Tongxinluo enhances the effect of atorvastatin on the treatment of atherosclerosis with chronic obstructive pulmonary disease by maintaining the pulmonary microvascular barrier

Abstract Atherosclerosis (AS) is a common comorbidity of chronic obstructive pulmonary disease (COPD), and systemic inflammation is an important mechanism of COPD with AS. Tongxinluo (TXL) improves the function of vascular endothelial cells. We aimed to prove that impairment of pulmonary microvascular barrier function is involved in COPD‐mediated aggravation of AS and investigate whether TXL enhances the effect of Ato (atorvastatin) on COPD with AS by protecting pulmonary microvascular endothelial barrier function. In vivo, a COPD with atherosclerotic apolipoprotein E knockout (AS ApoE−/−) mouse model was established by cigarette smoke combined with a high‐fat diet. The animals were administered TXL, Ato, and TXL + Ato once a day for 20 weeks. Lung function, lung microvascular permeability, lung inflammation, systemic inflammation, serum lipid levels, atheromatous plaque formation, and endothelial damage biomarkers were measured. In vitro, human pulmonary microvascular endothelial cells (HPMECs) were pretreated with TXL and incubated with cigarette smoke extract to establish the model. The permeability of the endothelial monolayer, inflammatory cytokines, endothelial damage biomarkers, and tight junction (Tj) proteins were determined. Cigarette smoking significantly exacerbated the high‐fat diet‐induced pulmonary function decline, pulmonary microvascular endothelial barrier dysfunction, inflammation, and atherosclerotic plaques. These changes were reversed by TXL–Ato; the combination was more effective than Ato alone. Furthermore, TXL protected the HPMEC barrier and inhibited inflammation in HPMECs. COPD aggravates AS, possibly through the destruction of pulmonary microvascular barrier function; thus, lung inflammation triggers systemic inflammation. In treating COPD with AS, TXL enhances the antiatherosclerotic effect of Ato, protecting the pulmonary microvascular barrier.


| INTRODUC TI ON
Chronic obstructive pulmonary disease (COPD) is characterized by an incomplete reversible airflow restriction, which gradually worsens and is associated with abnormal inflammatory responses to harmful gases or particles in the lungs, such as smoke from tobacco and biomass combustion (GOLD, 2019;Labaki & Rosenberg, 2020).
Over 300 million people suffer from COPD. Currently, this condition is the fourth leading cause of death worldwide and is predicted to be ranked third by 2030 (WHO, 2010). In addition to pulmonary symptoms, COPD is accompanied by extrapulmonary comorbidities, such as cardiovascular disease (CVD), metabolic syndrome, and diabetes (Choudhury et al., 2014;Wenjia et al., 2015). CVD is a common comorbidity and a major cause of death in patients with COPD (Curkendall et al., 2006;Huiart et al., 2005), indicating their epidemiological link (Eriksson et al., 2013;Mullerova et al., 2013).
Accumulating evidence has shown that systemic inflammation is associated with COPD, which may underlie the increased risk of COPD combined with atherosclerosis (AS; Fabbri et al., 2008;Fuschillo et al., 2012;Roversi et al., 2014;Sin & Man, 2003).
Previous studies have shown that increased pulmonary microvascular permeability may be a key factor for airway obstruction in patients with COPD (Kyomoto et al., 2019). The increased permeability of pulmonary microvascular endothelial cells (PMVECs) causes neutrophil accumulation and subsequent activation in the lungs.
These cytokines stimulate additional inflammatory cells and facilitate the release of inflammatory mediators, which in turn trigger inflammatory cascades and amplify injury signals to cause systemic inflammation. Importantly, systemic inflammation plays a crucial role in the pathogenesis of AS (Baumer et al., 2018). Therefore, maintaining the integrity of the pulmonary microvascular endothelial barrier and preventing pulmonary inflammation from leading to systemic inflammation are key factors in the treatment of COPD combined with AS. However, the role of pulmonary microvascular barrier function in the progression of COPD with AS has not yet been elucidated due to the lack of relevant studies. The treatment regimen for patients with COPD combined with AS involves the treatment of symptoms for a single disease, including inhalation of bronchodilators and antiinflammatory therapy for COPD (Halpin David et al., 2021), lipidlowering treatment (for example, statins), antihypertensive agents, or β-blockers for AS (Baigent et al., 2005). The symptoms of both diseases are treated as independent modalities, and the interactions between COPD and AS are not considered. Therefore, new treatment regimens for COPD combined with AS are required. Tongxinluo (TXL) is a Chinese herbal compound that has been widely used in the treatment of CVDs and cerebrovascular diseases in China (Chen et al., 2009;Zhang et al., 2009). TXL is composed ofRadix ginseng, Buthus martensii, Hirudo, Eupolyphaga seu steleophaga, Scolopendra subspinipes, Periostracum cicadae, Radix paeoniae rubra, Semen ziziphi spinosae, Lignum dalbergia odoriferae, Lignum santali albi, and Borneolum syntheticum (Chang et al., 2017). To date, approximately 6 million patients with CVDs and cerebrovascular diseases have been treated with TXL due to its beneficial effects against vascular diseases. Our previous studies showed that TXL not only relieves the symptoms of chest tightness and chest pain and significantly inhibits oxidized low-density lipoprotein (ox-LDL)induced apoptosis of macrophages by enhancing autophagy , but also alleviates the lung symptoms of dyspnea, significantly increasing the 6-min walk distance (6MWD) and the level of the lung marker Clara cell secretory protein-16 (CC-16;Liu et al., 2019) in patients with COPD with CVD. Previous studies have shown that TXL protects human cardiac microvascular endothelial cells from hypoxia/reoxygenation injury and alleviates cerebral microcirculatory disturbances against ischemic injury.
Furthermore, TXL improved the renal structure and function of individuals with diabetic nephropathy Liu et al., 2018;Wang et al., 2014). TXL-mediated improvement in microvascular blood flow perfusion is a common mechanism in the treatment of major diseases of the heart and brain and diabetic nephropathy, and its target is mainly microvascular endothelial cells. However, the role of TXL in PMECs has not been reported. The antiatherosclerotic effects and concomitant lipid-lowering effects of atorvastatin (Ato) have been demonstrated in previous studies (Meredith et al., 2005).
The present study aimed to determine whether the key mechanism of COPD exacerbation of AS is pulmonary microvascular barrier dysfunction, whether lung inflammation triggers systemic inflammation, and whether TXL can enhance the antiatherosclerotic effect of Ato in COPD combined with AS through pulmonary microvascular barrier protection.

| Animals and treatments
ApoE −/− mice with a C57BL/6N background were used as an animal model of human AS. Knockout of the ApoE allele results in hypercholesterolemia, increasing the susceptibility of the mice to AS when they are fed a high-fat diet. To eliminate the effect of a high-fat diet on the mouse lungs, we used C57BL/6N mice without spontaneous AS in this study. All mice were housed in an animal center under a 12-h light-dark cycle for 7 days to acclimatize to the environment prior to experimentation. The animals had free access to a standard rodent diet and water at all times. According to preliminary experiments, the dose of TXL was determined to be 1.5 g/kg/day (approximately 2 times the clinically equivalent dose; , and that of Ato was 10 mg/kg/day (Han et al., 2016). As previously described (Arunachalam et al., 2010;Florence et al., 2018), the smoke was pumped into a plastic box, measuring 42 cm (length) × 28 cm (width) × 27 cm (height), containing animals that passively inhaled the cigarette smoke. AS was induced in ApoE −/− mice fed a high-fat diet (SCXK2019-0003; Beijing Keao Xieli Feed Co., Ltd.). The detailed grouping and treatment of the animals are shown in Table 1.

| Preparation of TXL
Tongxinluo ultrafine powder (Shijiazhuang Yiling Pharmaceutical Co.) was dissolved in 0.5% sodium carboxymethyl cellulose (CMC) solution and stirred with a magnetic stirrer for 1 h. The suspension was intragastrically administered to the mice daily at a dose of 1.5 g/ kg (volume: 0.1 ml/10 g) per day by oral gavage.

| Mouse pulmonary functions
Mice were anesthetized with 50 mg/kg sodium pentobarbital (Sigma-Aldrich) via intraperitoneal injection and then tracheostomized and placed in a forced pulmonary maneuver system (Buxco Research Systems). For determination of the lung function of mice, Boyle's low functional residual capacity (FRC) was performed using the Buxco system (Vanoirbeek et al., 2010). The FRC was determined using Boyle's law FRC maneuver; the dynamic compliance (cdyn) was acquired by the quasistatic pressure-volume (PV) maneuver; and the forced vital capacity (FVC), forced expiratory volume (FEV) at 50 ms (FEV 50 ), and inspiratory resistances (RI) were recorded with the fast flow volume maneuver. Each maneuver was repeated at least three times. After the experiment, the mice were deeply anesthetized and decapitated.

| Lung microvascular permeability
Lung microvascular permeability was determined by the Evans blue dye method (Lv et al., 2020). Briefly, 50 μl of Evans blue dye (500 μg/50 μl phosphate-buffered saline [PBS]) per 20 mg/kg body weight was injected intravenously (iv, dorsal tail vein). Two hours after the Evans blue injection, the animals were anesthetized with sodium pentobarbital and perfused with 10 ml of 0.9% sodium chloride solution to remove the blood. Then, the lung tissue was weighed, incubated, and homogenized with formamide (Sigma-Aldrich) for 48 h at 37°C. The concentration of the dye (μg/mg tissue) was determined in the supernatant using a spectrophotometer at 620 nm against a standard curve.

| Tissue collection and morphological analysis
At the end of pulmonary function experiments, mice were sacrificed under deep anesthesia and decapitated to ensure death, and tissues were collected immediately. The mouse left lung without lavage and the total length of aortic arch were fixed in 4% phosphate-buffered paraformaldehyde (pH 7.4), embedded in paraffin, and sliced into 8μm-thick sections that were stained with hematoxylin and eosin (HE) solution. Alveolar enlargement was determined by the average linear intercept and the ratio of the total length of alveoli to the number of alveoli per field. Histological analysis was performed in 10 visual fields per mouse, and the average value of each mouse was calculated.

| Transmission electron microscopy
Fresh left upper lung tissues (1 × 1 × 1 mm) were collected for electron microscopy. The specimen was fixed in 2.5% glutaraldehyde (Sigma-Aldrich) and phosphate buffer. The specimen was then rinsed in phosphate buffer and postfixed with 1% osmic tetroxide (Sigma-Aldrich) in phosphate buffer. After graded dehydration in ethyl alcohol and propylene oxide, the specimen was embedded in Spurr resin.
Then, the embedded tissue was thin-sectioned, mounted on copper grids, and stained with uranyl acetate and lead citrate. Images were taken by electron microscopy (H-7700; Hitachi).

| Serum lipid analysis
Mice were fasted overnight, blood was taken from the retrobulbar venous plexus under deep anesthesia, and mice were sacrificed by decapitation. Serum total cholesterol (TC), triglyceride (TG), highdensity lipoprotein (HDL), and low-density lipoprotein (LDL) levels were determined using enzymatic kits from Jiuqiang Biotechnology Co., Ltd. according to the manufacturer's protocols.

| Preparation of CSE
Cigarette smoke extract (CSE) was generated as described previously (Lv et al., 2016). A syringe-driven apparatus device was designed and operated to allow a stream of smoke to flow into a tube-shaped trap. The smoke then entered a flask submerged in liquid nitrogen.
The amount of smoke obtained was calculated by the increase in the weight inside the flask. The smoke particulates were collected in dimethylsulfoxide (DMSO) at a concentration of 40 mg/ml, filtered through a 0.22μm pore filter, and stored at −80°C for subsequent use.
Preliminary experiments confirmed that DMSO had no effect on cell proliferation by comparing the DMSO versus no DMSO conditions.
HPMECs (1 × 10 5 cells/well) were plated in Transwell chambers with 500 and 1500 μl culture media in the upper and lower compartments,

| Enzyme-linked immunosorbent assays for inflammatory cytokines
Lungs were homogenized in a saline solution (0.9% NaCl), and the mouse serum and the cell culture supernatants were collected via centrifugation (159.84 g, 10 min, 4°C) to remove cell debris and stored at −80°C until subsequent analyses. The levels of inflammatory cytokines were quantified using enzyme-linked immunosorbent assays (ELISAs

| Western blotting analysis
The protein concentration of mouse lung tissue and HPMECs was determined using a bicinchoninic acid (BCA) protein assay kit before sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; GenScript). Then, the proteins were transferred to nitrocellulose blotting membranes (Life Sciences).

| Statistical analysis
Statistical significance of differences was assessed using one-way analysis of variance (ANOVA) or repeated-measures analysis, followed by post hoc analysis using Fisher's least significant difference (LSD) multiple comparison test. Differences at p < .05 were regarded as statistically significant. Data are presented as the mean ± SEM.  These data indicated that cigarette smoke exposure reduced lung function induced by a high-fat diet in mice. The TXL and Ato combination improved lung function compared with either drug alone.  Figure 2e). In the control group, the structure of the air-blood barrier was complete, the endothelial cells showed slight edema, the Tjs were dense, and the BM was smooth. The capillary En of the COPD + AS group had moderate edema, the cell membrane was incomplete, the Tjs between the cells were blurred and nearly disappeared, and the BM was blurred and discontinuous. The BM in the air-blood barrier of the TXL group was fuzzy and discontinuous, and the Tjs were fuzzy. The air-blood barrier in the Ato group showed edema, the BM was blurred, and the Tjs partially disappeared. The air-blood barrier damage in the TXL + Ato group was slightly weaker than that in the TXL group, and the Tjs were also slightly better than those in the TXL group. ELISA results showed that (Figure 3e,f) the IL-1β and TNFα levels in the COPD + AS group significantly increased compared to those in the AS group, and the TXL + Ato group showed significantly decreased IL-1β and TNFα levels compared to the TXL and Ato groups.

F I G U R E 1
Lung function tests' data revealed that Tongxinluo-atorvastatin (TXL-Ato) combination improved the lung function of COPD + AS mice. Cigarette smoke extract (CSE) combined with high-fat diet induced a significant increase in the functional residual capacity (FRC) (a) and inspiratory resistances (RI) (d) and a significant decrease in dynamic compliance (cdyn) (b) and forced expiratory volume (FEV) at 50 ms/forced vital capacity (FEV 50 /FVC) (c) in the COPD + AS group. The TXL-Ato combination improved the lung function, including reduced FRC, RI, and preserved cdyn and FEV 50 /FVC compared to the COPD + AS group, n = 8, *p < .05 versus control group; ▲ p < .05 versus AS group; # p < .05 versus COPD + AS group; △ p < .05 versus TXL + Ato group.
These data together with lung function data suggested that cigarette smoke aggravates high-fat diet-induced pulmonary inflammation in ApoE −/− mice. The TXL-Ato combination inhibited pulmonary inflammation in ApoE −/− mice treated with cigarette smoke combined with a high-fat diet, and the TXL-Ato combination exerted a better anti-inflammatory effect than TXL and Ato.

| Cigarette smoke aggravates high-fat diet-induced systemic inflammation in ApoE −/− mice, and the TXL-Ato combination attenuates systemic inflammation in ApoE −/− mice treated with cigarette smoke combined with a high-fat diet
To test the potential effect of cigarette smoke combined with a highfat diet on circulatory inflammation, we assessed the levels of different cytokines in the serum of the animal model.  Ato, and TXL-Ato groups had substantially reduced VCAM-1 and ICAM-1 mRNA levels compared to the COPD + AS group. Compared to that of the TXL group, VCAM-1 and ICAM-1 mRNA expression was significantly reduced in the TXL + Ato group. Compared to those of the Ato group, the mRNA levels of ICAM-1 and VCAM-1 were significantly reduced in the TXL + Ato group.

| Cigarette smoke aggravates high-fat dietinduced AS lesions in ApoE −/− mice, and the TXL-Ato combination attenuates AS lesions in ApoE
These data demonstrated that subjecting ApoE −/− mice to cigarette smoke aggravates high-fat diet-induced AS lesions and that the combined use of TXL with Ato enhances the antiatherosclerotic effect of Ato.

| TXL protects against CSE-induced pulmonary microvascular endothelial barrier dysfunction in HPMECs
Herein, we demonstrated that pulmonary microvascular barrier dysfunction is critical in COPD exacerbation of AS in vivo. Next, we verified whether the key mechanism by which COPD aggravates AS is pulmonary microvascular barrier dysfunction through in vitro experiments and whether TXL can protect the function of the pulmonary microvascular barrier. CSE was applied to HPMECs for 24 h to mimic pulmonary microvascular endothelial barrier dysfunction in vitro. The permeability of the endothelial monolayer was tested by measuring the influx of FITC-dextran and the TER across cells.
The influx of FITC-dextran was significantly decreased, and the TER across cells was significantly increased by TXL (Figure 6b).
Immunofluorescence staining revealed that CSE downregulated the expression of VE-cadherin and β-catenin in HPMECs (Figure 7), which was improved by TXL.
We also tested the changes in the expression of Tj proteins and the relative expression levels of VE-cadherin and β-catenin, as determined by Western blots (Figure 6c,d), indicating that CSE significantly decreased VE-cadherin and β-catenin protein levels and that TXL significantly increased the β-catenin protein level. TXL at 800 μg/ml upregulated the level of VE-cadherin; however, 400 and 200 μg/ml TXL did not show any significant effect on VE-cadherin.
Similarly, the RT-qPCR (Figure 6e-g) results showed that the VEcadherin, β-catenin, and Claudin-5 mRNA levels were downregulated in the CSE group, and TXL reversed this effect.
The effects of TXL against pulmonary microvascular barrier dysfunction induced by CSE were consistent with the finding that TXL attenuates pulmonary microvascular hyperpermeability under cigarette smoke combined with a high-fat diet in vivo.

| TXL protects against CSE-induced HPMEC inflammation
Next, we investigated the correlation between inflammatory signaling and increased permeability of the pulmonary microvascular barrier induced by CSE. ELISAs revealed that CSE exposure elevated the levels of IL-6, IL-1β, and high sensitivity C-reactive protein

| TXL protects HPMECs from endothelial damage
A previous study demonstrated that CSE mediates endothelial damage under various insults. Furthermore, TXL inhibits endothelial damage in HPMECs. Therefore, the present study evaluated the

F I G U R E 4 Inflammatory factors (interleukin 1β [IL-1β], interferon gamma [IFNγ]
and tumor necrosis factor alpha [TNFα]) in serum were detected by enzyme-linked assay (ELISA). Cigarette smoke exposure combined with high-fat diet induced a significant increase in the levels of IL-1β (a), IFNγ (b), and TNFα (c) in the COPD + AS group, and Tongxinluo-atorvastatin (TXL-Ato) combination decreased the inflammation level compared to that in the COPD + AS group, n = 6, *p < .05 versus control group; ▲ p < .05 versus AS group; # p < .05 versus COPD + AS group; △ p < .05 versus TXL + Ato group.
effects of TXL on the mRNA expression of ICAM-1 and VCAM-1.
The current results showed that the expression of these mRNAs was significantly upregulated after HPMECs were exposed to CSE, and pretreatment with TXL significantly counteracted this CSE-induced effect (Figure 9a,b).

| DISCUSS ION
The present study elucidated the effects of TXL on COPD com- According to the Global Burden of Disease report, approximately 3.2 million people died of COPD in 2017 (Li et al., 2020), accounting for 81.7% of all deaths from chronic respiratory diseases. CVD is the leading cause of death in patients with COPD, accounting for 25%-27% of patients with mild to moderate COPD (Calverley et al., 2007;Mannino et al., 2006), and the two diseases are often combined (Vestbo et al., 2013). COPD is known to be an independent risk fac- and systemic inflammation (Li et al., 2015). Pulmonary microvascular barrier dysfunction is caused by pulmonary inflammation that stimulates the apoptosis of PMVECs or changes in recombinant endothelial cytoskeleton and intercellular junction proteins, resulting in abnormal cell structure and function, impaired cell barrier function, and increased cell monolayer permeability (Wang et al., 2012). Thus, we hypothesized that pulmonary microvascular barrier dysfunction, followed by pulmonary inflammation leading to systemic inflammation, is the potential mechanism by which COPD aggravates AS.
Smoking is a common risk factor for both COPD and AS. Previous studies have shown that cigarette smoke causes systemic inflammation in mice, similar to that observed in COPD patients with AS.
F I G U R E 6 Tongxinluo (TXL) protects human pulmonary microvascular endothelial cells (HPMECs') pulmonary microvascular endothelial barrier dysfunction induced by cigarette smoke extract (CSE). The permeability of the endothelial monolayer was tested by measuring the influx of fluorescein isothiocyanate-dextran (FITC-dextran) (a) and the transendothelial electrical resistance (TEER) (b). The expression of Tj proteins vascular endothelial cadherin (VE-cadherin) (c) and β-catenin (d) were detected in HPMECs using Western blot. The expression of Tj proteins' messenger RNA (mRNA) levels of VE-cadherin (e), β-catenin (f), and Claudin 5 (g) was detected by real-time quantitative polymerase chain reaction (RT-qPCR). n = 3, *p < .05 versus Control group; # p < .05 versus CSE group.
In this study, ApoE −/− mice exposed to cigarette smoke combined with a high-fat diet for 20 weeks were used to establish a model of COPD combined with AS to explore the role of pulmonary microvascular barrier dysfunction in pulmonary inflammation leading to systemic inflammation, which results in COPD aggravation of AS.
We observed that peripheral airway inflammatory mediators (IL-1β and TNF-a) and lung tissue pathology were more severe in the ApoE −/− mice induced by smoking combined with a high-fat diet than  et al., 2000) and increased uptake of LDL by macrophages (Zwaka et al., 2001). Reportedly, C-reactive protein (CRP) has a direct effect on endothelial function (Verma et al., 2002(Verma et al., , 2004 and promotes the formation of atherosclerotic plaques (Eickhoff et al., 2008 (Colarusso et al., 2017). Inflammatory mediators, including CRP, TNFα, IL-6, and VCAM-1, are produced by various cells in response to stimulation by conserved tissue damage signals (Netea et al., 2017). These mediators contribute to the development of chronic inflammation and a proatherogenic lipid profile (Iqbal et al., 2017;Tamakoshi et al., 2003 of patients with stable COPD (Jiang et al., 2017). Our previous studies demonstrated that TXL protects cardiac microvascular endothelial cells (CMECs) against reperfusion injury , significantly increases the microvascular density of ischemic brain tissue (Chang et al., 2012), and improves the microangiopathy of diabetic nephropathy , revealing that the common target of TXL in the treatment of major diseases of the heart and brain and diabetic nephropathy is microvascular endothelial cells. To the best of our knowledge, the present study, for the first time, clarified the protective effects of TXL on PMVECs in cigarette smoke combined with high-fat diet-induced COPD in mice with AS. Statins are a commonly used lipid-lowering drug in clinical practice. Several studies have shown that statins also reduce inflammation (Ghobadi et al., 2014), inhibit oxidative stress (Ferreira et al., 2014), protect endothelial cells (Venturini et al., 2019), and stabilize plaques . The results showed that TXL combined with Ato inhibited serum lipid levels and aortic endothelial injury biomarkers in the COPD + AS group mice, reduced the expression of systemic inflammatory biomarkers, and inhibited the formation of atherosclerotic plaques better than Ato or TXL alone. Thus, TXL can enhance the antiatherosclerotic effect of Ato. On the anti-inflammatory side, Ato reduces the production of inflammatory mediators by reducing lipid production (Vadali & Post, 2014). In this study, we observed that the pathomorphologi-

CO N FLI C T O F I NTE R E S T
The authors declare that they do not have any conflict of interest.