The fruiting bodies of F. luteovirens were collected from Sêrxü County of Ganzi Autonomous Prefecture in Sichuan Province and identified by Professor Yu Li. The specimen was stored in the Herbarium of Mycology of Jilin Agricultural University (HMJAU) under specimen number HMJAU44964.

Fruiting bodies were placed in a drying oven, dried at 55 ℃ to a constant weight, crushed, and passed through an 80-mesh sieve. After degreasing with petroleum ether and 95% ethanol, crude polysaccharides were extracted using the classic water-extraction and alcohol-precipitation method [23, 24]. After the majority of proteins were removed using the papain enzyme-Sevag method, FLPs were obtained after dialysis (mw cutoff: 3500 Da) and lyophilization. The detailed extraction procedures are shown in Fig. 1.

Fig. 1.

Extraction and isolation of FLPs.

In this study, we used the phenol-sulfuric acid method [25], direct titration method [26], Kjeldahl determination (UDK169, VELP, Italy) [27], UV spectrophotometry [28], and periodate oxidation method [29] to analyze the levels of total sugars, reducing sugars, proteins, total flavonoids, and mannitol in FLP samples. The hydrolyzed amino acids in FLPs were determined by an automatic analyzer (Hitachi L-8900, Kyoto, Japan). The fatty acid levels were analyzed by the Gas chromatography mass spectrometry method (QP2010, Shimadzu, Kyoto, Japan) [30]. The mineral elements were analyzed using inductively coupled plasma optical emission spectrometry (ICP-OES) (Varian 720-ES, Varian, Palo Alto, CA, USA) and inductively coupled plasma mass spectrometry (ICP-MS) (BRUKER aurora M90, MA, USA) [30, 31].

Eight-week-old male db/m+ and db/db grade mice without specific pathogens (SPF) were provided by the Biomedical Research Institute of Nanjing University. The license of experimental animals was SCXK (Su) 2015-0001. The feeding temperature of the experimental animals was 23 $\pm$ 1 ℃, the relative humidity was 55 $\pm$ 5%, and the light-dark alternating time was 12 h. Animals were easily able to get food and water. All experiments were carried out in strict accordance with the Regulations on the Use of Experimental Animals and the China Animal Welfare Law, and were approved by the Experimental Animal Ethics Committee of Changchun University of Traditional Chinese Medicine.

After 1 week of adaptive feeding, all animals were used for experiments. A total of 10 db/m+ mice were considered as the controls (CK). The db/db mice with blood glucose $>$11.1 mmol/L were divided into 5 groups, 10 in each group. Drug administration was performed for 8 weeks, and the weight and blood glucose were measured weekly. The specific drug administration regimens were as follows: (a) model group–treatment with an equal volume of normal saline, n = 10; (b) positive control group – treatment with 0.10 g/kg metformin (Met) hydrochloride, n = 10; (c) low-dose group–treatment with FLP solution at 100 mg/kg, n = 10; (d) middle-dose group–treatment with FLP solution at 200 mg/kg, n = 10; (e) high-dose group–treatment with FLP solution at 400 mg/kg, n = 10; and (f) normal control group–treatment with an equal volume of normal saline, n = 10.

After drug administration for 8 weeks, db/db mice were fasted overnight (at least 12 h) after the last drug administration, drank water freely, and a OGTT was then performed. The weights and fasting blood glucose levels in mice were gauged. Mice in each group received intragastric administration of 2.0 g/kg glucose solution, and blood samples were obtained from the tail vein at 0, 30, 90, 120 and 240 minutes after intragastric administration to measure blood glucose levels. The area under the curve (AUC) of glucose was assessed by the trapezoidal method [32]: AUC = (blood glucose value at 0 min + blood glucose value at 30 min) $\times$ 0.25 + (blood glucose value at 30 min + blood glucose value at 60 min) $\times$ 0.25 + (blood glucose value at 60 min + blood glucose value at 120 min) $\times$ 0.5.

Serum collection: After 8 weeks of drug administration, mice were fasted for 12 h and drank freely. Blood specimens were collected from the retroorbital plexus and centrifuged rapidly for 10 min (3000 rpm). The supernatant was aspirated, and mouse serum specimens were obtained. For collection of organs: mice were sacrificed by cervical dislocation and rapidly dissected. Kidney, spleen, and liver tissues were collected and weighed. The remaining tissues were fixed in 4% paraformaldehyde or stored in a –80 ${}^{\circ }$C freezer for future use.

Glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), catalase (CAT), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), triglyceride (TG) and total cholesterol (TC) levels in serum samples were measured with ELISA kits. In addition, SOD, CAT, GSH-Px, ROS, tumor necrosis factor-$\alpha$ (TNF-$\alpha$), N-acetyl-$\beta$-D-glucosaminidase (NAG), interleukin-2 (IL-2), IL-6, interleukin-1$\beta$ (IL-1$\beta$), interferon-$\alpha$ (IFN-$\alpha$), IFN-$\gamma$, NF-$\kappa$B, and matrix metallopeptidase 9 (MMP-9) levels in kidney tissue were also measured.

The 4% paraformaldehyde was used to fix spleen and kidney tissues. After dehydration, paraffin was used for kidney tissues embedding, then cut into slices (thickness of approximately 5 $\mu$m), deparaffinized, subject to staining with periodic acid-Schiff (PAS) and hematoxylin-eosin (H&E), mounted, and observed under a microscope for pathological changes in kidney tissues.

The RIPA protein lysis buffer solution containing 1% protease inhibitor cocktail (cocktail P8340, Sigma-Aldrich, USA) and 2% PMSF (phenyl methyl sulfonyl fluoride) (P7626, Sigma-Aldrich, USA), which stored at low temperature for 10 minutes, were used to extract total protein from an appropriate amount of kidney tissue. After centrifugation, the protein content in supernatant was measured using the bicinchoninic acid protein assay kit (Merck Millipore, Darmstadt, Germany). Loading buffer was added into the protein sample. After denaturation at 95 ${}^{\circ }$C for 5 min, the specimens were aliquoted and storaged at –40 ${}^{\circ }$C. For all specimens, 40 $\mu$g was loaded. The gel was removed, and proteins were transferred onto a membrane at a low temperature for 120 min. The membrane was blocked in 5% BSA (A9647, Sigma-Aldrich, St. Louis, MO, USA) at 4 ${}^{\circ }$C for 4 h. The blocked polyvinylidene fluoride or polyvinylidene difluoride (PVDF) membrane was incubated with antibody, and the protein bands were visualized (UVP, Upland, CA, USA) [30]. Changes in protein expression of glyceralde-hyde-3-phosphate dehydrogenase (GAPDH), phosphor (P)-mTOR (S2448, ab109268; Abcam, Cambridge, MA, USA), total (T)-mTOR (ab83495; Abcam, Cambridge, MA, USA), P-GSK-3$\beta$ (Y216, ab75745), T-GSK-3$\beta$ (PK1111; Merck Millipore, Burlington, MA, USA), NRF2 (bs-1074R; Bioss, Beijing, China), CAT (ab52477), and HO-1 (ab137749) in mouse kidney tissue were assessed.

The results data were expressed as mean $\pm$ standard deviation (SD). The differences between groups were compared by ANOVA method in IBM SPSS 22.0 (IBM Corp., Armonk, NY, USA). Figures were plotted using GraphPad Prism 5.0 (GraphPad Software, San Diego, CA, USA).

Systemic composition analysis of the chemical composition of the FLPs samples indicated that FLPs comprised 65.0% total sugars, 7.2% reducing sugars, 7.93% proteins, and 0.36% total flavonoids, whereas mannitol was not detectable (Table 1). FLPs contained 17 types of amino acids including lysine at 0.949%, phenylalanine at 0.837%, glutamic acid at 0.753%, and aspartic acid at 0.674% (Table 2). The levels of 35 types of fatty acids in FLPs were measured, and the results indicated that FLPs contained 13 types of fatty acids (‰), such as oleic acid (43.50), palmitic acid (35.97), linoleic acid (25.47), stearic acid (13.19), elaidic acid (3.69) and arachidonic acid (2.48) (Table 3). FLPs contained 8 mineral elements. Potassium (K), calcium (Ca), and cupper (Cu); heavy metals were not detected (Table 4).

Compared with db/m+ mice, the body weight of untreated db/db mice was approximately 2–3 times higher and showed a significantly increasing trend; in addition, the fasting blood glucose level significantly increased, and liver, spleen, and kidney indicators significantly decreased (p 0.001, Table 5). After Met and FLPs administration in the positive control group for 8 weeks, mouse weights in Met group and FLPs drug administration groups had different levels of reduction compared with the model (db/db) group; however, their differences were not significant. The administration of FLPs (100 mg/kg) generated significant inhibitory effects on body weight during the last 4 weeks (p 0.001) (Table 5), indicating that FLPs inhibited excessive increases in mouse body weight and relieved obesity symptoms in db/db mice to some extent. Significant hypoglycemic effects were observed in the FLPs (200 mg/kg) drug administration group starting from week 6 (p 0.05, Table 5). The results showed that FLPs did not have significant effects on mouse liver and spleen indicators after 8 weeks of drug administration in the model group but significantly elevated the kidney indicators in model group (Table 5). Therefore, this research showed FLPs did not have toxic side effects on organs and generated certain improvements in kidney indicators in db/db mice.

One-hour plasma glucose (1-h PG), an accurate predictor of type 2 DM [33]. OGTTs were conducted in the mice in all groups. Fasting blood glucose levels at 0 min were measured first. After the intragastric administration of 2.0 g/kg glucose solution, the levels of glucose in all groups began to significantly increase. Compared to the model group, the AUC of the time-blood glucose curve showed a decreasing trend in the FLPs drug administration groups, indicating that FLPs accelerated glucose metabolism to reduce the blood glucose levels after intragastric administration of glucose (Fig. 2).

Fig. 2.

The AUC changes of oral glucose tolerance of db/db mice. ${}^{\mathrm{\#}\mathrm{\#}\mathrm{\#}}$p 0.001 versus db/m+ mice,*p 0.05 versus non-treated db/db mice.

Type 2 DM is usually accompanied by abnormal blood lipid metabolism, usually manifesting as significantly increased levels of LDL-C, TG, and TC, and significantly decreased levels of HDL-C [34]. The results showed that compared with db/m+ group, the serum TC and TG values of the db/db model group increased, while the HDL-C values decreased indicating that db/db mice had significant serum lipid metabolism abnormalities. After the administration of FLPs, serum TG and TC levels decreased significantly (p 0.05) (Fig. 3a,b). The FLPs (200 mg/kg) drug administration group exhibited increased HDL-C levels; however, the increase was not significant (Fig. 3c). These results indicated that FLPs reduced TC and TG; however, the effects on LDL-C and HDL-C were not significant.

Fig. 3.

Effects of FLPs on blood lipid metabolism in db/db mice. (a) The serum levels of TG; (b) the serum levels of TC, and (c) the serum levels of HDL-C in db/db mice compared to db/m+ mice. ${}^{\mathrm{\#}}$p 0.05 and ${}^{\mathrm{\#}\mathrm{\#}}$p 0.01 versus db/m+ mice; *p 0.05 versus non-treated db/db mice.

Oxidative stress is the unifying pathogenesis with respect to complications in DM. If the ability to clear ROS and RNS is weak, resulting a lack of homeostasis, a large amount of ROS will accumulate, causing cell injury. High blood glucose results in the excessive production of acetyl-CoA to produce more ROS and promote the progression of complications such as DM and DN [35, 36]. CAT is expressed in many tissues, such as serum, kidney and liver, and catalyzes the conversion of H${}_2$O${}_2$ into H${}_2$O and O${}_2$ to alleviate oxidative stress injury [37]. GSH-Px and SOD are also antioxidative stress enzymes; and function to clear free radicals such as H${}_2$O${}_2$ and O${}_2$${}^{-}$ [38].

3.4.1 Effects of FLPs on the Serum Antioxidant Ability of Mice

Compared with untreated db/db mice, in db/db mice, the serum CAT and GSH-Px values were increased after administration with 200 mg/kg FLPs (Fig. 4a,b), and the serum SOD values were increased after administered with 100 mg/kg and 400 mg/kg FLPs (p 0.01, Fig. 4c).

Fig. 4.

Effects of FLPs on the Serum Antioxidant Ability of Mice. (a) The serum levels of CAT; (b) the serum levels of GSH-Px, and (c) the serum levels of SOD in db/db mice compared to db/m+ mice. ${}^{\mathrm{\#}\mathrm{\#}}$p 0.01 and ${}^{\mathrm{\#}\mathrm{\#}\mathrm{\#}}$p 0.001 versus db/m+ mice; *p 0.05, **p 0.01 and ***p 0.001 versus non-treated db/db mice.

3.4.2 Effects of FLPs on the Kidney Antioxidant Ability in Mice

Compared with untreated db/db mice, the values of GSH-Px and SOD in the kidney were significantly increased in db/db mice administered with 200 mg/kg FLPs (p 0.05, Fig. 5a,b); CAT levels were slightly increased, but the increase was not significant (Fig. 5c). Compared with db/m+ mice, the ROS level in kidney tissues of untreated db/db mice increased, but not significantly. In addition, significant inhibitory effects on ROS levels were observed in the FLPs groups (Fig. 5d).

Fig. 5.

Effects of FLPs on the Kidney Antioxidant Ability in Mice. (a) GSH-Px, (b) SOD, (c) CAT, and (d) ROS in the kidney of db/db mice compared to db/m+ mice. ${}^{\mathrm{\#}}$p 0.05 and ${}^{\mathrm{\#}\mathrm{\#}\mathrm{\#}}$p 0.001 versus db/m+ mice; *p 0.05 and **p 0.01 versus non-treated db/db mice.

More and more evidence indicated that type 2 DM is an inflammatory disease. The low-grade inflammation in DN is different from classic inflammation and is often referred to as microinflammation. Many cytokines are closely related to the occurrence and progression of type 2 DM and its complications [39, 40, 41]. The levels of cytokines and inflammatory factors, i.e., TNF-$\alpha$, IFN-$\gamma$, IFN-$\alpha$, IL-2, IL-6, IL-1$\beta$, N-acetylglutamate (NAG), MMP-9, and NF-$\kappa$B, in kidney tissue were detected using ELISAs. The results indicated that compared to those in the model group, the levels of IL-2 and NAG in all groups administered FLPs were significantly decreased (p 0.001, Fig. 6a,b). In the FLPs group (200 mg/kg), the levels of IFN-$\alpha$ and IFN-$\gamma$ were significantly increased (p 0.01, Fig. 6c,d), with no significant effects on IL-6, MMP-9, IL-1$\beta$, TNF-$\alpha$, and NF- $\kappa$B. Staining of kidney tissue in db/db mice exhibited pathological changes in comparison to db/m+ mice, such as glomerular hypertrophy, dilation of the glomerular capillaries, inflammatory cell infiltration, and basement membrane thickening of renal tubular epithelial cells. After FLPs administration, these pathological changes in kidney tissues improved, and protective functions of the kidney were restored (Fig. 7). These results indicated that FLPs had certain inhibitory functions on some inflammatory factors in kidney tissue, enhanced immune functions, and alleviated the progression of DN complications.

Fig. 6.

Effects of FLPs on Kidney Inflammation. The levels of IL-2 (a), NAG (b), IFN-$\alpha$ (c), and IFN-$\gamma$ (d) in the kidney of db/db mice compared to db/m+ mice. ${}^{\mathrm{\#}}$p 0.05 and ${}^{\mathrm{\#}\mathrm{\#}}$p 0.01 versus db/m+ mice; * p 0.05, **p 0.01 and ***p 0.001 versus non-treated db/db mice.

Fig. 7.

The histopathological examination of kidney (A: H&E staining $\mathrm{\times }$ 200, B: PAS staining $\mathrm{\times }$ 200).

The potential mechanism of action of FLPs in DN was investigated using western blots. Compared to those in db/m+ mice, the expression levels of phosphorylated GSK-3$\beta$ and mTOR protein in kidney tissue of db/db mice significantly increased, while the expression levels of CAT, HO-1, and NRF2 protein were significantly decreased (p 0.01, p 0.001). After FLPs administration, compared with the db/db model group, the expression levels of GSK-3$\beta$ and mTOR aprotein significantly decreased, and all FLPs groups showed significant upregulation of NRF2 protein expression in the kidney of db/db mice. In addition, the levels of the antioxidases CAT and HO-1 in kidney tissue significantly increased (p 0.01, Fig. 8). These results showed that FLPs targeted and regulated GSK-3$\beta$ to further regulate the Keap1-independent nuclear factor erythroid 2-related factor 2/heme oxygenase 1 (NRF2/HO-1) signaling pathway to play an antioxidative and antiinflammatory role.

Fig. 8.

FLPs treatment regulated the expressions of P-mTor, P-GSK3$\beta$, HO-1, CAT and NRF2 in kidney. The data of quantitative protein expression were standardized to the levels of GAPDH and related total proteins. ${}^{\mathrm{\#}\mathrm{\#}}$p 0.01 and ${}^{\mathrm{\#}\mathrm{\#}\mathrm{\#}}$p 0.001 versus db/m+ mice; **p 0.01 and ***p 0.001 versus nontreated db/db mice.