Effect and Mechanism of Lycium Barbarum Polysaccharide on Hyperglycemia and Inflammation in Diabetic KKAy Mice

Scope The aim of this study is to examine whether lycium barbarum polysaccharide (LBP) supplementation improves hyperglycemia and inflammation in diabetic KKAy mice. Methods The successfully established diabetic KKAy mice are randomized into five groups: diabetic model, metformin, low‐dose LBP, middle‐dose LBP, and high‐dose LBP . C57BL/6J mice are fed a chow diet as normal control . The blood glucose and body weight of mice were measured at different time points. At the end of 90 days, serum inflammatory factors were determined with ELISA kits. The expression of TLR4, MyD88, TRAF6, IκKβ, IκB, P-IκB and nuclear NF-κB proteins in mouse peritoneal macrophages were detected by Western Blotting. Results Blood glucose decreased significantly after the intervention among low-, medium-dose LBP groups and Met group ( P <0.05). Met (40 mg/kg) inhibited the levels of IL-1β, TNF-α and IL-6 and elevated IL-10 level ( P <0.05). ELISA results showed that LBP promoted serum levels of IL-10 and decreased TNF-α level ( P <0.05). Compared with model group, KKAy mice in Met group expressed lower protein levels of MyD88, TRAF6, IκKβ, nuclear NF-κB and higher expression of IκB ( P <0.05); The expression of TLR4, MyD88, TRAF6, IκKβ and nuclear NF-κB protein in low- and medium-doses LBP groups were significantly declined ( P <0.05). Conclusion These findings indicate that dietary supplementation with LBP can improve hyperglycemia and inflammation in diabetic KKAy mice, which can be associated with potential benefits to human health. (HRP)-conjugated secondary anti-rabbit antibodies for 60 min at room After additional washing, bound conjugates were detected by ECL superSignalTM West Pico substrate Proteins were visualized by exposing the blot to X-ray film, photographed with a digital camera, and then the net intensities of the individual bands were measured using Bandscan 5.0 software. Rabbit anti-β-Actin monoclonal antibody (AbcamCompany, UK) was used as the loading control, and TLR4 protein expression was normalized to Actin. three doses of LBP can effectively increase IL-10 (P < 0.05) and reduce TNF - α level (P < low doses of LBP can also significantly inhibit the increase of IL-1 β level (P < 0.05); and these changes have no statistical difference compared with the metformin group (P > 0.05), which shows that LBP can effectively change the levels of IL-10, TNF - α and IL-1 β in serum, and its action Compared with metformin, there was no significant difference (P > 0.05). can of by macrophages by inhibiting the differentiation of 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 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 the TNF - α, T2DM rats,


Introduction
The global report on diabetes from WHO clarifies that in 2014, 422 million people in the world had diabetes with a prevalence of 8.5% among adult population. The prevalence in China was higher than the global average prevalence, total adult prevalence of 9.4% with most diabetic patients in the world [] . Compared to non-diabetic patients, diabetes patients lead to complications, such as blindness, kidney failure, low limb amputation and so on, which consequently, significantly impact on quality of life. Recent studies have shown that diabetes mellitus and its complications are closely related to inflammation.
The fruit of Lycium barbarum (Goji berry) has been commonly used as traditional Chinese medicine and herbal food for health promotion in countries. Goji berry is mainly comprised of polysaccharides, carotenoids, vitamin C, flavonoids, essential oil, fatty acids, glycerogalactolipids, free amino acids and miscellaneous compounds. Among these constituents, Lycium barbarumpolysaccharides (LBP) has been most widely researched and considered to be the main bioactive substance. The In a word, the beneficial effects of LBP on diabetes have been confirmed by animal and clinical studies. However, the effect of LBP on inflammation is unclear, which may provide the new evidence of protection mechanism of LBP on diabetes. Therefore, in the present study we attempted to explore the effects of LBP on hyperglycemia and inflammation in diabetic KKAy mice.

Materials And Methods
Preparation of LBP LBP was prepared as described previously [] . Dried L. barbarum was made into a powder and decocted with water (60 °C) by a traditional method used for Chinese medicinal herbs after degreasing. Then it was filtered by regenerated cellulose membranes of 300 kDa, 100 kDa, 80 kDa, 50 kDa and 30 kDa (0.2 MPa, 60 °C) after centrifuging. The resulting fraction was retained and vacuum-dried at 40 °C. Neutral sugars were determined by phenol-H 2 SO 4 , acidic sugars by carbazole and proteins by the Coomassie Brilliant Blue G-250 method .
LBP we prepared was a brown powder composed of neutral sugars (78.23%) and acidic sugars (14.83%). The protein content was < 6.92%.

Animals and Treatment
Five weeks old spontaneously diabetic female KKAy mice and age-matched female nondiabetic C57BL/6J mice were obtained from Beijing HFK Bioscience Co., Ltd (Beijing, China). All mice were housed one per cage at 22 ± 2 °C with a 12-h light/dark cycle. The KKAy mice were fed a high fat diet, which has applied for national invention patent of China (application number: CN201110127312.5).
The C57BL/6J mice (n = 10) were fed normal chow die as normal control (NC). The composition of the diets is shown in Table 1. After 5 weeks of high fat diet feeding when the fasting blood glucose was higher than 16.7 mmol L − 1 , the KKAy mice were randomly divided into five groups (n = 10 in each  photographed with a digital camera, and then the net intensities of the individual bands were measured using Bandscan 5.0 software. Rabbit anti-β-Actin monoclonal antibody (AbcamCompany, UK) was used as the loading control, and TLR4 protein expression was normalized to Actin.

Statistical analysis
Data were analyzed statistically using SPSS 16.0 for Windows and expressed as the mean ± SD of 10 mice per group. Experimental results were compared by one-way ANOVA with least significant difference (LSD) post-hoc tests used to compare individual means as appropriate. P < 0.05 or P < 0.01 were considered statistically significant.

Results
The weight changes of T2DM mice after LBP intervention As can be seen in Table 2, the weight of mice in each group increased significantly compared with normal control (P < 0.01). During the experiment, the weight of mice in each group increased significantly (P < 0.01), but during and after the intervention, there was no significant difference between the diabetic model group and other intervention groups (P > 0.05). Changes of blood glucose in T2DM mice after LBP intervention Table 3 shows that before the intervention, there was no statistical difference in blood glucose between the diabetic model group, metformin group and LBP low, medium and high dose groups (P > 0.05), and the difference was significant compared with normal control (P < 0.05). After 45 days of intervention, the blood glucose in the diabetic model group was significantly higher than that in metformin group and LBP low, medium and high dose group (P < 0.05). At the end of the intervention, there was a statistical difference between LBP low and middle dose group and diabitc model group (P < 0.05), no statistical difference between LBP high dose group and diabitc model group (P > 0.05), and there was a statistical difference between LBP high dose group and metformin group (P < 0.05).

Changes of serum inflammatory factors in T2DM mice after LBP intervention
It can be seen from Table 4 that compared with normal control, the levels of IL-1 β, IL-6, TNF -α in the serum of the diabetic model group increased (P < 0.05), and the levels of IL-10 and TGF -β 1 decreased (P < 0.05); the levels of IL-8 increased, but the difference was not statistically significant (P > 0.05). Compared with the diabetic model group, the levels of IL-1 β, IL-6, TNF -α decreased, IL-10 increased, TGF -β 1 and IL-8 had no statistical difference (P > 0.05), which indicated that metformin could effectively change the inflammatory state of T2DM model. After LBP intervention, compared with the diabetic model group, three doses of LBP can effectively increase IL-10 (P < 0.05) and reduce TNF -α level (P < 0.05); low doses of LBP can also significantly inhibit the increase of IL-1 β level (P < 0.05); and these changes have no statistical difference compared with the metformin group (P > 0.05), which shows that LBP can effectively change the levels of IL-10, TNF -α and IL-1 β in serum, and its action Compared with metformin, there was no significant difference (P > 0.05). Effects of LBP on TLR4, MyD88, TRAF6, I κ K β, I κ B, P-I κ B and NF -κ B protein expression in peritoneal macrophages of T2DM mice As shown in Fig. 1, compared with NC, the expression of TLR4, MyD88, TRAF6, I κ K β, P-I κ B in cytoplasm and NF -κ B in nucleus increased (P < 0.05) and I κ B in cytoplasm decreased (P < 0.05) in diabetic model group.
After the intervention, compared with the model group, the levels of MyD88, TRAF6, I κ K β and NF -κ B in the nucleus of metformin group decreased, the expression of I κ B in the cytoplasm increased (P < 0.05), TLR4 decreased, but the difference was not statistically significant (P > 0.05).
After LBP intervention, the expression of TLR4, MyD88, TRAF6, I κ K β and NF -κ B in the nucleus decreased significantly (P < 0.05). The expression of MyD88 and TRAF6 was even lower than that of metformin group (P < 0.05). The expression of I κ B in LBP group was higher than that of diabetic model group and metformin group (P < 0.05). The effect of LBP on TLR4, MyD88, TRAF6 and I κ K β protein was dose-dependent. The lower the concentration of LBP, the more obvious the inhibition.
However,the effect of middle dose LBP was the most significant of the effect on I κ B in cytoplasm and NF -κ B in nucleus.

Discussion
In this study, the body weight and blood sugar of KKAy mice were significantly increased compared 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 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