LTF, which consists of Radix Rhei Et Rhizome, Persicae Semen, Panax notoginseng (Burk.) F. H. Chen Ex C. Chow, and Hirudo has been used to treat diabetic secondary symptoms, such as DR, diabetic nephropathy, diabetic encephalopathy, and diabetic cardiomyopathy (Zhang, Zhang et al. 2018, Raafat, Aboul-Ela et al. 2021, Xiang, Zhou et al. 2021, Yang, Li et al. 2021). Previous studies from our lab have shown that LTF ameliorates DR via certain mechanisms, involving the miR-200b and the p38 MAPK/NF-κB pathway (Pang, Li et al. 2020, Pang, Ni et al. 2020). Nevertheless, the impact of LTF on the intestinal microbiota of diabetic rats, as well as the impact of altered microbiota on the host remains unclear. Based on one of our previous study, we hypothesized that LTF may modulate the gut microbiota of diabetic rats, which in turn might reduce inflammation. Our results indicated that LTF distinctly attenuated retinal damage, including retinal histology and BRB microvascular damage, while increasing tight junction protein expression and modulating gut microbiota, which in turn ameliorated systemic and local retinal inflammation.
Following 12 weeks of treatment with CaD or LTF, slightly decreased HbA1c and FBG levels were observed in diabetic rats, suggesting that both CaD and LTF prevented and delayed DR without apparently ameliorating hyperglycemia. This is consistent with our previous findings which indicated that herbs promoting blood circulation and preventing blood vessel blockage may delay the occurrence and development of microvascular complications in diabetes without apparently ameliorating hyperglycemia (Xiaolin Tong 2006). Xiaolin Tong, a member of the Chinese Academy of Sciences, stated that diabetes, a collateral blood glucose disease, can be defined as a collateral injury caused by hyperglycemia. Thus, curing diabetes-induced collateral injuries—including during its early stages—is vital. LTF is derived from the herb Di Dang Tang, which activates blood and resolves stasis, according to Shang Han Lun, a medical treatise compiled during the East Han Dynasty. Our previous studies have shown that herbs that promote blood circulation and remove obstruction in collateral injuries can alleviate fundus lesions and reduce microalbumin in the urine of diabetic patients. Additionally, these herbs reduced and delayed the occurrence and development of diabetic microangiopathy in diabetic rats, by repairing vascular damage and attenuating vascular leakage in the retina (Xiaolin Tong 2006, Xiaolin Tong 2007, Pang, Li et al. 2020).
To confirm whether LTF has an effect on the retinal histology of diabetic rats, we examined related indicators such as fundus oculi, retinal thickness, as well as microvascular and BRB permeability. Our FFA examination revealed yellowish-white spots, namely hyperfluorescence, contiguous patches, retinal veins with widened diameters, and diabetic ocular fundi exhibiting smaller arteriovenous ratios. Fundus oculi damage was effectively attenuated by CaD and LTF treatment. BRB permeability increased with high glucose levels, and although LTF significantly decreased BRB permeability compared to that observed in diabetic control rats, no significant decrease was observed in the CaD group. We also observed that retinal thickness was reduced in diabetic rats, and that both CaD and LTF significantly increased retinal thickness. Furthermore, acellular capillaries and the E/P ratio were increased in diabetic rats, whereas these two indicators were attenuated following CaD and LTF treatment. These results indicate that, although both CaD and LTF ameliorated retinal pathological damage, the effect exerted by LTF was superior to that exerted by CaD. This is in line with the results of our previous studies showing that LTF inhibits pathological changes in diabetic rats. Collectively, these findings clearly show that LTF plays an important role in protecting BRB and reducing pathological damage (Pang, Li et al. 2020).
Cell junctions, including tight junction proteins (e.g., occludin and claudins) and adherens junctions (e.g., VE-cadherin) strictly regulate the fluids, solutes, and cells that cross the BRB. A characteristic sealant ring, consisting of tight junction proteins and adherens junctions along the apical or basal perimeter of cells, restricts the access via paracellular routes. Peripheral proteins (e.g., ZO-1) may modulate the initial formation of tight junction proteins, while cell junctions and peripheral proteins regulate BRB and retinal vascular permeability (Rudraraju, Narayanan et al. 2020). Protein levels, including ZO-1, occludin, claudin-5, and VE-cadherin, as well as occludin and claudin-5 mRNA levels were significantly elevated in diabetic controls. CaD and LTF increased these indicators to varying degrees. These results indicated that LTF prevents BRB via a mechanism which enhances the expression of cell junctions and peripheral proteins.
Recent studies have shown that the gut microbiome is involved in the development as well as treatment of DR (Beli, Yan et al. 2018, Fernandes, Viana et al. 2019, Alarcón Yempén, Venzel et al. 2021, Huang, Wang et al. 2021, Khan, Sharma et al. 2021). However, only a few TCM studies have described the potential benefits of gut microbiota alteration as a therapeutic strategy against DR. Here, we observed obvious gut microbiota alterations in diabetic rats compared with normal rats. In diabetic rats, the α-diversity of the gut microbiota, including ACE richness and Chao’s diversity index, were significantly decreased. Meanwhile, PCoA based on Bray-Curtis distances indicated that vital microbiota at the phylum and genus levels, such as Firmicutes and Bacteroidetes, were distinctly altered in diabetic rats compared to those observed in normal rats. Murri et al. (2013) showed that children with T1DM displayed increased Bacteroidetes as well as decreased Firmicutes and Prevotella levels. Gao et al. (2019) and Yanni et al. (2020) discovered that Romboutsia was decreased in T1DM rats. These changes in the microbiota were consistent with our results. After 12 weeks of treatment, both CaD and LTF distinctly increased ACE richness and Chao’s diversity indices. Interestingly, LTF significantly changed the PCoA based on Bray-Curtis distance of the microbiota at the phylum level, increasing Firmicutes and decreasing Bacteroidetes. Furthermore, the LTF treatment also affected vital microbiota at the genus level such as Romboutsia, Enterorhabdus, and Prevotella, which were not significantly altered in the CaD group. Wei et al. (2020) suggested that sennoside A, the main active ingredient of rhubarb, elevated the levels of Firmicutes and reduced the levels of Bacteroidetes, which are associated with intestinal permeability and inflammation, in diabetic rats. These data suggest that LTF may ameliorate DR mainly via the modulation of gut microbiota.
To further investigate whether LTF treatment alleviates inflammation in diabetic rats, several inflammatory markers from sera and tissues were measured. We also analyzed the correlation between inflammation and the microbiome. We discovered that pro-inflammatory cytokines including ICAM1, IL-6, IL-8, MCP1, VCAM1, VEGF, and IL-1β from the serum of diabetic rats, as well as NF-κB and TNF-α in the retina, were all increased. Four cytokines were significantly decreased following the CaD treatment, and seven were decreased following the LTF treatment; thus, suggesting that LTF effectively alleviated systemic inflammation. There was a positive correlation between IL-6, IL-8, IL-1β, and VEGF levels, which were significantly decreased by the LTF treatment, and several microorganisms, including Enterobacteriales, Enterobacteriaceae, Prevotellaceae, Bacteroides, and Klebsiella, which were altered by the LTF treatment. Yan et al. (2019) reported that Lactobacillus acidophilus KLDS1.0901 supplementation decreased the abundance of Bacteroides and lowered pro-inflammatory cytokines such as IL-8, IL-1β, and TNF-α, in diabetic rats. Furthermore, Prevotella has been shown to be positively correlated with IL-6, IL-8, and TNF-α in diabetic rats (Xu, Ge et al. 2021). Moreover, a previous study showed that Bacteroides vulgatus, Bacteroides rodentium, and Bacteroides xylanisolvens abundances as well as IL-6 levels were significantly increased in T1DM patients (Higuchi, Rodrigues et al. 2018). Overall, these data indicate that LTF alleviated inflammation in diabetic rats by modulating gut microbiota.
Our study was affected by several limitations. First, only the overall therapeutic effect of LTF against DR, rather than that of its individual components, was evaluated. The main chemical components of LTF that contribute to the prevention and cure of DR were not explored in this study. Thus, further studies aimed at clarifying the exact roles of the main LTF components are necessary. Lastly, the SCFA levels in the feces from animals of different groups were not measured.
In conclusion, we revealed the mechanisms underlying the therapeutic effects of LTF against DR development and progression. Our findings indicate that the therapeutic formula of LTF, which is intended for promoting blood circulation and removing obstructions in collateral injuries, distinctly attenuates retinal damage during the early stages of diabetes. Our results also suggest that LTF potentially attenuates systemic and local retinal inflammation by regulating gut microbiota. Finally, our study provides reliable evidence that LTF may ameliorate DR during the early stage of DM via the “microbiota-inflammation-retina” axis.