Ligustrazine alleviates pulmonary arterial hypertension in rats by promoting the formation of myocardin transcription complex in the nucleus of pulmonary artery smooth muscle cells

Abstract

Pulmonary arterial hypertension (PAH) is a pathophysiological state of abnormally elevated pulmonary arterial pressure caused by drugs, inflammation, toxins, viruses, hypoxia, and other risk factors. We studied the therapeutic effect and target of tetramethylpyrazine (tetramethylpyrazine [TMP]; ligustrazine) in the treatment of PAH and we speculated that dramatic changes in myocardin levels can significantly affect the progression of PAH. In vivo, the results showed that administration of TMP significantly prolonged the survival of PAH rats by reducing the proliferative lesions, right ventricular systolic pressure (RVSP), mean pulmonary arterial pressure (mPAP), and the Fulton index in the heart and lung of PAH rats. In vitro, TMP can regulate the levels of smooth muscle protein 22‐alpha (SM22‐α), and myocardin as well as intracellular cytokines such as NO, transforming growth factor beta (TGF‐β), and connective tissue growth factor (CTGF) in a dose‐dependent manner (25, 50, or 100 μM). Transfection of myocardin small interfering RNA (siRNA) aggravated the proliferation of pulmonary artery smooth muscle cells (PSMCs), and the regulatory effect of TMP on α‐smooth muscle actin (α‐SMA) and osteopontin (OPN) disappeared. The application of 10 nM estrogen receptor alpha (ERα) inhibitor MPP promoted the proliferation of PSMCs, but it does not affect the inhibition of TMP on PSMCs proliferation. Finally, we found that TMP promoted the nucleation of myocardin‐related transcription factor‐A (MRTF‐A) and combined it with myocardin. In conclusion, TMP can inhibit the transformation of PSMCs from the contractile phenotype to the proliferative phenotype by promoting the formation of the nuclear (MRTF‐A/myocardin) transcription complex to treat PAH.

STUDY HIGHLIGHTS.

• WHAT IS THE CURRENT KNOWLEDGE ON THE TOPIC?

The incurability of pulmonary arterial hypertension (PAH) leads to a sharp contradiction between prolonging patients' survival time, improving patients' quality of life, and relatively harsh surgical conditions. China is rich in natural medicinal resources; therefore, screening active substances among natural compounds is also an effective measure to find drugs for the treatment of PAH.

• WHAT QUESTION DID THIS STUDY ADDRESS?

In this study we first determined the inhibitory effect of the natural product tetramethylpyrazine (TMP; ligustrazine) on the proliferation of the pulmonary artery smooth muscle layer in PAH rats. Subsequently, we identified the targets of TMP in vivo and in vitro. Finally, we explored the potential mechanism by which TMP regulates α‐smooth muscle actin (α‐SMA) and osteopontin (OPN) in human pulmonary artery smooth muscle cells (PSMCs) with a proliferative phenotype.

• WHAT DOES THIS STUDY ADD TO OUR KNOWLEDGE?

Our study confirmed that the inhibition of TMP on the proliferation of vascular smooth muscle depends on the activation of myocardin in vivo and in vitro.

• HOW MIGHT THIS CHANGE CLINICAL PHARMACOLOGY OR TRANSLATIONAL SCIENCE?

In brief, our research, as a part of preclinical research, clarified the potential target and mechanism of TMP on PSMCs, and provided a preliminary research foundation for the clinical application of TMP. In addition, developing targeted drugs to activate myocardin is a potential means to treat PAH.

INTRODUCTION

Pulmonary arterial hypertension (PAH) is a pulmonary vascular disease with a very high rate of mortality and disability. ¹ PAH is difficult to cure, and the main purpose of drug therapy is to reduce the clinical response of patients and prolong their survival time. In recent years, it has been reported that the drugs screened from natural compounds can significantly improve many refractory diseases. Therefore, pharmaceutical researchers try to screen natural compounds with positive effects on the treatment of PAH. Epidemiology shows that female puberty and perimenopause aggravate PAH. ² One of the characteristics of these two stages is the instability of estrogen levels in the body. However, the mechanism of estrogen in PAH is still unclear. Some reports have confirmed that myocardin is a very powerful transcriptional coactivator and is specifically expressed in the cardiovascular system. ³ Myocardin, myocardin‐related transcription factor‐A (MRTF‐A), and MRTF‐B can all be associated with serum response factor (SRF) and participate in synergistic activation. ⁴ The cytoskeleton and the differentiated transcription regulatory element CARG box induce the gene expression of downstream α‐smooth muscle actin (α‐SMA), smooth muscle protein 22‐alpha (SM22‐α), and other targets. ⁵ However, whether estrogen regulates myocardin remains to be further studied. Unlike MRTF‐A and MRTF‐B in the cardiomyocyte protein family of transcriptional cofactors, myocardin only exists in the nucleus, and there is no clear report confirming its upstream target. Consequently, we wanted to confirm the regulatory effect of estrogen receptor alpha (Erα), which is the site of estrogen action, on myocardin. Then some research reported that MRTF‐A and MRTF‐B respond to Rho signaling and actin homeostasis, and by entering the nucleus from the cytoplasm they can directly cause the activation of SRF‐directed target genes, and it has been speculated that MRTF‐A and MRTF‐B nuclear translocation can indirectly regulate the binding ability of myocardin and SRF. Furthermore, no targets or drugs have been reported to play their role by promoting the nucleation of MRTF‐A/B and the combination of myocardin to form a transcription complex. ⁶

Vascular remodeling caused by the proliferation and hypertrophy of pulmonary artery smooth muscle cells (PSMCs) is the most common pathological feature of PAH patients. ⁷ The contraction and relaxation of PSMCs are influenced by both the heart and lung, so whether it is congenital physiological defects or acquired diseases of heart and lung, it will induce changes in the morphology and function of PSMCs. Generally, the differentiation/maturation state of vascular smooth muscle cells (VSMCs) is highly plastic. Complex factors in the local environment such as ischemia, hypoxia, the drug, and mechanical damage can affect the final state of VSMC phenotypes, and the transition from the differentiation/maturation state to the synthesis/proliferation state of VSMCs is called phenotypic conversion. ⁸ Classic research has confirmed that myocardin is a key cytokine that maintains the contractile phenotype of VSMCs, and it can regulate cell proliferation, migration, and myogenesis through synergy with MRTFs and SRFs. ⁹ In addition, researchers have been trying to find other targets and ways to regulate myocardin. For example, Yang et al.'s research has shown that the lack of acetaldehyde dehydrogenase 2 (ALDH2) can upregulate the mRNA level of myocardin, thereby significantly inhibiting the proliferation of VSMCs and preventing the occurrence of aortic aneurysm/dissection (AAD) in humans and mice. ¹⁰ A study led by Wen et al. showed that TEAD1 (TEA domain transcription factor 1) can promote the differentiation of VSMCs by activating myocardin and maintaining the normal development of the mouse heart and blood vessel wall. ¹¹ In this study we wanted to prove the effectiveness of the phenotypic transformation of PSMCs on PAH and identify the target of drugs on PSMCs.

Ligustrazine, also known as tetramethylpyrazine (TMP), is an alkaloid extracted from the Chinese herbal medicine Ligusticum chuanxiong Hort and food natto. ¹² In China, drugs containing TMP have been used in the treatment of cerebral thrombosis, coronary heart disease, vasculitis, and other occlusive vascular diseases. ¹³ In recent years, a number of meta‐analyses on the clinical efficacy of TMP in treating diabetic nephropathy, ischemic stroke, and stable angina pectoris have confirmed the therapeutic effect of TMP. ¹⁴ Preclinical studies have also shown that TMP and some of its derivatives have pharmacological activities, such as inhibiting the proliferation of colorectal cancer cells, inducing early apoptosis of HepG2 cells, ¹⁵ and inhibiting the contraction of vascular endothelial cells (VECs) and VSMCs. ¹⁶ In addition, recent clinical studies have shown that TMP can significantly extend the Six‐Minute Walk Test (6MWT) distance between patients with chronic thromboembolic pulmonary hypertension (CTEPH) and patients with PAH induced by other reasons. ¹⁷

In vitro, our results confirmed that TMP can inhibit the proliferation of PSMCs through the myocardin‐α‐SMA/OPN pathway. We used a mature PSMC in vitro proliferation model to evaluate the regulatory effect of ERα on myocardin and to further clarify that the mechanism by which TMP inhibits PSMCs is mediated by myocardin. In brief, the mechanism of TMP inhibiting the proliferation of PSMCs is to promote MRTF‐A to enter the nucleus and form a transcription complex with myocardin, to participate in regulating the transcription activity of downstream target genes.

MATERIALS AND METHODS

Animals and treatments

Female Sprague‐Dawley (SD) rats (200–220 g) were obtained from Nanjing Qinglong Mountain Animal Breeding Center (Nanjing, Jiangsu, China). All animal experiments were approved by the Animal Care and Use Committee of Nanjing University of Traditional Chinese Medicine (reference number: 2019AE01073) and were conducted in accordance with institutional guidelines. All SD rats were raised in the SPF animal room of the Animal Experiment Center of Nanjing University of Traditional Chinese Medicine. The indoor temperature is controlled at 22–26°C, and the humidity is lower than 50%. The light–dark cycle was 12 h, and drinking water and food were freely available. After the SD rats were adaptively fed for 1 week, they were randomly divided into three groups according to their body weight. The operation was performed according to the group, and postoperative observation was performed for 1 week. The estrus cycle of the rats was observed through vaginal smears. The rats were fed continuously for 6 weeks, including the Normal group (removal of the fat on both sides of the ovary only); Model group (Model‐1 group [monocrotaline (MCT), 50 mg·kg⁻¹·day⁻¹, i.h.]; Model‐2 group [removal of both ovaries; MCT, 50 mg·kg⁻¹·day⁻¹, i.h.]); Estrogen (E2) group (removal of both ovaries; MCT, 50 mg·kg⁻¹·day⁻¹, i.h.; E2, 10 mg·kg⁻¹·day⁻¹, i.g.); TMP‐L group (removal of both ovaries; MCT, 50 mg·kg⁻¹·day⁻¹, i.h.; TMP, 25 mg·kg⁻¹·day⁻¹, i.g.); and TMP‐M group (removal of both ovaries; MCT, 50 mg·kg⁻¹·day⁻¹, i.h.; TMP, 50 mg·kg⁻¹·day⁻¹, i.g.); TMP‐H group (removal of both ovaries; MCT, 25 mg·kg⁻¹·day⁻¹, i.h.; TMP, 100 mg·kg⁻¹·day⁻¹, i.g.). Intraperitoneal injection of pentobarbital sodium anesthesia (45 mg·kg⁻¹, 1 mL·100 g⁻¹) was performed to minimize pain. Rats were sacrificed by intraperitoneal injection of five times the anesthesia volume of sodium pentobarbital (45 mg·kg⁻¹, 5 mL·100 g⁻¹).

Hemodynamic testing

The rats in each group were weighed weekly, and their survival was recorded. According to the feeding status and survival of the rats, the two groups of rats were tested on the 56th and 42nd days of the feeding cycle. The anesthetized rat was fixed on a special experimental table, and the posture of the rat was adjusted. Then, the position of the catheter was adjusted according to the pressure value of the blood pressure waveform measured by the main pulmonary artery (MPA) cardiac function analysis system and the catheter entry length so that the catheter passed through the superior vena cava and right. The atrium, right ventricle, and pulmonary artery were used to measure the right ventricular systolic pressure (RVSP) and mean pulmonary arterial pressure (mPAP).

PSMC culture and model evaluation

PSMCs were obtained from Lianmai Biotechnology Co. Ltd. Then, 15% fetal bovine serum (FBS) and 1% penicillin/streptomycin DMEM high glucose medium were added and cultured in a cell incubator at 5% CO₂ and 37°C. To induce the proliferation of PSMCs, we incubated the cells with cobalt dichloride (CoCl₂, 100 μM) in DMEM high glucose medium containing 5% FBS for 24 h to induce the hypoxic proliferation model of PSMCs.

Statistical analysis

Student's t‐test was used for comparisons between the two groups. The log‐rank test was used to compare the survival rates of the two groups. Values of p < 0.05 were considered statistically significant. Data are expressed as the mean ± SD of at least three independent experiments.

RESULTS

TMP alleviated the process of PAH induced by MCT in female rats with ovaries removed

First, due to some studies having shown that a decrease in E2 level can reduce the incidence of PAH, in order to clarify the effect of E2 on PAH, we observed the difference between PAH rats induced by MCT (Model‐1) and PAH rats induced by ovariectomy and MCT (Model‐2). Then we observed the therapeutic effects of E2 and different doses of TMP on PAH. By drawing the Kaplan–Meier survival curve, we found that compared with Model‐1 group rats, the median survival time of Model‐2 group rats was further shortened to only 4 weeks, and all rats in the the Model‐2 group died in 8 weeks. By plotting the weight change trend of rats in each group, we found that after applying MCT, the weight of rats in each group showed a downward trend; and compared with the rats in the Normal group, the weight of the rats in the two Model groups were significantly reduced. The weights of ovariectomized PAH rats that were given TMP simultaneously recovered significantly (Figure 1a). After intragastric administration of TMP, the median survival time of ovariectomized PAH rats was extended to 8 weeks (Figure 1b).The results of hemodynamic testing showed that rats in the two Model groups had dangerous RVSP and mPAP levels, while the above two parameters were significantly reduced after the administration of TMP (Figure 1c–e). Not only that, compared with Normal group rats, the Fulton index of the two Model groups rats increased significantly, suggesting that ovariectomy and MCT aggravated the right ventricular hypertrophy of PAH rats, and the administration of TMP can significantly alleviate right ventricular hypertrophy and reduce the risk of PAH‐induced heart failure (Figure 1f). Similarly, by hematoxylin and eosin (HE) staining and Masson staining of lung tissue, we found that ovariectomy and MCT can lead to a significant increase in the thickness of the pulmonary artery wall and fibrosis grade of rats in the two Model groups. After simultaneous administration of TMP, the proliferation and fibrosis of pulmonary artery smooth muscle in PAH rats with ovaries removed were significantly alleviated (Figure 1g–i). First of all, we proved that ovariectomy will not improve the symptoms of PAH. We found that TMP improved PAH in Model rats in a dose‐dependent manner. However, the mechanism by which ovarian removal aggravates PAH and the target of TMP is not yet fully clear. Although exogenous E2 supplementation has improved the indexes of PAH rats, it is not clear whether its target is related to ERα. We speculated that the sharp drop in endogenous E2 leads to the inhibition of pulmonary artery smooth muscle ERα expression, thereby accelerating the process of PAH, but whether TMP plays a role by activating ERα remains to be further explored.

FIGURE 1.

The therapeutic effect of tetramethylpyrazine (TMP; ligustrazine) on monocrotaline (MCT)‐treated ovariectomized female pulmonary arterial hypertension (PAH) rats. (a) Kaplan–Meier survival curve of each group of rats in the experimental period (n = 10). #p < 0.05 vs. Normal group, *p < 0.05 vs. Model group, obtained by log‐rank test. (b) The weight change trend of SD rats in each group during the experimental period (n = 5–10). Data are expressed as the mean ± SD, #p < 0.05 vs. Normal group, *p < 0.05 vs. Model group, obtained by Student's t‐test. (c) After carotid artery intubation, the right ventricular systolic pressure (RVSP, red line) and mean pulmonary artery pressure (mPAP, blue line) are representative images of each group of rats fed back by the hemodynamic detection system. (d, e) Nondiscrimination is the statistics of RVSP and mPAP of rats in each group (n = 5). The data are expressed as the mean ± SEM, and the p value was obtained by t‐test. (f) Statistics of the [right ventricle/(left ventricle + ventricular septum)] ratio (Fulton index) of each group of rats (n = 5). The data are expressed as the mean ± SD, and the p value was obtained by Student's t‐test. (g) Representative images of hematoxylin and eosin (HE) staining (1 row, scale bar = 100 μm; 2 rows, scale bar = 50 μm) and Masson staining (3 rows, scale bar = 100 μm; 4 rows, scale bar = 50 μm) of rat lung tissue in each group (n = 5). (h, i) The statistics of the thickness of the pulmonary artery wall and the proportion of pulmonary artery fibrosis in each group of rats (n = 5). The data are expressed as the mean ± SD, and the p value was obtained according to Student's t‐test.

TMP improved myocardin expression in the pulmonary artery smooth muscle of female PAH rats

To clarify the target of TMP in PAH rats, we observed the representative protein expression as α‐SMA, OPN, smoothelin, SM22‐α, ERα, and myocardin in the smooth muscle of the pulmonary artery by Western blotting (WB) and immunohistochemistry (IHC) (Figure 2a–d). The final results showed that the protein expression of α‐SMA, smoothelin, ERα, and myocardin in the pulmonary artery smooth muscle of Normal group rats was significantly inhibited, and the protein expression of OPN was significantly upregulated. Compared with the Normal group rats, the protein expression of α‐SMA, smoothelin, ERα, and myocardin in the pulmonary artery smooth muscle of rats in the two Model groups was reduced, and the protein expression of OPN and SM22‐α was increased. The application of TMP significantly upregulated the protein expression of α‐SMA, smoothelin, and myocardin in the pulmonary artery smooth muscle of two groups of PAH rats, while it significantly downregulated the protein expression of OPN and SM22‐α but had no significant effect on the protein expression of ERα (Figure 2a–d). Based on the above results, we confirmed that ERα directly participates in the transformation of rat pulmonary artery smooth muscle from a contraction phenotype to a proliferation phenotype, and inhibition of ERα can lead to abnormal proliferation of pulmonary artery smooth muscle. However, it is not clear whether ERα is involved in the regulation of myocardin. In addition, although TMP did not directly activate ERα, it can act by upregulating myocardin and other targets involved in smooth muscle phenotype transformation. We speculated that TMP can directly activate the protein expression of myocardin to affect the protein expression of α‐SMA, OPN, smoothelin, and SM22‐α, and ultimately inhibit the proliferation of pulmonary artery smooth muscle in ovariectomized PAH rats.

FIGURE 2.

The intervention effect of tetramethylpyrazine (TMP; ligustrazine) on the targets involved in the phenotypic transformation of pulmonary artery smooth muscle in pulmonary arterial hypertension (PAH) rats. (a) Representative images of Western blot of α‐smooth muscle actin (α‐SMA), osteopontin (OPN), smoothelin, smooth muscle protein 22‐alpha (SM22‐α), estrogen receptor alpha (ERα), and myocardin in the pulmonary arteries of rats in the Normal, Model, E2, TMP‐L, TMP‐M, and TMP‐H groups (n = 3). (b) Representative images of immunohistochemical staining of α‐SMA, OPN, smoothelin, SM22‐α, ERα, and myocardin in the pulmonary arteries of rats in the Normal, Model, E2, TMP‐L, TMP‐M, and TMP‐H groups (n = 5). Scale bar = 20 μm. (b) Statistics of the Integrated optical density (IOD) of the abovementioned protein immunohistochemical active area in the pulmonary artery of each group of rats (n = 5). (C) Statistics of the protein expression of the abovementioned protein immunohistochemical active area in the pulmonary artery of each group of rats (n = 3). (D) Statistics of the IOD of the abovementioned protein immunohistochemical active area in the pulmonary artery of each group of rats (n = 5). The data are expressed as the mean ± SD, and the p value was obtained by Student's t‐test.

TMP inhibited the abnormal proliferation of human PSMCs induced by cobalt dichloride

We hoped to confirm that TMP can also inhibit the abnormal proliferation of human PSMCs and explore the mode of action and target of TMP through in vitro experiments. Before that, we first tested the effect of TMP on cell viability. PSMCs were treated with TMP at a concentration ranging from 0 to 400 μM for 24 h. The 3‐(4:5‐dimethylthiazol‐2‐yl)‐2:5‐diphenyltetrazolium bromide (MTT) results showed that when the concentration was lower than 100 μM, TMP did not have a significant impact on the cell survival rate of PSMCs (Figure 3a,b). Based on these findings, the concentration of TMP used in subsequent experiments was not higher than 100 μM. Subsequently, we performed HE staining of PSMCs induced by 50 μM cobalt dichloride (CoCl₂) for 24 h. The results showed that the PSMC cells induced by cobalt dichloride were densely arranged, with increased cell volume and number (Figure 3c). The results of immunofluorescence staining (IF) showed that the protein expression of α‐SMA in the Model group cells was significantly reduced, and the protein expression of OPN was significantly increased (Figure 3d,e). The aforementioned results suggested the successful construction of a PSMC proliferation model. Next, we added different concentrations of TMP (0, 25, 50, or 100 μM) and 50 μM CoCl2 to PSMCs and incubated them for 24 h. We found that TMP reduced the number of cells in the field of view in a dose‐dependent manner and made some PSMCs present a contracted morphology (Figure 3f). In addition, TMP significantly reduced the levels of proliferation‐promoting cytokines such as transforming growth factor beta (TGF‐β) and connective tissue growth factor (CTGF) in PSMCs induced by CoCl₂ and increased the level of the cytokine NO, which inhibits proliferation (Figure 3g–i). Finally, consistent with the trend we observed in the SD + MCT group in the overall experiment, the WB results showed that the application of CoCl₂ led to a significant downregulation of the protein expression of α‐SMA, smoothelin, ERα, and myocardin in PSMCs, while the protein expression of OPN and SM22‐α was significantly upregulated. TMP has a significant activating effect on the expression of α‐SMA, smoothelin, and myocardin proteins in cells and can significantly inhibit the protein expression of OPN but has no significant effect on ERα. In conclusion, we confirmed that TMP also has a significant inhibitory effect on the proliferation of PSMCs in vitro, and the effect on the protein expression of ERα and myocardin was consistent with the results in vivo.

FIGURE 3.

The inhibitory effect of tetramethylpyrazine (TMP; ligustrazine) on cells in the human pulmonary artery smooth muscle cell (PSMC) proliferation model induced by cobalt dichloride (CoCl₂). (a) The 3‐(4:5‐dimethylthiazol‐2‐yl)‐2:5‐diphenyltetrazolium bromide (MTT) method shows the survival rate of PSMCs after applying different concentrations (0, 1, 10, 100, 200, and 400 μM) of TMP (n = 6). (b) The MTT method shows the survival rate of PSMCs (n = 6) after applying different concentrations (0, 25, 50 and, 100 μM TMP). (c) Representative images of hematoxylin and eosin (HE) staining of PSMCs in the control group and proliferation model group (50 μM CoCl₂ 24 h) (n = 5). (d, e) Representative images of α‐smooth muscle actin (α‐SMA) and osteopontin (OPN) protein immunofluorescence in the PSMCs of the control group and the proliferation model group and the statistics of the active area Integrated optical density (IOD) (n = 5). (f) Representative images of HE staining of PSMCs in the control group and each proliferation model administration group (0, 25, 50, and 100 μM TMP) (n = 5). (g–i) These are the relative concentrations of NO, transforming growth factor beta (TGF‐β), and connective tissue growth factor (CTGF) in each group of PSMCs. (j–k) Representative Western blot images and quantitative statistics of α‐SMA, OPN, smoothelin, smooth muscle protein 22‐alpha (SM22‐α), estrogen receptor alpha (ERα), and myocardin in each group of PSMCs (n = 5). The data are expressed as the mean ± SD, and the p value was obtained by Student's t‐test.

Inhibitory effect of TMP on the transformation of PSMCs to a proliferative phenotype depends on the activation of myocardin

We confirmed that the protein expression of myocardin was significantly reduced under proliferative conditions in vitro and vivo and that TMP can upregulate the protein expression of myocardin. However, it is not clear whether the ability of TMP to inhibit the proliferation of PSMCs depends on myocardin activation. To further clarify whether TMP inhibits the transformation of PSMCs from the contractile phenotype to the proliferative type mediated by myocardin, we verified this in PSMCs transfected with myocardin small interfering RNA (siRNA). The results of HE staining showed that the inhibition of myocardin aggravated the abnormal proliferation of PSMCs and reversed the improvement effect of TMP on the number and morphology of PSMCs (Figure 4a). WB and reverse transcription‐polymerase chain reaction (RT‐PCR) results showed that myocardin siRNA transfection led to a significant reduction in the levels of α‐SMA, smoothelin, and myocardin protein and mRNA in PSMCs with a CoCl₂‐induced proliferation phenotype, and OPN and SM22‐α protein and mRNA levels significantly increased. In addition, we found that the inhibition of myocardin had no significant effect on ERα protein and mRNA, and TMP‐mediated regulation of α‐SMA and OPN protein and mRNA was significantly inhibited in PSMCs transfected with myocardin siRNA (Figure 4b–d). Then, we further observed the position and expression of α‐SMA, OPN, Erα, and myocardin in PSMCs through IF. The results were consistent with the WB and RT‐PCR results (Figure 4e–i). The aforementioned results indicated that TMP can inhibit human PSMCs from the contraction phenotype to the proliferation phenotype by activating myocardin to adjust α‐SMA/OPN.

FIGURE 4.

Myocardin participates in the regulation of tetramethylpyrazine (TMP; ligustrazine) on the inhibition of the proliferation of human pulmonary artery smooth muscle cells (PSMCs) induced by cobalt dichloride (CoCl₂). (a) Representative images of hematoxylin and eosin (HE) staining of PSMCs in each group before and after the application of myocardin small interfering RNA (siRNA) (n = 5). (b, c) Representative Western blot images and quantitative statistics of α‐smooth muscle actin (α‐SMA), osteopontin (OPN), smoothelin, smooth muscle protein 22‐alpha (SM22‐α), estrogen receptor alpha (ERα), and myocardin in each group of PSMCs before and after the application of myocardin siRNA (n = 3). (d) Quantitative statistics of the relative mRNA content of α‐SMA, OPN, smoothelin, SM22‐α, ERα, and myocardin in PSMCs of each group before and after myocardin siRNA application (n = 3). (e–i) Representative images of immunofluorescence of α‐SMA, OPN, ERα, and myocardin proteins in PSMCs before and after myocardin siRNA application and statistics of active area Integrated optical density (IOD) (n = 3). The data are expressed as the mean ± SD, and the p value was obtained by Student's t‐test.

TMP regulates the myocardin‐α‐SMA/OPN pathway by stimulating MRTF‐A to enter the nucleus in a way independent of ERα

Similar to the role played by myocardin in maintaining the phenotype of VSMCs, some studies have confirmed that activated ERα can inhibit the proliferation of VSMCs under pathological conditions. Next, we explored whether the regulation of PSMC phenotypic transformation by ERα in the proliferative state depends on the activation of myocardin. We first investigated the effects of the CoCl₂‐induced PSMC proliferation model on the number and morphology of cells in human PSMCs under the condition that ERα is inhibited. Consistent with previous reports, the results of HE staining showed that administration of MPP, a specific inhibitor of ERα, can aggravate the proliferation of PSMCs induced by CoCl₂. Simultaneous administration of TMP significantly reduced the number of cells, restored cell morphology, and inhibited the proliferation of PSMCs (Figure 5a). Therefore, we have determined that TMP can inhibit the proliferation of human PSMCs under the condition of inhibited ERα activity, which is also consistent with the results of animal experiments. Subsequently, the results of WB and reverse transcription‐quantitative real‐time PCR (RT‐qPCR) showed that the administration of MPP significantly inhibited the protein and mRNA expression of myocardin, α‐SMA, and smoothelin in PSMCs. Although the protein and mRNA expression levels of ERα, OPN, smoothelin, and SM22‐α were not significantly changed after the simultaneous administration of TMP, the protein and mRNA expression levels of α‐SMA and myocardin were significantly restored (Figure 5b–d). We separated the proteins in the nucleus and cytoplasm of cells in the blank group and the administration group and carried out Co‐Immunoprecipitation with MRTF‐A antibody and myocardin antibody, respectively. The results showed that TMP could promote MRTF‐A to enter the nucleus and combine with myocardin to form a transcription complex. As MRTF‐A/B and myocardin can participate in the regulation of downstream targets in the form of the transcription complex, we think that the pharmacological activity of TMP is mediated by this pathway.

FIGURE 5.

Tetramethylpyrazine (TMP; ligustrazine) promotes myocardin‐related transcription factor‐A (MRTF‐A) to enter the nucleus to form a complex with myocardin. (a) Representative images of hematoxylin and eosin (HE) staining of pulmonary artery smooth muscle cells (PSMCs) in each group before and after the application of 10 nM of the estrogen receptor alpha (ERrα) activity inhibitor 3‐(4:5‐dimethylthiazol‐2‐yl)‐2:5‐diphenyltetrazolium bromide (MPP) (n = 5). (b, c) Representative Western blot images and quantitative statistics of α‐smooth muscle actin (α‐SMA), osteopontin (OPN), smoothelin, smooth muscle protein 22‐alpha (SM22‐α), ERα, and myocardin in each group of PSMCs before and after the application of MPP (n = 3). (d) Quantitative statistics of the relative mRNA content of α‐SMA, OPN, smoothelin, SM22‐α, ERα, and myocardin in each group of PSMCs before and after the application of MPP (n = 3). (e) Combination of MRTF‐A and myocardin in nucleus and cytoplasm before TMP application. (f) Combination of MRTF‐A and myocardin in nucleus and cytoplasm after TMP application.

DISCUSSION

The pulmonary artery is responsible for transporting the venous blood pumped out of the right ventricle into the lung and plays a key role in blood circulation. ¹⁸ PSMCs in the pulmonary artery will proliferate and suppress apoptosis, ² resulting in blockage of the pulmonary artery lumen and an increase in pressure in the lumen, eventually resulting in cardiac, lung, and systemic vascular dysfunction. ¹⁹ In addition, PAH is considered related to connective tissue disease, HIV infection, portal hypertension, congenital heart disease, schistosomiasis pulmonary venous‐occlusive disease, pulmonary capillary haemangiomatosis, and congenital factors. ²⁰ Previous studies have confirmed that MCT has the effect of inducing pulmonary vascular inflammation in rats, and the pathological features are similar to PAH caused by hypoxia. Consequently, we used MCT to induce PAH in rats.

A forecast from Australia on the treatment cost of patients with systemic sclerosis‐related pulmonary hypertension showed that the average annual simulated cost per patient in the monotherapy and combination treatment groups was US$16,080 and US$19,982, respectively. ²¹ Such expenses are unacceptable for low‐income individuals. China is rich in natural medicinal resources; therefore, screening active substances among natural compounds is also an effective measure to find drugs for the treatment of PAH. Studies have confirmed that TMP inhibits the proliferation of vascular smooth muscle. ²² TMP preparations have been widely used in the adjuvant treatment of various cardiovascular and cerebrovascular diseases in China. ¹² , ²³ Before clarifying whether ERα can regulate myocardin, we hoped to confirm the therapeutic effect of TMP in PAH rats. Our research confirmed for the first time in PAH rats that TMP improved the symptoms of PAH by inhibiting the proliferation of PSMCs and provided a basis for clinical use of TMP to treat PAH.

Some results also suggested a potential correlation between myocardin and ERα. Li et al. confirmed that steroid receptor coactivator 3 (SRC3) can act as a transcriptional coactivator of ERα and myocardin in VSMCs through a pull‐down test, suggesting the possibility of ERα regulating myocardin. ⁴ In previous studies, Meng et al. ²⁴ confirmed that the protein levels of ERα and myocardin in the aortic VSMCs of atherosclerotic ApoE^−/− mice are positively correlated, but it is not clear whether myocardin is directly regulated by ERα in PSMCs. ERα is a receptor located in the nucleus, corresponding to changes in estrogen levels, affecting the state of the human cardiovascular system, bones, and gonads. ²⁵ In the absence of ligands, ERα is located in a heat shock protein complex in the nucleus. After binding to estrogen, ERα undergoes a conformational change, depolymerizes with heat shock proteins, and promotes the interaction of receptor dimers in the regulatory regions of target genes. The interaction of ERα with target gene promoters can occur directly through specific estrogen response elements (EREs), or indirectly through exposure to other DNA‐binding transcription factors to affect target genes at the transcriptional level. ²⁶

The results of echocardiography performed on 3381 volunteers from Rotterdam, The Netherlands showed that the prevalence of PAH among people aged 65–70 years was 0.8%, and the prevalence of PAH among people aged over 85 years was as high as 8.3%. ¹⁹ In addition, a study from Sweden showed that the 5‐year survival rate of patients with idiopathic pulmonary hypertension (IPAH) in the 18–45 years age group was as high as 88%, while the survival rates dropped by 25%, 32%, and 52% in the 46–64, 65–74 and ≥75 years age groups, respectively. ²⁷ These results indicated that aging is an independent risk factor for the onset and poor prognosis of PAH. Menopause is a sign of female physiological aging. ²⁸ With the decline in ovarian function, the level of endogenous estrogen (E2) in the body decreases sharply, ²⁵ and the anti‐inflammatory, antiproliferative, and lipid metabolism‐mediated effects of estrogen receptor alpha (ERα) are gradually lost after menopause. ²⁹ There is no dispute that postmenopausal women have a significant increase in the probability of cardiovascular diseases such as coronary heart disease, hyperlipidemia, and atherosclerosis. ³⁰ Although clinical studies have shown that higher estrogen levels are positively correlated with the risk and severity of PAH in postmenopausal women, there is no direct evidence that inhibition of ERα can reduce the incidence of PAH in postmenopausal women. In this study, we induced a low expression model of ERα at the animal and cellular levels successfully by removing the ovaries of rats or administering the Erα‐specific inhibitor, MPP. First, we found that the protein expression of myocardin was abnormally decreased in the pulmonary artery smooth muscle of MCT‐PAH rats, and the expression of the corresponding proliferation marker protein and matrix protein showed the characteristics of a proliferation phenotype. Subsequently, we found that the protein expression of myocardin was further reduced by the removal of the ovary. However, the regulatory relationship between ERα and myocardin is still unclear. We interfered the protein expression of myocardin by transfecting myocardin siRNA, and found no significant changes in the mRNA and protein levels of ERα. Therefore, we judged that ERα is not the target of myocardin. Then we confirmed that the inhibition of ERα protein activity can significantly downregulate the protein expression of myocardin. We speculate that ERα is involved in the regulation of the downstream target myocardin in the form of an upstream transcription complex.

As regards whether ERα plays a positive or negative role in PAH, it seems difficult for us to give a clear conclusion. On the one hand, some reports have shown that activation of ERα directly upregulates the proliferation and migration rate of VECs and VSMCs, ³¹ and some evidence has shown that the prevalence of PAH in young women is significantly higher than that in men of the same age. ² , ³² On the other hand, our results showed that the reduction in ERα protein expression caused by ovarian removal undoubtedly aggravated the condition of PAH rats, and the smooth muscle of the Model group rats showed a more pronounced proliferative phenotype. It seems that we should conclude that inhibition of ERα promotes the proliferation of PSMCs. We speculated that maintaining the normal expression level of ERα is an important way to maintain the phenotype of PSMCs, and the role of ERα is different in the proliferation models of different induction conditions. Although some researchers believe that ERα regulates the phenotype of VSMCs differently in rodents and humans, the results of our in vivo and in vitro experiments at least confirmed that the protein expression trends of ERα and myocardin were synchronized. This confirmed the positive regulation of myocardin by ERα. In conclusion, although ERα cannot be used as a direct target for the treatment of PAH, its important role in the phenotypic transformation of VSMCs cannot be ignored. Our results clarified the positive effect of the ERα‐myocardin pathway on inhibiting the excessive proliferation of PSMCs. At the same time, we confirmed that the activation of the myocardin‐α‐SMA/OPN pathway by TMP inhibits the transformation of PSMCs from a contractile phenotype to a proliferative phenotype. Although we have clarified the regulatory effect of ERα on myocardin, the specific regulatory sites and mechanisms still need to be further explored and we aim to elucidate the binding site where TMP acts on myocardin in future studies.

AUTHOR CONTRIBUTIONS

X.Y., Y.L., and X.Q. wrote the manuscript, designed the research, performed the research, and analyzed the data. M.W., Q.M., and W.Z. designed the research, performed the research, and analyzed the data. C.Z., B.L., Y.Q., S.G., X.W., and J.W. analyzed the data.

FUNDING INFORMATION

This work was sponsored by the General Project of Jiangsu Provincial Health Commission (H2019029); Jiangsu province ‘Six One Project’ (LGY2018054); ‘Six talent peaks’ high‐level talents level B (WSN‐015); 333 High‐level personnel Training Program.

CONFLICT OF INTEREST STATEMENT

The authors declared no competing interests for this work.

Supporting information

Data S1:

Yu X, Wu M, Meng Q, et al. Ligustrazine alleviates pulmonary arterial hypertension in rats by promoting the formation of myocardin transcription complex in the nucleus of pulmonary artery smooth muscle cells. Clin Transl Sci. 2023;16:1369‐1380. doi: 10.1111/cts.13534