Effects of the Mechanism of Traditional Chinese Medical Prescription-Yiqihuoxue Formula on Vasculopathy with Systemic Sclerosis

Vasculopathy is a prevalent complication that may appear in the early stage of Systemic Sclerosis (SSc). Traditional Chinese medical prescription-Yiqihuoxue formula (YQHX) has been used as a therapeutic option for SSc in our department, which has acquired a significant treatment effect. We interrogated the role of the YQHX on Vasculopathy with SSc by investigating its effects on the HDMECs from H₂O₂ injury. The viability of the HDMECs which were induced by H₂O₂ and then impacted by the YQHX was noticeably more than that of the H₂O₂-induced HDMECs, while the apoptosis cell percentage was significantly lower. The YQHX could promote cell proliferation and inhibit apoptosis which meant it could protect HDMECs from H₂O₂ injury. And the tube formation assay shown that the YQHX may have a promoted effects on angiogenic properties of HDMECs. Our data demonstrated that the YQHX is helpful on alleviating vasculopathy. These data help in directing personalized therapeutic approaches in SSc.

Systemic Sclerosis (SSc) is a chronic autoimmune disease characterized by functional and structural vasculopathy, dysimmunity and fibrosis of the skin and internal organs [1,2]. Although the pathogenesis of SSc remains elusive, it is generally accepted that vasculopathy and immune activation lead to activation of fibroblasts and fibrosis as the end effect [3]. The initial clinical situation of SSc is mostly Raynaud's Phenomenon (RP) caused by microvascular abnormalities, and most SSc patients show a very characteristic combination of capillary abnormalities [4]. The damage of internal organs such as pulmonary hypertension, heart failure and renal crises [5] may occur early or late in the disease course. These internal organ complications, especially pulmonary and cardiac involvement, cause morbidity and premature death in SSc [6]. To date, therapeutic options of many SSc patients are limited that treatment with immunosuppression can increase quality of life, decrease progression of organ involvement and increase survival [7,8].

In diagnostics of traditional Chinese medicine, SSc has been regarded as the impediment disease related to immune function. A growing body of evidences has pointed that traditional Chinese medicine has multiple roles in the treatment of systemic sclerosis [9]. The patient with SSc treated with integrated traditional Chinese and Western medicine showed ideal therapeutic effects in controlling the progression of the disease and improving symptoms [10]. In our department, the traditional Chinese medical prescription-Yiqihuoxue formula (YQHX) has been used to relieve the affection including vascular compromise, inflammation and fibrosis of SSc nearly 20 years, which has acquired a significant treatment effect [11].

The YQHX is composed of 10 herbs, including Radix Salviae Miltiorrhizae (Danshen in Chinese), Radix Astragali (Huangqi in Chinese), Angelica Sinensis (Danggui in Chinese), Caulis Spatholobi (Fuling in Chinese), Peach Kernel (Taoren in Chinese), ,em>Szechuan Lovage Rhizome

This article mainly researched the effect of the mechanism of the YQHX on vasculopathy in SSc, which could provide future clinical guidance in the therapeutic options for SSc patients.

Cell culture

Human Dermal Micro-Vascular Endothelial Cells (HDMECs) were obtained from Shanghai Dongjian Biotechnology Center. Cells were cultured in Endothelial Cell Medium (ECM) at 37°C under a humidified 5% (v/v) CO2 atmosphere and the medium was renewed every 1 or 2 days. Adherent cells were detached by treatment with phosphate-buffered saline, pH 7.2, 0.02% (w/v) EDTA, 0.25% (w/v) trypsin.

Construction of cellular model

We used the non-interventional HDMECs as a blank group, the HDMECs from H₂O₂ injury as a control group [13]. And the experimental group would be the HDMECs induced by H₂O₂ and then impacted by the YQHX.

Preparation of the prescription-Yiqihuoxue formula

The granules of the YQHX (Neo-Green Pharmaceutical Technology Development Co., Ltd., Sichuan) were dissolved in distilled water to obtain a made-up liquid with a higher concentration, which would be placed at 4°C, and dilute when using.

The toxicity of the prescription-Yiqihuoxue formula

We determined the dosage of the YQHX with Cell Counting Kit-8 (CCK-8, Beyotime biotechnology, Shanghai). The HDMECs were cultured in 96-well plates with different concentrations of the YQHX and Dulbecco’s Modified Eagle's Medium (DMEM), and CCK-8 were applied according to the manufacturer’s instructions. The absorbance was measured at 450nm by SpectraMax ID 3 (Molecular Devices, USA) as the result. The cell viability was calculated according to the formula: cell viability (%) = average A450 nm of the treated group/average A450 nm of the distilled-water group*100%. Each experiment was performed in quadruplicate and repeated at least three times.

Cell proliferation assay

The effect of the YQHX on cell proliferation were measured using CCK-8 too. The blank group, the control group and the experimental group were cultured in 96-well plates with DMEM, and CCK-8 were applied according to the manufacturer’s instructions. The absorbance was measured as above. The cell viability was calculated as above. Each experiment was performed in quadruplicate and repeated at least three times.

Cell apoptosis assay

The effect of the YQHX on cell apoptosis were measured using AnnexinV-FITC/PI Apoptosis Assay Kit (Sunbio Technology, Nanjing). The blank group, the control group and the experimental group were cultured in 6-well plates with (DMEM, and AnnexinV-FITC/PI Apoptosis Assay Kit were applied according to the manufacturer’s instructions. The result of apoptosis assay was measured at Ex = 488 nm and Em = 530nm by FACSCalibur Flow Cytometer (BD Biosciences, USA). Each experiment was performed in quadruplicate and repeated at least three times.

Tube formation assay

The effect of the YQHX on angiogenic properties was measured using Matrigel Basement Membrane Matrix (BD Biosciences, USA). The blank group, the control group and the experimental group were cultured in 96-well plates with DMEM and Matrigel Basement Membrane Matrix was applied according to the manufacturer’s instructions. The tube formation was observed under Leica DMi8 (Leica, German) and the result was photographed by Leica DFC310FX (Leica, German). Each experiment was performed in quadruplicate and repeated at least three times.

Statistical analysis

All data were presented as with at least three independent experiments, each performed at least in triplicate. One-way analysis of variance was used to determine the statistical significance between different groups. Means were compared using Student’s t-tests, and p < 0.05 was accepted as statistically significant. All data were analyzed with SPSS 25.0 and GraphPad Prism 8 software.

The appropriate dosage of the prescription-Yiqihuoxue formula

To determine the appropriate dosage of the YQHX, HDMECs were cultured in 96-well plates with different concentrations of the YQHX and DMEM. As shown in figure 1, after 24 h, the rate of cell viability in the group of the 68.1 ug/ml-YQHX (107.07 ± 12.46)% was significantly higher than that in the 272.4ug/ml-group (44.49 ± 4.52)%. After 48 h, the rate of cell viability in the 68.1 ug/ml-group (87.98 ± 8.36)% was higher than that in the 34.05 ug/ml-group (75.71 ± 5.37)%. After 72h, the rate of cell viability in the 68.1 ug/ml-group (67.80 ± 7.32)% was higher than that in the groups of other concentrations of the YQHX. In summary, the YQHX with the concentration of 68.1 ug/ml was less toxic to HDMECs, and the appropriate dosage of the YQHX would be 68.1 ug/ml in latter assays.

Figure 1: The appropriate dosage of the prescription-Yiqihuoxue formula would be 68.1 ug/ml.
The non-interventional HDMECs as the blank group and the HDMECs impacted by different concentrations of the YQHX were cultured in 96-well plates and cell proliferation rates were determined with CCK-8 Assay. Bars indicate the values expressed as. *p < 0.05, ####p < 0.0001 versus vector controls. Data were expressed as from three independent experiments. Each experiment was performed in triplicate.

Effects of the prescription-Yiqihuoxue formula on proliferation and apoptosis of HDMECs

In order to verify the protective role of the YQHX for the HDMECs from H₂O₂ injury, we compared the cell proliferation and apoptosis in different groups including the blank group, the control group and the experimental group. The CCK-8 results showed that proliferation was promoted significantly in cells of the experimental group as compared with that of the control group. The rate of cell viability in the experimental group (86.68 ± 9.59)% was remarkably higher than that in control group (15.93 ± 18.86)% (Figure 2A). Consistent with cell proliferation assay results, the YQHX could inhibit apoptosis in HDMECs from H₂O₂ injury (Figure 2B); the apoptosis cell percentage in the experimental group (12.34 ± 1.37) was significantly lower than that in the control group (71.05 ± 4.53), but it was close to that in the blank group (11.33 ± 2.22). Taken together, these results indicated that the YQHX could promote cell proliferation and inhibit apoptosis which meant it could protect HDMECs from H₂O₂ injury.

Figure 2: The prescription-Yiqihuoxue formula promoted cell proliferation and inhibited apoptosis.
A): The non-interventional HDMECs, the H₂O₂-induced HDMECs and the HDMECs which were induced by H₂O₂ and then impacted by the YQHX were cultured in 96-well plates and cell proliferation rates were determined with CCK-8 Assay. Bars indicate the values expressed as. ****p < 0.0001 versus vector controls.
B): The non-interventional HDMECs, the H₂O₂-induced HDMECs and the HDMECs which were induced by H₂O₂ and then impacted by the YQHX were cultured in 6-well plates, suspended in binding buffer containing AnnexinV-FITC and PI, and analyzed by flow cytometry.
C): Data were expressed as from three independent experiments. Each experiment was performed in triplicate.

Effects of the prescription-Yiqihuoxue formula on angiogenic properties of HDMECs

To investigate the effect of the YQHX on the angiogenic ability of HDMECs, tube formation assay was utilized. As shown in figure 3Ba,c, after the HDMECs cultured on Matrigel Basement Membrane Matrix for 6h, numbers of nodes and branches of the cells in experimental group were more than that in the control group, while numbers of meshes and total length of branches of the cells in the experimental group were also significantly more than that in the control group after 12h (Figure 3Bb,c). These results suggested that the YQHX had a promoted effects on angiogenic properties of HDMECs from H₂O₂ injury.

Figure 3: The prescription-Yiqihuoxue formula had promoted effects on the angiogenic properties of HDMECs.
A). The non-interventional HDMECs, the H₂O₂-induced HDMECs and the HDMECs which were induced by H₂O₂ and then impacted by the YQHX were cultured in 96-well plates on Matrigel Basement Membrane Matrix. The tube formation was observed under Leica DMi8 and the result was photographed by Leica DFC310FX.
B) (a). The numbers of nodes of the cells in the experimental group were more than that in the control group.
(b). Numbers of meshes of the cells in the experimental group were more than that in the control group.
(c). Numbers of branches of the cells in the experimental group were more than that in the control group.
(d). Total length of branches of the cells in the experimental group was more than that in the control group. Bars indicate the values expressed as. *p < 0.05, ****p < 0.0001 versus vector controls. Data were expressed as from three independent experiments. Each experiment was performed in triplicate.

Microcirculation impairment and related vasculopathy are hallmarks of Systemic Sclerosis (SSc) which are usually thought to have a key role in the early pathogenesis [15,16]. Evidence suggests there is an early insult to the microvasculature, followed by an ongoing chronic process that can result in profound vascular damage in a subset of patients who develop severe events such Digital Ulcers (DU) and Pulmonary Arterial Hypertension (PAH) [17]. The extant therapy for vascular SSc is limited, especially at the less severe stages of vasculopathy. Previous clinical studies have verified that the prescription-Yiqihuoxue formula (YQHX) has a significant treatment effect of relieving the affection including vascular compromise, inflammation and fibrosis of SSc [11]. Therefore, we aimed to research the effect of the mechanism of traditional Chinese medical YQHX on vasculopathy in SSc.

Endothelial cells, as central constituents of the vascular system, play a key role in all aspects of vascular homeostasis [18]. It was found that incubation of endothelial cells with SSc-immune complexes results in modulation of several molecules involved in the cardinal scleroderma pathophysiological processes [19]. Based on that, our experiment mainly focused on effects of the YQHX on human dermal micro-vascular endothelial cells (HDMECs). We used HDMECs from H₂O₂ injury to simulate the damaged cells of vascular SSc. The YQHX could promote cell proliferation and inhibit apoptosis. It shown that the YQHX could protect HDMECs from H₂O₂ injury.

In addition, we found the YQHX could promote the angiogenic ability of HDMECs from H₂O₂ injury which may involve the mechanism of the therapeutic on SSc by promoting angiogenic. The onset of pathophysiological processes in SSc results in impaired vascular homeostasis, because of which angiogenesis is dysregulated and efficient vascular recovery is impaired, while the damage of the vessels evolves progressively from early to late stages and is characterized by different morphological aspects [20,21]. In line with this, the YQHX was shown to alleviate Raynaud's Phenomenon (RP) and DU in our preliminary clinical study [11].

In summary, there were few reports in the literature on effects of the traditional Chinese medical YQHX on vasculopathy in SSc. This study provided that which associated with protecting HDMECs from H₂O₂ injury, and promoting the angiogenesis. The results of the study suggested that the YQHX could be of great help in the treatment on vasculopathy in Systemic Sclerosis. Further investigations are still needed to determine the mechanisms which the YQHX effects on vasculopathy in Systemic Sclerosis.

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