Treatment with thyroid hormones and grape juice in a model of pulmonary hypertension: the response of apoptosis and inflammationAbstract
This study explored the association of cardioprotective role of thyroid hormones (TH) and benefit effects of grape juice (GJ) on vascular function against the apoptosis and inflammation associated with monocrotaline (MCT)-induced pulmonary hypertension (PH). Wistar rats were distributed into five groups: control; PH (MCT 60mg/kg i.p.); PH+GJ (GJ-7 µL/g / day, gavage for 14 days); PH+TH (T3, 2μg/100g/day, and T4, 8μg/100g/day, by gavage for 14 days); and PH+TH+GJ. Echocardiographic, morphometric measurements and expression of proteins associated with apoptosis and inflammation were evaluated. The reduced cardiac output (35%) in PH was attenuated in the PH+GJ, PH+TH, and PH+TH+GJ groups (P<0.05). Both GJ and TH had no effect on the PH-induced hypertrophy index. The PH + TH group showed reduced BAX (~90%) and increased Bcl2 levels (~80%) as compared to PH group (P<0.05). The PH-induced increase in caspase 3 was diminished (~92%) in the PH+TH (P<0.05). Both GJ and TH, isolated and combined, reduced caspase 9 levels (~70%). PH-provoked elevated NLRP3 levels was decreased (~90%) in PH+TH (P<0.05). The therapeutic approach showed cardioprotective effects in the PH model which may be mediated by mitigation of apoptosis and inflammation.
Key words
NLRP3; BAX; Bcl2; caspases 9 and 3; monocrotaline
INTRODUCTION
Pulmonary hypertension (PH) is a disease that affects both the lung and the heart in which changes in the pulmonary vascular bed is the major pathological condition (Poch & Mandel 2021). The adverse vascular remodelling of lung and increased resistance to flow in PH may lead to the augmented afterload of the right ventricle (RV). Over time, this provides RV hypertrophy and progression to heart failure (Maron et al. 2021). Al-Qazazi et al. (2022) suggest that the activation of the nucleotide-binding oligomerisation domain-like receptor (NLR) family pyrin domain containing 3 (NLRP3) inflammasome, in the RV, plays a critical role in the development of this heart failure. In this context, apoptosis mechanisms play a key role in cardiac dysfunction. The downregulation of anti-apoptotic proteins such as Bcl-x L and upregulation of the caspase-3 represent important apoptotic markers in adverse cardiac remodelling (Zungu-Edmondson et al. 2016). Therefore, the search for therapeutic approaches that act on the targets of inflammation and apoptosis appears to be promising.
In fact, several studies demonstrate a cardioprotective effect of thyroid hormones in heart disease (De Castro et al. 2014). De Castro et al. (2016) showed that TH treatment improved cardiac remodelling and decreased the Bax/Bcl-2 ratio in the infarcted rats. In parallel, Ortiz et al. (2023) observed that inflammatory signalling was reduced when infarcted rats were treated with TH. Although the relationship between reduced TH levels and pulmonary hypertension is not fully understood, it is thought that hypothyroidism may contribute to the development or progression of PH through various mechanisms, such as increased pulmonary vascular resistance and impaired right ventricular function (Scicchitano et al. 2016). Nevertheless, thyrotoxic hyperthyroidism appears to be related to the development of PH. According to literature data, an accepted hypothesis understands that the autoimmune response in Graves’ disease could be the cause of the injury to the pulmonary vasculature, provoking the PH condition (Sugiura et al. 2015, Scicchitano et al. 2016). In this sense, the control of thyroid hormones within homeostatic levels means to be the most appropriate way for protecting the vascular bed of the lung.
On the other hand, flavonoids from grape juice (GJ), which are a group of compounds with various structural conformation, such as flavones, flavonols, dihydroflavonoids, isoflavones, and chalcones, also have potential for treating PH. These compounds can protect against PH by inhibiting pulmonary vascular remodelling (Wang et al. 2021). Ludke et al. (2010) showed that treatment with GJ attenuated the decreased pulmonary endothelial nitric oxide synthase and thickening of tunica media. Moreover, Mosele et al. (2012) explored the antioxidant role of phenolic compound from GJ in the PH experimental model. These authors verified that pre-treatment with GJ improved adverse cardiac remodelling and augmented antioxidant proteins, such as thioredoxin -1. Ashoori et al. (2023), in a meta-analysis study, showed that whole grape products improved vascular function (such as vasodilatation) and reduced arterial pressure. Therefore, GJ presents a protective role that can be useful against PH-provoked vascular remodelling.
Considering the cardioprotective and anti-inflammatory role of TH, as well as the antioxidant and vasodilatory role of phenolic compounds from grape juice, this study aimed to investigate both therapeutic strategies (thyroid hormones and grape juice), isolated and combined, on proteins of apoptosis and the inflammasome pathway in the RV of animals with PH.
MATERIALS AND METHODS
Ethical
The experimental protocols followed the guidelines established by the Legislation of the National Council for the Control of Animal Experimentation (CONCEA), in accordance with Ethical Principles in Animal Experimentation, formulated by the Brazilian College of Animal Experimentation; as well as those contained in the International Guidance Principles for Biomedical Research Involving Animals from the Council for International Organizations of Medical Science (CIOMS). It was approved by the Federal University of Rio Grande do Sul Animal Use Ethics Committee (CEUA-UFRGS) (Protocol number: 37372).
Experimental groups
Wistar rats, age 45 days and weighing 180 ±20 g, were numbered from 1 to 46 and randomly divided into the following experimental groups: Control (n=10), received an intraperitoneal injection of saline and received water by gavage; PH (n=10), received monocrotaline (MCT) (60mg/kg, single dose, intraperitoneal); PH+GJ (n=9), received MCT (60mg/kg, single dose, intraperitoneal) and grape juice (7 µL/g / day, gavage); PH+TH (n=7) received MCT (60mg/kg, single dose, intraperitoneal) and T3 and T4 (T3, 2μg/100g/day and T4, 8μg/100g/day, by gavage); and PH+TH+GJ (n=10) received MCT, thyroid hormones and, one hour after this administration, grape juice, as described above. The administrated doses are per de Castro et al. (2014) and Dani et al. (2008), respectively. The analyses were performed in a double-blinded manner. The calculation of the sample size was performed using the Sigma Plot 11.0 program, with a probability of error α = 0.05 and statistical test power (1−β error probability) of 0.95.
Experimental design
The animals received treatment with thyroid hormones (Sigma Aldrich) and grape juice (Company Uva’só Organic Products) 7 days after the single dose of MCT and were euthanised 14 days after the beginning of the treatments, totalling 21 days of experimental protocol (Dani et al. 2008; De Castro et al. 2014). Before euthanasia, the animals were submitted to echocardiographic evaluation; subsequently, the right ventricle, lung, and liver were removed for morphometrics and western blot analyses (Figure 1).
The animals received treatment with thyroid hormones and grape juice after 7 days of the single dose of MCT, being euthanized 14 days after the beginning of the treatments, totaling 21 days of experimental. Before euthanasia, the animals were submitted to echocardiographic evaluation, subsequently, the collection of the right ventricle, lung and liver was performed to morphometrics and western blot analyses. Parts of the figure were drawn by using: pictures from Servier Medical Art. Servier Medical Art by Servier is licensed under a Creative Commons Attribution 3.0 Unported License.
Echocardiography of right ventricle
Animals were anesthetized with ketamine (90 mg/kg i.p.) and xylazine (10 mg/kg i.p.), submitted to trichotomy of the thoracic region, and placed in the lateral decubitus position. Echocardiography images were obtained through two-dimensional mode and pulsed Doppler (Philips HD7 225 Ultrasound System), using an S12-4 (21780A) transducer (Philips). The following RV systolic parameters were evaluated: cardiac output (mL/min), fractional shortening (%), stroke volume (mL). Cardiac output and stroke volumes were calculated using cardiac volumes obtained through Simpson’s rule (De Castro et al. 2014). Fractional shortening was calculated following equation: RVFS = diastolic diameter − systolic diameter/diastolic diameter × 100 (Tavares et al. 2012).
Morphometric evaluation
The hypertrophy index of the right ventricle (RV) was calculated dividing the RV weight by tibial length. The organ congestion was calculated as the wet weight/dry weight ratio of the lungs and liver.
Western blot analyses
Part of the RV, 100 mg, was homogenized with the cell lysis buffer RIPA (25 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS). The homogenate was centrifuged at 1000 x g and supernatant was used for electrophoresis. Electrophoresis in polyacrylamide gel and transfer to a polyvinylidene difluoride membrane (Immobilon-P transfer membrane; Millipore) were performed. The immunodetection was performed to according to Table I. Secondary antibodies (anti-mouse or anti-goat or anti-rabbit radish peroxidase conjugate) were used for detection by chemiluminescence. The quantification was performed with ImageJ software. The Ponceau method was used for normalisation (Klein et al.1995). The sample size for each protein was four animals per group and they were randomly chosen.
Statistical analysis
Data distribution was determined using the Shapiro Wilk test. For data with a normal distribution of homogeneous variance, values were represented as mean ± standard deviation, one-way analysis of variance (ANOVA) was used, followed by Tukey’s post-test (F). For data with a normal distribution and non-homogeneous variance, Welch’s analysis of variance (ANOVA) was used, followed by the Games Holmes post-test (W). For data that did not have a normal distribution, values were expressed as median and 25th and 75th percentiles, Kruskal-Wallis analysis with Dunn’s post-test was used (K). Values of P<0.05 were considered statistically significant. All analyses were performed using SPSS Statistics version 18 software.
RESULTS
Treatment with both thyroid hormones and grape juice improves the cardiac output of the right ventricle
There was a reduction in cardiac output of around 35% in PH compared to the control group. However, the PH+GJ, PH+TH, and PH+TH+GJ groups were not different compared to the control group, showing a mitigation of this dysfunction. Shorting fraction and stroke volume were not significantly different between the groups (Table II).
The therapeutic approach did not reduce the right ventricle hypertrophy index
The right ventricle mass and right ventricle hypertrophy index, given by the weight/tibia length ratio, was increased in the PH group (~47 and 52%, respectively) as compared to control animals (P<0.05). Nevertheless, both thyroid hormones and grape juice treatments did not reduce the right ventricle mass and hypertrophy index in PH+GJ, PH+TH, and PH+TH+GJ groups. Additionally, the PH+TH group showed both increased right ventricle mass and right ventricle hypertrophy (~26% and 29%, respectively) as compared to the PH+GJ group (P<0.05). Regarding lung congestion index, there was no changes among experimental group (Table II).
The thyroid hormone treatment reduced PH-induced BAX elevation and increased Bcl2 protein expression
BAX protein expression was increased in the PH group compared to the control group. Nevertheless, the levels this of protein were normalized in PH+TH. The PH+GJ and PH+TH+GJ groups had augmented BAX protein expression compared to the control and PH+TH groups [F(4,15) = 10.765, P= <0.001] (Figure 2a). BCL2 levels were increased in PH+TH compared to other groups [F(4,15) = 5.587, P=0.006] (Figure 2b). The BAX/BCL2 ratio was elevated in the PH group compared to other groups [W(4,6.850) = 5.838, P=0.023] (Figure 2c). Regarding JNK protein expression, PH+GJ, PH+TH, and PH+TH+GJ showed reduced levels compared to the control group [F(4,15) = 5.557, P=0.006] (Figure 2d).
Representative images of western blot (sample RV). (a) BAX (b) Bcl2 (c) BAX/Bcl2 ratio (d) Total JNK. Data were expressed as mean and standard deviation. (*) significant difference compared to the control group. (#) significant difference in relation to the PH group. ($) significant difference in relation to the PH+GJ group. (@) significant difference in relation to the PH+TH group.
PH-induced-augmented caspase levels are attenuated by thyroid hormone treatment
There were increased levels of caspases 3 and 9 in the PH group compared to the controls. Caspase 3 immunocontent was reduced in the PH+TH and PH+TH+GJ groups compared to the PH group [F(4,15) = 9.045, P = <0.001] (Figure 3a). However, there were lower levels of Caspase 9 in the PH+GJ, PH+TH, and PH+TH+GJ groups compared to the PH group [F(4,15) = 8.574, P= <0.001] (Figure 3b).
Representative images of western blot (sample RV). (a) CASPASE 3 (b) CASPASE 9. (*) significant difference compared to the control group. (#) significant difference in relation to the PH group. ($) significant difference in relation to the PH+GJ group.
The combination of thyroid hormones and grape juice treatment reduced MyD88 protein expression
There were no significant difference in the immunocontent of NFκB [F(4,15) = 2.859, P= 0.061] and Toll Like Receptor 4 [F(4,15) = 3.139, P= 0.046] among the experimental groups (Figure 4a and 4b). On the other hand, the PH+TH+GJ group showed reduced MyD88 levels compared to other groups [F(4,15) = 6.304, P=0.003] (Figure 4c).
Representative images of western blot (sample RV). (a) NFkB (b) TLR4 (c) MyD88. Data were expressed as mean and standard deviation. (*) significant difference compared to the control group. (#) significant difference in relation to the PH group.
Thyroid treatment reduced the PH-induced level of the NLRP3 inflammasome
The NLRP3 inflammasome levels in the PH group were lower than in the PH+TH group, which had lower levels than in the control, PH+GJ, and PH+TH+GJ groups [K(4,20)= 14.257, P= 0.007] (Figure 5a). There was no difference in COX2 protein expression between the experimental groups (Figure 5b).
Representative images of western blot (sample RV). (a) NLRP3 (b) COX2. Data were expressed as median and 25th and 75th percentiles (*) significant difference compared to the control group. (#) significant difference in relation to the PH group. ($) significant difference in relation to the PH+GJ group. (&) significant difference in relation to the PH+TH+GJ group.
DISCUSSION
This study aimed to explore the therapeutic potential of TH and GJ on the cell death and inflammation induced by PH. The data suggest that isolated treatment with thyroid hormones or grape juice has a positive effect on the cardiac output of the right ventricle. In parallel, the TH treatment provided reduction in BAX and increase in Bcl2 levels. Similarly, increased caspases 3 and 9 levels were reduced by TH and GJ treatment. In the PH experimental protocol, cell death activation can be associated with the inflammatory process. On the other hand, the combination TH and GJ treatment led to a reduction in the expression of a protein called MyD88. Moreover, the NLRP3 inflammasome, a protein complex that plays a critical role in the inflammatory response, was reduced with TH treatment.
The PH model induced by the administration of monocrotaline causes numerous alterations in the structure and function of the RV. Cardiac output represents a relevant echocardiographic parameter to assess the functional status of the RV. Our results show that cardiac output was reduced in PH group, indicating ventricular dysfunction that can be associated with increased pulmonary pressure (Ley et al. 2023). Treatment with phenolic compounds has shown promising results of cardiac functional improvement in experimental models of PH. Mosele et al. (2012) showed that pretreatment with GJ improved pulmonary vascular resistance and hemodynamic parameters in the RV. Lacerda et al. (2020) reported amelioration of myocardial performance index and stroke volume of PH rats which were treated with pterostilbene, a blueberry-derived phenolic compound. Our findings suggest that GJ mitigates ventricular impairment, given that the cardiac output of the PH+GJ and PH+TH+GJ groups was not different to the control group. Vázquez-Garza et al. (2020) attribute the cardioprotective effects of phenolic compounds, especially resveratrol, given that this flavonoid causes a reduction in ventricular remodelling markers and increased sirtuin1-mediated deacetylation.
On the other hand, TH treatment showed similar action to the GJ administration on cardiac output. De Castro et al. (2014), in a model of acute myocardial infarction, also observed an improvement in cardiac performance and oxidative stress. Corssac et al. (2016) showed that TH treatment leads to the augmented angiogenesis in the RV of infarcted rats, suggesting a relevant protective effect of TH on ventricular tissue. The development of TH-induced cardiac hypertrophy was also reported by Corssac et al. (2016) and de Castro et al. (2014). Our results showed that the TH treatment-provoked improvement of ventricular function was in parallel to the RV hypertrophy. Even though, in our study, histological analysis was not performed, TH administration could reduce fibrosis and provide a ameliorate functional and structural of RV, suggesting a beneficial ventricular remodelling (Teixeira et al. 2018). A reduction in apoptosis signalling may also contribute to adaptive remodelling of the RV.
There is increased apoptosis signalling in the PH-induced heart failure (Campos-Carraro et al. 2018). Apoptosis is a programmed and physiological cell death mechanism. However, the apoptotic mechanism is also associated with many cardiovascular pathological events. Ortiz et al. (2020) demonstrated that maladaptive cardiac remodelling post-infarction is mediated by activation of BAX. In our study, the PH group showed increased ventricular BAX levels, suggesting an apoptotic signalling associated with pulmonary hypertension. Moreover, the BAX/Bcl2 ratio was augmented in the PH group, which reinforces the apoptotic character of ventricular remodelling in this model. Zungu-Edmondson et al. (2016), who used a PH model based on administration of SU5416 (vascular endothelial growth factor inhibitor) and hypoxia (10% O2) for 3 weeks, also found apoptosis activation and fibrosis in the RV. However, the authors showed that PH provoked reduced Bcl-XL levels. Our data showed that only TH treatment elevated anti-apoptotic protein Bcl-2 expression, as well as mitigating the augmentation of BAX levels. As consequence, BAX/Bcl-2 ratio was diminished in the PH+TH group, suggesting that TH treatment can mitigate apoptosis signalling. In fact, an anti-apoptotic mechanism of thyroid hormones involves the control of gene expression of proteins such as Bcl2 (Shen et al. 2022). Apoptosis also implies the role of the caspase family in the progression of right heart failure.
Caspases 9 and 3 play a relevant role in maladaptive remodelling induced by PH. Colombo et al. (2015), using monocrotaline-induced PH as an experimental model, found increased caspase 3 levels in PH animals. Our results also showed increased caspase 9 and 3 protein expression. The activation of the BAX and BAK proteins leads to the release of cytochrome c from the mitochondria which, in turn, activates caspase 9. Once activated, caspase 9 cleaves caspase 3 which initiates the DNA fragmentation phase and the apoptotic process (Tu et al. 2022). Both TH and GJ treatments were able to reduce caspase 9 protein expression, and TH administration was successful in diminishing caspase 3 in the PH, as our study showed. The hypotheses of the mechanisms of action of TH and GJ on the control of these apoptotic proteins involve the regulation of caspases gene expression and mitigation of the release of cytochrome c by free radicals, avoiding caspase activation (Zhang et al. 2022). Vince et al. (2018) showed that apoptotic protein, such as BAX and caspase 3 can be involved with NLRP3 inflammasome protein.
Indeed, inflammation plays a relevant role in PH-induced maladaptive ventricular remodelling (Zimmer et al. 2020). Moreover, Tang et al. (2015) demonstrated that monocrotaline-induced PH leads to the increased NLRP3 levels in their pulmonary tissue. Our results are according to Tang et al study, since we showed augmented NLRP3 protein expression in the PH group. Inflammatory and fibrotic processes may mediate NLRP3 activation in heart failure, which, in turn, reinforces the progression of cardiac dysfunction (Wang et al. 2020). In our study, only TH treatment was able to reduce NLRP3 protein expression, as observed in the PH+TH group. Ortiz et al. (2023), in the infarction model, showed that infarcted rats treated with TH had a reduction in NLRP3. Similarly, Vargas & Videla (2017) showed that increased NLRP3 levels in ischaemia-reperfusion hepatic model were reduced by T3 treatment. Therefore, TH had a more significant anti-inflammatory action in HP than GJ in our experimental model.
Limitations of this study
Although the MCT-induced PH model shows many conditions that mimic the characteristic signs of this disease in humans, it may feature some biases that could be circumvented by testing other experimental PH protocols. Histological evaluations of the RV could also be an approach to explore the dynamics of apoptosis and inflammation. Another limitation, it was the red ponceau as a load control in the Western blot. There are two justifications of this choice: the first is based on the alteration of the expression of housekeeping proteins (HKP) in conditions of the progression of hypertrophy to heart failure, a situation that has been brought to the agenda by the current literature (Colombe et al. 2022); the second is related to the interference that thyroid hormones exert in the structural proteins commonly used as load controls (Ortiz et al. 2023). The absence of previous levels of thyroid hormones is also a limitation of this study. However, using the same model, thyroid hormones levels have already been tested in Proença et al. (in press).
CONCLUSIONS
Even though further studies are necessary to explore the mechanisms involved in this cardioprotective effects of TH and GJ, our data pave the way to study the benefits and adverse effects of this therapeutic approach combining TH and GJ.
ACKNOWLEDGMENTS
Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). The authors thank the Universidade Federal do Rio Grande do Sul – UFRGS. This study was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brasília, DF, Brazil (grant number 301403/2018-0).
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