Jiangtang decoction ameliorate diabetic nephropathy through the regulation of PI3K/Akt-mediated NF-κB pathways in KK-Ay mice

DN is a leading cause of end-stage renal disease with limited effective therapies, and affects up to 40% of patients with type 1 or type 2 DM [15]. As DN is the strongest predictor of mortality in diabetic patients worldwide [15], it is critical to study the mechanism of DN in conjunction with strategies for prevention and treatment. Although knowledge regarding the molecular mechanisms of DN has progressed in recent years [16‐18], efficient therapeutic approaches that prevent or reverse the progression of DN are lacking, highlighting the need to identify new treatment strategies.

In China, TCMs are known to produce remarkable results and some TCMs are recommended for the treatment of DN [19‐21]. Unlike single compounds with only one active chemical ingredient, TCMs are typically complex combinations of chemicals that act in concert. Moreover, any one of the active components of a TCM alone cannot reflect the ultimate therapeutic effect of the entire medicine [22, 23]. Previous studies show that TCMs that exhibit heat- and toxin-clearing effects can down-regulate blood glucose, promote circulation, and relieve the clinical indicators of DN [20]. Several studies suggest that TCMs might exert hypoglycemic effects through anti-inflammatory mechanisms, including the modulation of inflammatory factors, AGEs, renin-angiotensin systems, and lipid metabolism [11]. In addition, JTD contains various plant components that are widely used in TCM, many of which are effective in reducing both glucose and inflammation (Fig. 8). Euphorbia humifusa Willd is one of the elements of JTD thought to reduce FG and enhance insulin sensitivity in KK-Ay mice [24]. Additionally, it can inhibit NF-κB activity and down-regulate the production of inflammatory mediators, such as nitric oxide (NO) and TNF-α [25, 26]. Salvia miltiorrhiza Bunge has been reported to improve the quantity and amount of pancreatic islet cells, repair their structure and function, increase glucose transporter 4 (GLUT4) and glycogen synthase protein expression in the skeletal muscle, as well as suppress the secretion of NO, TNF-α, and IL-6 in RAW264.7 macrophages stimulated with LPS [12]. Additionally, it has been reported that S. miltiorrhiza Bunge inhibits platelet activation and arterial thrombosis via regulation of the PI3K and NF-κB signaling pathways [24]. Raw A. mongholicus Bunge, which tonifies qi, was commonly used clinically to reduce diabetic symptoms, such as fatigue; it was also found to increase serum levels of total adiponectin, as well as alleviate hyperglycemia, glucose intolerance, and IR in obese mice [27]. It was also reported to attenuate DN by reducing endoplasmic reticulum stress [28]. Similarly, A. asphodeloides Bunge was reported to reduce FG and improve impaired glucose tolerance in type 2 DM rats, and the flavonoids of A. asphodeloides Bunge are thought to reduce glucose and TGs [13]. Furthermore, TB-II, a main ingredient of A. asphodeloides Bunge, notably ameliorated IR and inflammation, and significantly improved cell viability decreased TNF-α and IL-6 levels, and the expression of p-NF-κBp65, p-IKKβ, p-IRS-1, p-PI3K, and p-Akt [13]. Coptis chinensis Franch was confirmed to significantly inhibit all the three periods of nonenzymatic protein glycation in vitro, including amadori products, dicarbonyl compounds, and AGEs formation [29]. It is also reported to attenuate myocardial ischemia–reperfusion by regulating the PI3K/Akt signaling pathway and the subsequent reduction of inflammatory factors, including IL-6, IL-18, and TNF-α [30]. In our previous random control trials and animal experiments, we confirmed that JTD is safe and effective at reducing glucose and MA/UCREA in both patients and KK-Ay mice (accepted). In this study, we found that JTD can reduce the morphological changes in renal tissue, ameliorate glucose and lipid metabolism dysfunction, as well as decrease MA, UN, and Cr. Moreover, JTD can also decrease the enhanced levels of blood glucose and the accumulation AGEs and RAGE, thereby normalizing IR. In our study, the increased phosphorylation of PI3K-Akt and IκBα expression decreased IKKβ, and reduced phosphorylation of NF-κB was observed in the kidneys of JTD-treated KK-Ay mice. In addition, we found that alterations in a number of indexes of renal inflammation (e.g., increased ICAM-1, TNF-α, and IL-6 expression) in KK-Ay mice were alleviated by JTD treatment. These results suggest that the activation of PI3K/Akt, inhibition of the NF-κB signaling pathway, and NF-κB-dependent inflammation may be a key mechanism by which JTD attenuates DN (Fig. 9).

DM is considered to be a group of metabolic diseases characterized by a high concentration of blood glucose, and glucose glycates proteins that give rise to AGEs, which induce cellular effects through interacting with specific cellular receptors. RAGE is an important signal transduction receptor known to activate an array of signal transduction cascades in response to the binding of AGEs [31]. Moreover, AGEs and RAGE play critical roles in the pathogenesis of DN [31]. AGEs result in cellular oxidative stress and IR, which have been implicated as causative factors in diabetic complications [4]. The accumulation of AGEs and RAGE can induce harmful changes to some protein structures, thereby decreasing their enzymatic activity and interfering matrix protein connection, which can cause dysfunction in renal cells and induce histological changes, including inflammation, focal glomerulosclerosis, mesangial expansion, tubulointerstitial fibrosis, and epithelial- to- mesenchymal transdifferentiation [3]. It has been confirmed that elevated levels of AGEs and RAGE have been found to activate pro-inflammatory NF-κB, an initial step in type 2 DM evolution that contributes to both β-cell apoptosis and IR [32], and induces the release of inflammatory factors [33]. IRS-1 and PI3K are key insulin signaling molecules critically involved in glucose metabolism and IR [34, 35]. One of the key mechanisms observed in the tissues impacted by type 2 DM is that the phosphorylation of serine residues in the insulin receptor and IRS-1 molecule results in diminished enzymatic activity in the PI3K/Akt pathway [33]. When insulin binds to the insulin receptor (InsR), it stimulates InsR intrinsic kinase activity and subsequently activates PI3K and Akt in succession [33]. Therefore, due to an increase in insulin receptor activity, PI3K/Akt signaling is activated in the renal cortex of db/db mice during the early phase of DM [33]. Additionally, PI3K/Akt signaling was found to be lower in the glomeruli of 12-week-old db/db mice, which has been linked to the death of podocytes in db/db mice [36]. The down-regulation of PI3K/Akt signaling contributes to renal tubular apoptosis in the kidneys of STZ-induced diabetic mice and apoptosis in the proximal tubular cells of the kidney [37]. Mounting evidence has highlighted that DM activates NF-κB signaling, which regulates the expression of numerous genes that play key roles in the inflammatory response during kidney injury [5, 38, 39]. Moreover, the inhibition of NF-κB has been shown to result in the significant improvement of DN [40, 41]. Therefore, we postulated that the inactivation of NF-κB was involved in the JTD-mediated protection against DN. Under steady-state conditions, NF-κB is present in the cytoplasm and bound to its inhibitory protein, IκB; however, when IκB is phosphorylated by IKKβ, NF-κB is then released from the inhibitory unit, translocated into the nucleus, and undergoes phosphorylation. Finally, NF-κB promotes the transcription of pro-inflammatory cytokines, such as TNF-α, IL-6, and ICAM-1 [42]. A growing number of studies have revealed that the serum and urinary concentrations of TNF-α in patients with DN are substantially higher than that found in non-diabetic individuals, and TNF-α levels are implicated in the process of renal hypertrophy and hyperfunction during the initial stages of DN [43]. IL-6 is confirmed to be increased in patients with DN, and its levels are higher in patients with overt proteinuria compared to those exhibiting microalbuminuria or normoalbuminuria [44]. Additionally, recent work also found that increased IL-6 is associated with glomerular basement membrane (GBM) thickening and mesangial expansion in the kidney biopsies of DM patients [45]. Produced by renal mesangial cells, IL-6 is thought to mediate endothelial permeability and mesangial proliferation [46]. Additionally, an ICAM-1 deficiency in transgenic mice results in a substantial decrease in macrophage accumulation in the glomeruli and a reduction in glomerular hypertrophy [42].

In our study, we found that the concentration of AGEs and RAGE in a KK-Ay mouse model were higher than that of the normal controls. In addition, the expression of IRS-1, PI3K/Akt, and NF-κB, as well as the histological changes observed in the KK-Ay model group, are consistent with previous reports [3, 24, 36]. The findings of the present study suggest that JTD can relieve DN by down-regulating increased levels of blood glucose, so as to reduce the accumulation of AGEs and RAGE, as well as alleviate inflammation via activation of the PI3K/Akt signaling pathways and inhibiting NF-κB signaling. Additionally, this study demonstrates that PI3K/Akt-mediated NF-κB signaling might be a mechanism for the treatment of DN, and use of multiple herbal compounds may be a useful therapeutic approach. While unlike single compounds with only one active chemical ingredient, Traditional Chinese Medicine, especially compounds are typically complicated combinations of chemicals that act in concert. The ultimate therapeutic effect of the compound is multi-targets and the effect might vary as the concentration change. Thus the effect of compound might not all be in a dose dependent manner. However, since the evidence demonstrating the effect of JTD on DN was obtained from studies using animal models, these findings should be confirmed with further research in diabetic patients.