-Zulu Natal, for his help, guidance, and continued supervision for the entirety of this study, Professor M. L. Channa, for his co-supervision during the early stages of the study, as well as Mr. D. Makhubela of the Physiology department for his assistance and guidance with experimental laboratory work. I would also like to acknowledge previous Honour’s students; Tasmyn Arumugam, Siyethaba Bhengu, and Silindile Ndziba for their assistance at the BRU during the duration of the trial, as well as current Honour’s students; Dre Naidoo and Kiara Singh for their assistance with laboratory work. Lastly, I would like to thank Dr. N. C. A. Jaca, Dr. L. Bester, and Mr. D. Mompe of the biomedical resource unit who assisted me in the caretaking, dissections, and animal handling and training respectively. This study would have not been successful without the continuous help and support of everyone involved.