Effects of Cana on hyperglycemia and IR in T2DM mice
T2DM is related to impaired glucose tolerance and IR[57]. Chronic hyperglycemia and IR are known to play a significant role in the development of CVD among patients with T2DM [58]. SGLT2i are newly developed antidiabetic agents via inhibiting glucose reabsorption in the kidney [59]. Thus, we investigated the effect of Cana on blood glucose control, insulin sensitivity, and pancreatic protection in T2DM mice. Fasting blood glucose levels of the HFD-fed groups were markedly increased compared with that of the Control group (Fig. 1A, showed in blue color) at the 24th week, which indicated that T2DM was successfully induced by HFD-feeding. As shown in Fig. 1A and B, Cana significantly decreased fasting hyperglycemia and HbA1c level compared with the Model group (p < 0.05), suggesting that Cana has a long-term effect on alleviating hyperglycemia. OGTT and IPITT assays at the end of the trial demonstrated that Cana treatment efficiently increase glucose tolerance (Fig. 1C and D) and improve insulin sensitivity (Fig. 1E&F) in mice with T2DM (p < 0.05). The suppressed hyperinsulinemia and the lowered HOMA-IR index in Cana group compared with the Model group (Fig. 1G&H) (p < 0.05) indicate that Cana can effectively improve insulin homeostasis.
In addition, it has been demonstrated that as diabetes developed the proliferation of pancreatic α cells increased, which resulted in an elevated plasma glucagon level and hyperglycemia [60]. Furthermore, we found that the amount of β cells was obviously decreased, while α cells were more or less evenly distributed in the islets of T2DM mice compared with the Control group by immunofluorescence staining of insulin and glucagon (Fig. 1I). Treatment with Metf or Cana not only increased β cells percentage and β cells mass (Fig. 1I), it also drove α cells to the periphery of islets (Fig. 1I), which show excellent islet protection in T2DM mice. All of the above analyses confirm the anti-diabetic effects of Cana as demonstrated by regulation of blood glucose, insulin homeostasis, insulin sensitivity, and relieving pancreatic disorder in mice with T2DM.
Effects of Cana on energy metabolism in T2DM mice
After 30-weeks feeding trial, significantly different degrees of obesity were observed among various groups. The waistlines of the Model group and the Cana group were larger than the Control group but were significantly reduced after Metf treatment for six weeks (Fig. 2A), which indicate that Metf treatment can conspicuously reduce obesity degree of T2DM mice by qualitative observation while Cana treatment can’t. In addition, the T2DM mice took in much more energy and gained more weight compared with the normal mice (p < 0.05) (Fig. 2B–D). However, Metf administration markedly prevented HFD-induced increase of body weight, body weight gain, and body mass index (BMI) (Fig. 1B–E) (p < 0.05), while Cana treatment couldn’t. It is noteworthy that the Cana group took in much more energy compared with both the Control and the Model groups (p < 0.05) (Fig. 1B) in the state of taking food freely, while the energy efficiency of the Cana group was much lower than that of the Model group (p < 0.05) (Fig. 1F). It has been confirmed that Cana delay the rate of intestinal glucose absorption via intestinal SGLT1 inhibition and increase the urinary glucose excretion via renal SGLT2 inhibition [61], thus reducing the available energy intake. And, clinical research demonstrated that Cana can induce body weight reduction in patients with T2DM in the state of taking food with limitation [62].
Effect of Cana on hematological parameters in T2DM mice
Studies demonstrate the strong association between hematological parameters and risk of CDV, during to systemic inflammation and hypoxemia implicated in the pathophysiology mechanisms of CVD [63]. Our results (Table 1) showed that white blood cell count (WBC), platelets, and mean platelet volume (MPV) levels were conspicuously increased in T2DM mice compared with the normal mice, while Cana or Metf treatment effectively reversed this adverse situation. There is an significant association of increased MPV and platelet counts with diabetes related to endothelial dysfunction, coronary artery disease, and its vascular complications [64, 65]. Moreover, elevated WBC is a classical inflammatory marker and associated with several CVD risk factors [66]. The above results indicate that Cana can conduct anti-CVD effect via regulating the disorderly hematological parameters in T2DM mice.
Table 1 Effect of the Cana treatment on hematological parameters
Variable
|
Control
|
Model
|
Metf
|
Cana
|
WBC (x109/L)
|
4.6±0.5
|
14.1±0.2*
|
8.1±0.9*#
|
10±1.1*#
|
Monocytes (x109/L)
|
0.2±0.03
|
0.3±0.02*
|
0.6±0.04*#
|
0.4±0.02*#&
|
Monocytes (%)
|
5±0.3
|
3.8±0.25*
|
7.4±0.5*#
|
2.5±0.3*#&
|
Lymphocytes (x109/L)
|
3.2±0.24
|
11.9±0.9*
|
4.9±0.35*#
|
11.2±1.3*&
|
Lymphocytes (%)
|
67.4±5.6
|
76.6±4.3
|
60.8±5.7#
|
83.7±7.6*#&
|
Granulocytes (x109/L)
|
1.3±0.09
|
2.3±0.14*
|
2.6±0.15*
|
3.5±0.41*#&
|
Granulocytes (%)
|
27.5±2.7
|
19.5±1.5*
|
31.8±2.9#
|
13.7±1.1*#&
|
Platelets (x109/L)
|
1940.5±124.2
|
3307±32.3*
|
2467±250.4*#
|
2707±265.1*#
|
PDW
|
17.4±1.2
|
17.4±1.3
|
18.0±1.5
|
19.3±1.2
|
RBC (x1012/L)
|
4.1±0.4
|
5.1±0.3*
|
4.49±0.39
|
1.4±0.11*#&
|
HCT (%)
|
29.3±2.1
|
24.5±1.7
|
21.1±1.3*
|
7.3±0.5*#&
|
HGB (g/L)
|
115.3±10.5
|
138.7±14.2*
|
123.7±13.2
|
139.7±14.1*
|
MCV (fL)
|
49.5±5
|
48.5±5.0
|
48.7±3.6
|
52.7±4.8
|
RDW (%)
|
37.8±3.2
|
38.5±2.8
|
38.4±3.4
|
37.0±2.8
|
MPV (fL)
|
4.75±0.5
|
7.56±0.8*
|
6±0.7*#
|
6.3±0.7*#
|
PDW: platelet distribution width; RBC: red blood cell count; HCT: hematocrit; HGB: hemoglobin; MCV: mean corpuscular volume; RDW: red cell distribution width. Data are expressed as mean ± SEM. * p < 0.05, compared with the Control group; # p < 0.05, compared with the Model group; & p < 0.05, the Cana group compared with the Metf group.
Effects of Cana on hyperlipemia in T2DM mice
Evidences demonstrate that increased concentration of cholesterol and TG is an causal risk factor for CVD [67]. And, hyperlipidemia is characterized by increased lipid accumulation in serum, which is a strong risk factor for CVD and T2DM related coronary artery disease [68, 69]. Thus, managing hyperlipidemia is an effective way to prevent CVD. Our results showed that HFD feeding significantly increased the levels of TC, TG, and LDL-C in the serum compared with the NCD-fed mice (p < 0.05) (Fig. 3A–C). After treatment for six weeks, the serum TG, TC, and LDL-C levels in Cana group were markedly reduced compared with the Model group (p < 0.05), and the therapeutic effect was comparable to the Metf group (Fig. 3A–C). In addition, serum HDL-C level increased in the Cana and the Metf groups compared with the Model group (Fig. 3D) (p < 0.05). Atherogenic index of plasma (AIP) and arteriosclerosis index (AI) values increase with the elevated CVD risk, which are also highly sensitive markers of lipoprotein profiles in CVD patients [70]. And, Cana decrease the AIP and AI levels in T2DM mice (Fig. 3E&F), which indicate that Cana can effectively alleviate CVD induced by hyperlipidemia in mice with T2DM. In addition, Oil red O staining indicated that abnormal lipid accumulation was observed in the Model group as compared to the Control group, while treated with Cana alleviated the lipid accumulation in cardiac tissues (Fig. 3G). Taken together, the above results demonstrate that Cana can perform anti-CVD effect via suppressing lipid accumulation in serum and cardiac tissue in T2DM mice.
Effects of Cana on oxidative stress and systematic inflammation in T2DM mice
Oxidative stress was increased in T2DM and this appeared to underlie the development of T2DM and diabetic complications such as CVD [71]. Oxidative stress is known as a major contributor to endothelial dysfunction [72], which is a key precursor to the development of CVD [73]. As shown in Fig. 4A, the area and intensity of fluorescence from DHE oxidation were notably increased in the Model group compared to that of the Control group (p < 0.05), while Cana or metformin treatments markedly reversed this situation (p < 0.05). Notable decreases in the activities of SOD and GSH, while increasing MDA content in the heart of T2DM mice compared with the normal mice (Fig. 4B–D). However, the above situation was conspicuously reversed by Cana supplementation, implying that Cana can effectively relieve myocardial oxidative stress (Fig. 4B–D). Importantly, TEM evaluation revealed marked cardiomyocyte dissolution, muscular fiber twisting, intercalated disc blurred, Z line disappearance, and some myofilaments were flocky in appearance and showed no cross striations in the myocardial tissue of diabetic mice. In addition, mitochondrial crista deformation and damage, mitochondrial autophagy (red arrows presented in Fig. 4E, vacuoles with black residue), and fat drop (yellow arrows presented in Fig. 4E, vacuoles without black spots) were significantly presented in the Model group compared with the Control group. And, the effects of which were greatly attenuated by Cana or Metf treatment (Fig. 4E), which are beneficial for the suppression of mitochondrial ROS production and subsequently oxidative stress [74].
Chronic inflammation is also an established mediator of vascular dysfunction in individuals with T2DM [75-77]. Through the inflammatory processes, the initial lesion of atherosclerosis and CVD is formed [78]. We found that HFD feeding elevated systematic inflammation state as demonstrated by increased serum levels of inflammatory cytokines including TNFα, MCP-1, and IL-6 compared with the Control group, whereas Cana administration markedly suppressed this increase of pro-inflammatory cytokines (Fig. 4F–H). Besides, HO-1 level was markedly decreased in the Model group compared with the Control group (p < 0.05), while Cana or Metf treatment effective elevated the HO-1 level in T2DM mice (p < 0.05). Upregulation of HO-1 expression plays an important role in the protective response against oxidative injury and inflammatory effects [79], which proposes that HO-1 is a promising target protein for therapeutic intervention in CVD. In general, the above results indicate that Cana can effectively alleviate oxidative stress and systematic inflammation by protecting myocardial structural and mitochondrial homeostasis.
Effects of Cana on cardiovascular abnormalities in T2DM mice
Normal vasculature is crucial to cardiac function and CVD, which is susceptible to hyperglycemia [80]. CD31 plays an important role in endothelial protection, which alleviates the apoptosis of vascular endothelial cells [81]. HFD induced T2DM triggered an overtly drop in the number of CD31 positive microvessels compared with the normal mice (Fig. 5A), while the unfavorable state was reversed by Cana or Metf treatment so as to play a good role in protecting the vascular endothelium. In addition, diabetes-induced changes in microvascular morphology including fibrosis (Fig. 5B) and basement membrane thickening (Fig. 5C), which were largely ameliorated by Cana administration. These data demonstrated a cardioprotective property of Cana by maintaining cardiovascular structural homeostasis in T2DM mice.
Effects of Cana on myocardial injury in T2DM mice
Damage to microvessel integrity and cardiac microvascular endothelial cell are considered the initial step in vascular complications in diabetes [82]. Diabetes-induced effects (such as turbulent blood flow and capillary blockage) raised the accumulation of erythrocytes in the microvessel [74], while Cana considerably retarded this negative situation (red arrows presented in Fig. 6A). Additionally, Metf or Cana treatment inhibited the diabetes-induced TUNEL positive cells increasing (red arrows presented in Fig. 6B), supporting the pro-survival capacity of Cana on hyperglycemia-mediated cardiac microvascular endothelial cell apoptosis. These data revealed the beneficial effects of Cana on microvessel integrity in diabetes, favoring a significant decrease for the risk of cardiac microvascular endothelial cell dysfunction or death in diabetic hearts.
Cardiac troponin is used to diagnose myocardial infarction, and the increased concentration of cardiac troponin indicates the elevated risk for adverse outcome in individual [83]. In addition, cTn I, an important subunits of cardiac troponin, is recognized as marker of myocardial damage [84]. Studies also demonstrated that high level of sCD40L indicates enhanced inflammatory responses, heightened risk of death and myocardial infarction in patients [85-87]. In our study, levels of serous cTn I and sCD40L in T2DM mice were significantly increased compared with that in the Control group (Fig. 6 C&D), while Cana treatment markedly improved the situation. The above results demonstrate the salient effects of Cana on alleviating myocardial injury in T2DM mice.
Cana modulated gut microbiota at different taxonomic levels
High-throughput 16S rDNA sequencing was applied to elucidate the effect of Cana on colonic microbiota in HFD-induced T2DM. As shown in Table S1, α-diversity (e.g. Good’s coverage, Chao1, Shannon and Simpson indices) of the colonic flora in Cana-treated group was not significantly different compared with that in the Control and the Model groups. In addition, evaluation of β-diversity based on nMDS ordination plot (Fig. S2A), weighted UniFrac heatmap (Fig. S2B), and weighted PCoA analysis (Fig. 7A) showed that there were significant differences in colonic flora between the Cana treatment group and the Model group, and the microbial community of the Cana group was similarly to that of the Control group. At the phylum level (Fig. 7B), the Cana group had a higher abundance of Bacteroidetes with a lower abundance of Firmicutes, and an increased ratio of Firmicutes/Bacteroidetes (Fig. 7C) compared with that of the Model group. A significant increase in the abundance of Proteobacteria was induced in the Model group and was significantly reduced in the Cana group, however, not reversed by Metf treatment (Fig. 7D). In addition, administration of Cana remarkably enriched the abundance of Actinobacteria and decreased the abundance of Deferribacteres in mice with T2DM (Fig. 7E&F). The abundance analysis at genus level showed that there were significant differences in flora types and abundance in T2DM mice after Cana treatment (Fig. 7G). Furthermore, Cana regulated the disorder of microbial community in genus level as follows: 1. increasing the relative abundance of Olsenella, Alistipes, and Alloprevotella (Fig. 7I, J&K), which may be associated with a healthier microbiome, as suggested previously [88]; 2. decreasing the relative abundance of Helicobacter and Mucispirillum (Fig. 7H&L), which was positively correlated with TNFα and LPS contents [89]. Altogether, the above results indicated that intestinal ecosystem in mice with diabetic CVD were adjusted to a relatively normal state by oral administration of Cana.
Key phylotypes of gut microbiota in response to Cana treatment
To further elucidate the effects of Cana treatment on the regulation of various bacterial taxa in colon, a pairwise comparison between the Model and the Cana groups was conducted using LEfSe analysis. It revealed that Cana decreased the level of Firmicutes, including Sporacetigenium, Veillonella, and Clostridium XI, while markedly increasing Bacteroidetes, including short-chain fatty acid producers such as Alloprevotella and Bacteroides, compared with the Model group (p < 0.05) (Fig. 8A&B). And, the Cana-treated group was characterized by a higher amount of Porphyromonadaceae, Rikenellaceae, and Corynebacteriaceae, while a decreased in Desulfovibrionaceae and Veillonellaceae. At the genus level, the Cana group was marked by a dramatic increase in Alistipes, Roseburia, and Corynebacterium (Fig. 8B), which can increse the production of short-chain fatty acids (SCFAs) [90, 91]. Meanwhile, Paraprevotella, Veillonella, Sporacetigenium, and Intestinimonas were significantly reduced after Cana treatment (Fig. 8B). In general, the taxonomical distribution within groups at phylum, family, and genus levels for the colonic samples revealed the divergent composition of communities after Cana treatment in mice with diabetic CVD.
Using the Spearman’s correlation analysis, relationships of physiological index and bacterial abundance were clarified (Fig. 8C–E). At the genus level, Parabacteroides and Alistipes were positively correlated with CAT and SOD, while negatively correlated with MDA, ROS, IL-1β, TNFα, LBP, and MCP-1 contents, and their relative abundances were raised after Cana treatment compared with the Model group. In addition, Clostridium XI, Veillonella, and Paraprevotella were negatively correlated with CAT and SOD, while positively related to MDA, ROS, and the above inflammatory cytokines levels. Meanwhile, Alistipes and Roseburia were negatively correlated with LDL-C, TC and TG contents, while positively related to HDL-C level. And, Cana treatment caused an increase in the relative abundances of Alistipes and Roseburia in mice with diabetic CVD. Moreover, Paraprevotella, Veillonella, Clostridium XI, Barnesiella presented positively correlated with LDL-C, TC and TG contents, while negatively related to HDL-C level. These findings suggest that alterations of the gut microbiome after Cana treatment lead to the decrease of oxidative stress level, systemic inflammation, and lipid accumulation in mice with diabetic CVD. Collectively, these data reinforce the link between gut dysbiosis and diabetic CVD, and indicate that Cana can manipulate gut microbiota and attenuate the CVD complications of T2DM.