T2DM is a metabolic disease characterized by chronic hyperglycemia with impaired insulin secretion and/or utilization. We evaluated the effects of PCP on blood glucose and lipids, IR, inflammatory cytokines, pancreatic tissue damage, and gut microbiota in db/db mice. It was found that PCP was effective in suppressing hyperglycemia, hyperinsulinemia, and the expression of inflammatory cytokines (IL-6 and TNF-α), and boosting glucose tolerance in db/db mice. These health-promoting effects of PCP may be related to the modulation of the structure and composition of the gut microbiota.
Growing evidence shows that dysbiosis of the gut microbiota is an underlying causative agent of T2DM and plays a potential regulatory role in its pathogenesis and progression [23]. The structure and composition of the intestinal flora play an essential role in the host's health status. Previous studies have reported that PCP can significantly change the composition of the gut microbiota in healthy mice, common carp, and broilers by upregulating the relative abundance of Bacteroidetes and decreasing the amount of Firmicutes, contributing to a health-promoting intestinal environment [20, 21, 24]. Gut microbial changes characterized by a higher abundance of Firmicutes and a lower abundance of Bacteroidetes are frequently reported in patients with T2DM [25, 26]. Firmicutes are beneficial in stimulating host appetite and energy metabolism, and their abnormal increase may lead to obesity and IR in hosts. Bacteroidetes are considered to be the most common beneficial bacteria in the gut. The ratio of Firmicutes to Bacteroidetes is positively correlated with body weights and negatively associated with glucose tolerance [27]. In this study, PCP decreased the abundance of Firmicutes and increased the abundance of Bacteroidetes in db/db mice as expected, which was consistent with the trend in their body weight, lipids, blood glucose, and glucose tolerance. In addition, we found that PCP significantly raised the relative abundance of Mucinivorans at the genus level. It had been reported that Mucinivorans not only alleviated obesity and improved insulin sensitivity in hosts, but also regulated lipid metabolism and inhibited cholesterol synthesis [28].
Chronic inflammation induced by high glucose can lead to impaired islet β-cell function and worsen IR [29, 30]. As shown in the results, PCP reduced the expression of IL-6 and TNF-α, significantly improved pancreatic tissue edema, and further attenuated islet β-cell mitochondrial damage. Previous studies have proved that PCP can suppress inflammation in the body by activating the Nrf2/HO-1, AMPK/p62/Nrf2/mTOR, or SIRT1/AMPK signaling pathways [31–33]. In particular, AMPK/SIRT1 and AMPK/mTOR have been known to be key signaling pathways for regulating glucose and lipid metabolic homeostasis and insulin sensitivity. In this study, PCP treatment significantly depressed the expression of IL-6 and TNF-α as well as lowered the levels of FINS and HOMA-IR in db/db mice. It was further observed under TEM that PCP significantly improved pancreatic tissue edema and β-cell mitochondrial swelling, indicating that PCP could attenuate pancreatic injury and enhance insulin sensitivity in db/db mice. LPS, made up the outermost layer of the cell wall of Gram-negative bacteria, is an endotoxin that can lead to chronic inflammation and IR [34]. SCFA can increase the expression of intestinal peptide YY and Glucagon-like peptide-1 through depletion of hunger-promoting hormones to reduce appetite and enhance insulin sensitivity [35]. A lack of SCFA-producing bacteria and an increase in LPS-producing bacteria are common manifestations of gut microbiota dysbiosis in patients with T2DM. Interestingly, SFCA-producing bacteria are considered to inhibit the rise of LPS-producing bacteria and further inflammation induced by them [36].
In the present study, PCP treatment significantly decreased the relative abundance of Desulfovibrio and Lactobacillus, and increased the relative abundance of Muribaculum, Prevotella, and Barnesiella in db/db mice at the genus level. Desulfovibrio is the representative LPS-producing bacterium that converts sulfate into hydrogen sulfide, which disrupts the gut barrier and increases gut permeability [37]. Muribaculum and Prevotella are known to produce butyrate primarily, which is known to inhibit the expression of proinflammatory cytokine genes in adipocytes and contribute to preserving the integrity of the intestinal barrier [38, 39]. In addition, PCP was significantly enriched for the anti-inflammatory bacteria Barnesiella and Muribaculum. Specifically, Muribaculum is an important mucin monosaccharide forager that inhibits Clostridium difficile colonization of the intestine, contributing to the reduction of intestinal inflammation and the maintenance of intestinal microecological stability [40]. Barnesiella is associated with some immunomodulatory factors, such as IL-10, which can suppress inflammation in the gut [41, 42]. Ye's study finds that IL-10-/- mice with higher abundance of Barnesiella developed lower levels of colitis disease [43]. In summary, we hypothesized that PCP suppressed intestinal inflammation and maintained intestinal homeostasis by increasing the abundance SCFAs-producing and anti-inflammatory bacteria while decreasing the abundance of LPS-producing bacteria to ameliorated hyperglycemia and IR in db/db mice.
Notably, the relative abundance of Lactobacillus showed a significant decrease after PCP treatment, which may be contrary to some studies[44, 45]. Some ideas were that the increase of Lactobacillus, such as Lactobacillus johnsonii and Lactobacillus rhamnosus, can inhibit the growth of pathogens by secreting lactic acid to decrease the pH of the gut, maintain the intestinal barrier and repair the impaired glucose tolerance [46]. However, Karlsson et al. [47] had reported that Lactobacillus, classified to the phylum of Firmicutes, were notably enriched in T2DM individuals and positively correlated with FBG and HbA1c. Additionally, Li et al. [22] similarly found that the PCP significantly reduced the relative abundance of Lactobacillus in NAFLD mice. They proposed that PCP could accelerate cholesterol metabolism by reducing the abundance of bile salt hydrolase (BSH)-producing bacteria, including Clostridium_IV, Clostridium_XIVb, and Lactobacillus, modulating FXR signaling in the intestine and liver and inhibiting taurine-coupled BA conversion. Besides its role in lipid metabolism, BA is also involved in the regulation of glucose, energy metabolism, and insulin sensitivity by binding to FXR, TGR5, and other signaling molecules [2]. Therefore, we conjectured whether the improvement of glycemia and IR in db/db mice by PCP was also mediated through the gut microbiota-bile acid-FXR axis. However, there was insufficient evidence for this assumption and further exploration will be needed.
The increased diversity of intestinal flora is beneficial for maintaining the stability of gut function [48]. Based on the results of α diversity analysis, we found that Chao1, PD, Simpson, and Shannon indexes were increased to some extent in db/db mice after PCP treatment. Interestingly, the PD and Shannon indexes were significantly lower in the NC group than those in the DC group. This was contrary to the results of previous studies, which overwhelmingly concluded that individuals with T2DM should have lower intestinal flora diversity than normal individuals [49]. Further analysis revealed that the results of Sun's study were in agreement with ours, that db/db mice had higher α diversity of gut microbiota than db/m mice, and the experimental animals for both studies were from the same institution [50]. Therefore, we hypothesized that the results above may be related to the breeding environment and individual differences of animals.
To the best of our knowledge, this is the first time to investigate the effects of PCP on gut microbiota in db/db mice. Our findings provided a new perspective on the possible mechanism of PCP action in diabetes. However, there are some limitations to our study. Firstly, PCP, as a new novel food, was only approved to be consumed in the form of tea bags in China. Therefore, db/db mice were selected to be intervened with an aqueous extract of P. chinense to simulate the way of human consumption. Secondly, the markers of PCP are not well-defined yet, and its active ingredients may act on different targets. Therefore, this study only describes the hypoglycemic effect of PCP at the level of the animal as a whole. In addition, whether the active ingredients of PCP exert hypoglycemic effects through the gut-liver axis is a direction we would like to investigate deeply in the future.