In this study, the body weight and blood sugar of KKAy mice were significantly increased compared with normal control after high-fat feed, showing the characteristics of abdominal obesity and hyperglycemia. This is consistent with the experimental results of Liu min []. Previous studies have shown that LBP can reduce body weight by increasing the hypothalamic leptin level, reducing appetite and accelerating fat metabolism in SD obese rats. But after LBP intervention, the weight of KKAy mice did not change significantly. This is not consistent with the result that LBP can reduce the body weight of obese SD rats. This may be related to the functional defect of melanocortin receptor-4 (MC4R) in the brain of KKAy mice. MC4R is a kind of peptide secreted by hypothalamus. It can affect the function of leptin by binding with melanocortin (MC), agouti protein and agouti related protein (AgRP). It plays an important role in regulating the energy balance of the body [ ]. In KKAy mice, agouti protein encoded by ay gene will bind to melanocortin receptor MC4R to antagonize the effect of leptin []. Therefore, the effect of LBP on the body weight of KKAy mice was not observed.
In this study, after the intervention of LBP, the blood glucose of mice in the low, medium and high dose groups was significantly lower than that in the diabetic model group, which was consistent with the research results of Zhao R. et al [], further verifying the hypoglycemic effect of LBP. At present, it is believed that the hypoglycemic effect of LBP is not only related to the up regulation of glucokinase (GK) and pyruvate kinase (PK) expression, but also related to the up regulation of GLUT4 and the increase of glucose uptake and utilization [ ]. Metformin can improve hyperglycemia by inhibiting gluconeogenesis of liver and increasing glucose intake of muscle. Its molecular mechanism is related to activation of adenylate activated protein kinase, protein kinase A and inhibition of mitochondrial respiratory enzyme [ , ]. The results of this experiment showed that compared with the diabetic model group, the blood glucose in the met group decreased significantly, and there was no significant difference between the blood glucose levels in the low and medium dose LBP groups and that in the metformin group, which indicated that the hypoglycemic effect of the low and medium dose LBP was similar to that of the metformin group, which provided a basis for the later application of LBP in the clinical treatment of T2DM.
In the long-term pathological process of T2DM, in addition to IR and islet B cell dysfunction, it is also accompanied by the activation of tissue and cell immune inflammation. Inflammatory factors secreted by activated monocyte macrophages, lymphocytes and other immune cells are the pathogenic factors of inducing IR and T2DM. Compared with NC mice, the levels of IL-1 β, IL-6 and TNF - α in serum of KKAy mice were significantly increased. IL-1 β is an important promoter of inflammatory cascade and an independent risk factor for T2DM. It is suggested that IL-1 β can activate NF - κ B and MAPKs signaling pathway to induce islet inflammatory response and apoptosis of B cells [], while the apoptosis of B cells induced by high glucose decreased when IL-1 β was knocked out [].
Inflammatory marker IL-6 is also involved in the development of T2DM. It has been reported that chronic IL-6 exposure can induce the over expression of insulin inhibitor SOCS-3, damage insulin signal transmission, and cause IR. At the same time, excessive IL-6 in the islet will cause the over activation of killer T cells, which, together with other cytotoxic effects, will cause apoptosis of islet β cells [ , , ]. TNF - α, as a kind of non glycosylated protein, can induce the body to produce IR by promoting the decomposition of fat granules, increasing FFA, reducing the activity of insulin receptor tyrosine kinase and interfering with normal insulin signal transduction. Therefore, this study suggests that there is a high level of inflammatory response in T2DM, and these inflammatory factors are related to the occurrence of T2DM, which is consistent with the research results of chanchira phosat et al. [ ] on T2DM population.
The study also found that compared with NC mice, the level of IL-10 in the serum of KKAy mice decreased. IL-10 is an anti-inflammatory factor. The decrease of serum IL-10 level is significantly related to glucose intolerance [ ]. This is consistent with the low reactivity of IL-10 under the stimulation of high glucose in vitro experiments by Julianne C. Barry et al. TGF - β 1 plays an important role in the regulation of cell proliferation, apoptosis and immune response. At present, whether TGF - β 1 plays an anti-inflammatory or pro-inflammatory role in vivo remains controversial [ ]. Yoshikazu naiki et al. [ ] found that TGF - β 1 can promote the ubiquitination and proteasome degradation of MyD88, reduce the level of MyD88 protein and delay the activation of NF - κ B by blocking the activation of TLR4 ligands, indicating that TGF - β 1 has certain anti-inflammatory effect. In this experiment, the decrease of TGF - β 1 in KKAy model mice may be related to the activation of NF - κ B signal pathway in T2DM mice, which further verified the anti-inflammatory effect of TGF - β 1.
In addition to improving the parameters of hyperglycemia, IR and other metabolic abnormalities in the body, some studies have suggested that metformin can also inhibit the secretion of pro-inflammatory molecules by macrophages by inhibiting the differentiation of monocytes into macrophages. At the same time, it can also inhibit the ratio of neutrophils and lymphocytes, the markers of inflammation to a certain extent [ , , ]. In this study, compared with the diabetic model group, the serum levels of IL-1 β, IL-6, TNF - α in the metformin group were significantly reduced, and the levels of anti-inflammatory factor IL-10 were significantly increased. The results were consistent with those of bobae Hyun et al. [ , ], indicating that metformin can improve the level of inflammation in T2DM mice and has a certain anti-inflammatory effect. After treatment with different doses of LBP, serum IL-10 increased, TNF - α decreased, and IL-1 β decreased in the low dose group. Some studies have shown that the increase of IL-10 can inhibit macrophages' secretion of proinflammatory factors, alleviate obesity mediated inflammatory response, and improve insulin sensitivity of skeletal muscle [ , ]. When IL-6 is deficient, it can alleviate the condition of T2DM patients to a certain extent, while for TNF - α knockout mice, IR is also relieved [ , ]. This shows that LBP can inhibit the production of inflammatory factors, improve IR and alleviate T2DM, and the effect of LBP on inflammatory factors is similar to metformin. This result is consistent with the results reported by Ge JB et al. [ ] that LBP can reduce the expression of TNF - α, IL-6 and CRP in the retina of T2DM rats, play an anti-inflammatory role and alleviate the condition of T2DM rats, which provides a theoretical basis for the application of LBP in anti-inflammatory treatment of T2DM in the future.
IL-8 is an immunosuppressive factor secreted by activated monocytes. High concentration of IL-8 in serum is an important cause of late T2DM vascular complications, and also a marker of early diagnosis of diabetic nephropathy [ , ]. It is suggested that high glucose can stimulate the rapid rise of IL-8, which in turn can activate monocytes and neutrophils and other immune molecules, and aggravate the inflammatory damage of endothelial cells [ ]. In this study, the level of IL-8 did not change significantly. It has been suggested that in the process of TNF - α induced IR formation, IL-6 can promote insulin secretion, synergistically accelerate hyperinsulinemia, and produce toxic effects on B cells. At the same time, when there is a high level of IL-1 β in the body, it will release a large number of inflammatory factors such as TNF - α and IL-6 through the activation of inflammatory pathway to intensify the inflammatory response [ ]. However, IL-10 can inhibit NF - κ B activity by inhibiting the I κ B kinase complex, thereby reducing the expression of IL-8 [ ]. This shows that all kinds of cytokines in the body do not play a biological role alone, but there is a certain synergistic interaction, and they participate in the occurrence and development of diseases. This may also be the main reason that no obvious change of IL-8 was found in this study.
As a family member of transmembrane glycoprotein, TLR4 plays an important role in the innate immune response and the development of various diseases by mediating the release of inflammatory factors, chemokines and adhesion molecules through NF - κ B signaling pathway. TLR4 / NF - κ B is an important signaling pathway in inflammation. The activation of TLR4 / NF - κ B signaling pathway has been confirmed to be involved in the induction of adipocyte inflammation in T2DM patients. Blocking NF - κ B receptor activation signal can improve the IR state of the liver and prevent the occurrence of T2DM [].
Du Mingzhao et al. [] used 100, 250 and 500 mg / kg LBP to intervene STZ combined with high-fat diet induced diabetic SD rats, and found that compared with the normal group, NF - κ B in the kidney of the model group was highly activated, accompanied by the high expression of TNF - α, IL-6, IL-2 and other inflammatory factors in the serum. After three doses of LBP intervention, NF - κ B activation could be significantly inhibited. It is suggested that LBP can improve inflammation by inhibiting the activation of NF - κ B in T2DM rats. However, whether the inhibition is related to the inhibition of TLR4 / MyD88 dependent pathway upstream of NF - κ B has not been reported. In this study, we found that the protein levels of TLR4, MyD88, TRAF6, I κ K β, P-I κ B and nuclear NF - κ B in the model group were increased, and I κ B in the cytoplasm was decreased, suggesting that TLR4 mediated MyD88 dependent signaling pathway was activated in T2DM mice, which was consistent with the research results of Lin J and Han LP [ , ]. Duan D et al. [ ] mentioned that metformin can inhibit the activation of TLR4 / MyD88 / NF - κ B by activating AMPK signal pathway, and play an anti-inflammatory role. In this study, it was found that the expression level of I κ B protein in the cytoplasm of KKAy mice increased and the expression level of other key proteins decreased after the intervention of metformin, which indicated that metformin could inhibit the TLR4 / MyD88 dependent pathway activated in T2DM mice. After the intervention of LBP, the expression of TLR4, MyD88, TRAF6, I κ K β and NF - κ B protein in the nucleus can be significantly down regulated by low and medium doses of LBP. The level of I κ B in the cytoplasm of the three dose groups is increased, and the level of P-I κ B and NF - κ B in the nucleus can be significantly inhibited by high doses of LBP, which shows that LBP, like metformin, has a certain inhibitory effect on TLR4 / MyD88 dependent signaling pathway activated in T2DM.