The number of diabetes patients is expected to increase from 415 million in 2015 to 640 million by 2040 globally (Gao et al., 2016). Vascular complication of diabetes is one of the most serious manifestations of the disease (Rask-Madsen and King, 2013), and the leading cause for this condition is vascular dysfunction including impaired vascular endothelial vasodilation, reduced vascular compliance, and slowed blood flow (Shi and Vanhoutte, 2017). The most well-established clinical advances in the prevention of vascular complication include the control of glucose, cholesterol and blood pressure, which can slow the progression of diabetic microvascular pathology and reduce the risk of cardiovascular disease (Ong et al., 2008). However, the effect of the above-mentioned treatment on vascular dysfunction is not ideal (Grunberger, 2017; Ali et al., 2018). Therefore, there is an urgent need for the discovery and development of new drugs for the treatment of diabetic vascular complication.

Traditional Chinese medicine has long been involved in the treatment of cardiovascular diseases. HuangqiGuizhiWuwu Decoction (HGWWD), originated from “Synopsis of the Golden Chamber,” is one of the main prescriptions for treating vascular disease in ancient China. Recent clinical evidence-based studies have shown that HGWWD has a positive therapeutic effect on various cardiovascular and cerebrovascular diseases. For example, HGWWD can improve cerebral blood flow in stroke patients and alleviate the subjective symptoms of diabetic peripheral neuropathy (Baiqing, 2015; Pang et al., 2016). Similarly, a recent study reported that HGWWD effectively treated lower extremity macroangiopathy in diabetic patients (Hu et al., 2018). However, the underlying mechanism behind the HGWWD prevention of diabetes-induced vascular complication is poorly understood.

Studies from diabetic mice models and human patients with cardiovascular disease have reported abnormal arginase activation in the vascular endothelium (Romero et al., 2008, 2012; Beleznai et al., 2011; Shemyakin et al., 2012). Arginase, a urea cycle enzyme, can reciprocally regulate nitric oxide (NO) production by nitric oxide synthase (NOS) through competition for their common substrate, L-arginine (Bhatta et al., 2017). There are two isoforms of arginases: arginase 1, located in the cytoplasm and arginase 2, largely present in the mitochondria. We and other previous research found that elevated vascular arginase activity, especially arginase 1, can impair normal vascular endothelial function in various cardiovascular disease models, for example, diabetes (Elms et al., 2013), atherosclerosis (Rabelo et al., 2015), hypertension (Toque et al., 2013), aging (Santhanam et al., 2007), coronary artery disease (Shemyakin et al., 2012), and ischemia-reperfusion (Jung et al., 2010). We recently reported that knocking out endothelial arginase 1 completely prevented obesity-induced vasculopathy, including endothelial-dependent dysfunction, arterial stiffening and vascular inflammation (Bhatta et al., 2017; Yao et al., 2017). All of these findings indicate that arginase 1, especially in endothelial cells, is a valuable target for the treatment of vascular disease.

In this study, we evaluated the therapeutic effect of HGWWD on diabetes-induced vascular dysfunction and investigated whether the ameliorative effect of HGWWD on diabetic vascular endothelial dysfunction is associated with endothelial arginase 1-NO signaling.

Arginase1 knockout specific in endothelial cells mice (EC-A1^–/–) are obtained from Dr. W.R. Caldwell Laboratory (Augusta University, United States). As described previously (Bhatta et al., 2017), EC-A1^–/– mice were generated by mice expressed Cre-Cadherin 5 (C57BL/6J × 129S1/SvImJ, Stock No. 017968, Jackson Laboratory) crossed with mice carrying floxed arginase 1 alleles (A1^loxp/loxp,C57BL/6J xB6.Cg-Thy1^a,Stock No. 008817, Jackson Laboratory). Littermate A1^loxp/loxp mice were used as control mice. C57BL/6J Wild type male mice (WT) were obtained from Jinan Peng Yue Experimental Animal Breeding Co., Ltd. (Jinan, China). All mice were housed under a 12 h light/dark cycle with ad libitum access to water and food. All experimental procedures in this study were performed according to Institutional Animal Care and Use Committee of Guangzhou University of Chinese Medicine (GZUCM). Diabetic mice were used in a model of streptozotocin (STZ, 50 mg/kg)-induced diabetes (Yao et al., 2013). After 8 weeks of diabetes progression, the mice from the Chinese formula treatment groups received HuangqiGuizhiWuwu Decoction (HGWWD) by daily gavage at different doses (60 g/kg/d of crude drugs) for another 2 weeks with or without co-treatment with L-Name (NOS inhibitor) (Anea et al., 2012). Blood glucose and body weight levels of each animal were measured at the first day, second, eighth, and tenth weeks of STZ injection. Both hemodynamic function and vascular wall function were assessed by ultrasound as previously described (Bhatta et al., 2015; Kenwright et al., 2015; Faita et al., 2018; Esfandiarei et al., 2019). Vascular endothelial function was measured by myograph as previously described (Yao et al., 2013). Vascular NO production was determined using the fluorescent NO indicator4, 5-diaminofluorescein diacetate (DAF-2 DA) (Yao et al., 2013). Arginase activity in aortic lysates or plasma samples was determined as previously described (Yao et al., 2013). The mRNA levels of arginase 1 and arginase 2 were measured by quantitative reverse transcription PCR (Q-PCR) as previously described (Bhatta et al., 2017). Data are shown as mean ± SEM. The number of experiments is indicated by “n.” Statistical differences were determined using analysis of variance (ANOVA) followed by Tukey post-test. P < 0.05 were taken as significant. Detailed methods are provided in Supplementary Material and Methods.

In this study, we found that (1) treatment with HGWWD for 2 weeks protected against diabetes-induced impairment of both hemodynamic function and vascular wall function, but did not alter blood glucose levels and body weight in diabetic mice; (2) HGWWD prevented diabetes-caused vascular endothelial dysfunction through arginase 1-NO signaling; (3) endothelial arginase 1 is a critical target for the prevention of HGWWD on vascular endothelial dysfunction in diabetic models.

HGWWD is used in the treatment of various cardiovascular and cerebrovascular diseases, including diabetic lower extremity macroangiopathy (Hu et al., 2018), diabetic peripheral neuropathy (Liu et al., 2019), and stroke (Li, 2015). Similarly, we found that HGWWD significantly ameliorated vascular wall and hemodynamic dysfunction, especially endothelial dysfunction in STZ-induced mice (Figures 1, 2), suggesting that HGWWD is effective in treating the disorder of vascular system caused by diabetes. Controlling blood glucose levels is one of the most common therapies for slowing the progress of vascular disease in diabetic patients (Zhang et al., 2019). However, our present study shows that HGWWD has no effect on blood glucose in diabetic mice (Figures 1D,E), which is supported by several clinical studies (Liu, 2005; Bian, 2010; Lu and You, 2010; Pang et al., 2016). One study reported that HGWWD can reduce the glucose levels of diabetic patients (Chen, 2019), but the reasons for this discrepancy are unclear. We speculate the different effects of HGWWD on blood glucose may be attributable to differences in dosage or duration of treatment. Nevertheless, this study provides evidence to support that this traditional Chinese formula can significantly attenuate vascular complications in diabetes patients.

In this study, we reported that HGWWD treatment significantly restored aortic vascular endothelial dysfunction through NO signaling (Figure 3). Abnormal NO levels have been reported in vascular pathological conditions (Bondonno et al., 2016), and intervention in NO signaling can effectively prevent various vascular disorders, including endothelial dysfunction, vascular inflammation and vascular stiffness (Bondonno et al., 2016; Batko et al., 2019; Chen et al., 2019). Furthermore, excessive arginase activation can down-regulate NO production by reducing NOS availability through competition for their common substrate L-arginine (Bhatta et al., 2017; Zhou et al., 2018). This notion is supported by our results showing that HGWWD decreased endothelial arginase activity and aortic arginase 1 expression in STZ mice (Figure 4). Moreover, overexpression of arginase 1 in the aorta can block the effect of HGWWD in diabetic mice (Figure 5) and the lack of endothelial arginase 1 cannot further improve the vascular endothelial function in the HGWWD-treated diabetic group (Figure 6). Thus, for the first time, we demonstrated that HGWWD prevented STZ-induced vascular dysfunction through arginase 1-NO signaling in vascular endothelial cells. Similarly, some other traditional medicines treated the vascular pathology involved in mechanisms with regulating vascular endothelium dysfunction and NO production, including Yangxin Decoction (Li et al., 2011), Taohong Siwu Decoction (Zhang, 2014), Shixiao San (Wang X. et al., 2015), Ling-Yang-Gou-Teng-decoction (Zhao et al., 2018). Moreover, our previous study also found that Xiao-Shen-Formula prevented the impairment of renal microvascular remodeling via inhibiting arginase activation (An et al., 2018). Altogether, our study further confirms that endothelial Arginase-NO signaling is a valuable therapeutic target in the invention of vascular disease.

There is wide belief that traditional Chinese multi-component formulas act on multiple targets. Besides arginase 1-NO signaling in endothelial cells, our results revealed that there should be other mechanisms involved the effect of HGWWD on the protection of vascular impairment in the STZ model. We found that HGWWD has better effect in preventing vascular endothelial function of diabetes than knocking out endothelial arginase 1 alone (Figure 6). Furthermore, there are other pharmacological effects of HGWWD, including the reduction of oxidative stress levels in diabetic neuropathy and myocardial ischemia-reperfusion injury in rats (Qi et al., 2013; Wang Y.-C. et al., 2015; Zhang et al., 2017), and anti-inflammation in nephropathy mice or arthritis in rats (Huang et al., 2009; Li et al., 2016; Liu, 2017; Liu et al., 2018). It is conceivable that the findings of this paper could shed light on the pharmacological mechanisms of HGWWD treatment, particularly in targeting endothelial arginase 1.

The datasets generated for this study are available on request to the corresponding author.

The animal study was reviewed and approved by the Institutional Animal Care and Use Committee of Guangzhou University of Chinese Medicine (GZUCM).

LY, YC, WS, CL, and WY conceived and designed the protocol. HC, TL, JW, YX, XCha, MZ, YY, NZ, SZ, and XChe performed the experiments. LY, YC, HC, and TL analyzed the data. LY and YC wrote the manuscript. All the authors reviewed and approved the submitted version of the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.