The Regulatory Effect of Lycium Barbarum Polysaccharide on I κ B Phosphorylation and NF - κ B Nuclear Translocation in RAW264.7 Cells

Scope: RAW264.7 cells was used to study the regulatory effect of LBP on I κ B phosphorylation and NF κ B nuclear translocation. Methods: (1)After 5, 10, 20 and 30 minutes of LBP intervention, Western blotting was used to detect the expression of I κ B and P-I κ B in each group, (2)After 5 minutes of intervention with different doses of LBP,Western blotting was used to detect the expression of P-I κ B and I κ B in the cells of each group, (3)After 30 minutes of LBP intervention, the distribution of NF - κ B nuclear translocation was observed by immunouorescence, and the number of NF - κ B nuclear translocation was detected by Western blotting. Results : The results of the time-dependent regulation of I κ B phosphorylation by LBP showed that when stimulated by LPS, I κ B phosphorylated rapidly, the level of P-I κ B increased and I κ B decreased, and the level of I κ B increased at each time point in the combined treatment group of LPS (1 μ g / ml) + LBP (25 μ g / ml). in cytoplasm increased (P < 0.05), and NF - κ B level in nucleus decreased (P < 0.05).


Introduction
NF -κB is a key regulatory factor in the in ammatory mechanism of T2DM. Both NF -κB and its inhibitor I κB are key molecules of TLR4 / MyD88 dependent pathway, and play an important role in mediating the activation of in ammatory cytokines transcription [] . Under normal conditions, NF -κB and inhibitor I κB are stably bound in the cytoplasm and exist in the inactive form. When the cells are stimulated to produce stress, phosphorylation of I κB can release the inhibition of NF -κB in the cytoplasm, and make NF -κB shift to the nucleus to regulate the transcription and expression of a variety of in ammatory factors, such as TNF-α, IL-1 β, IL-6, etc. In turn, the high expression of in ammatory factors will further increase the activation of this signaling pathway, up regulate the activity of NF -κB, and aggravate the in ammatory cascade reaction [] .
RAW264.7 cells are derived from mouse peritoneal macrophages. It has been con rmed that RAW264.7 cells stimulated by lipopolysaccharide (LPS) are one of the classical cell models to study in ammation [, ] . LPS can induce the formation of M1 macrophages, activate NF -κB signaling pathway, induce IL-1β, TNF-α, IL-6, IL-8, nitric oxide (no) and inducible nitric oxide synthetase (inducible nitric oxide) Synthase, iNOS and so on [, ] , and the activation of NF -κB signaling pathway and the generation of a large number of in ammatory factors have been con rmed to be closely related to the pathogenesis and in ammatory response of T2DM.
The fruit of Lycium barbarum (Goji berry) has been commonly used as traditional Chinese medicine and herbal food for health promotion in countries. Lycium barbarumpolysaccharides (LBP) has been most widely researched and considered to be the main bioactive substance. The monosaccharide composition of LBP contained rhamnose, arabinose, xylose, mannose, glucose, galactose, galacturonic acid [] .
In this study, LPS stimulated RAW264.7 macrophages were used to study the regulatory effect of LBP on subsequent I κ B phosphorylation and NF -κ B nuclear translocation in MyD88 dependent pathway. The effects of LBP on the phosphorylation of I κ B in dose-response, time-dependent and nuclear translocation of NF -κ B were studied.

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 ltered 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%.

Cell lines and reagents
The mouse macrophage RAW264.7 cells were purchased from China Center for Type Culture Collection (Shanghai, China).
Rabbit anti-β-Actin monoclonal antibody, rabbit monoyclonal antibody against NF-κB p65, IκB etc. were purchased from Abcam (Cambridge, MA). Secondary antibodies were obtained from Boster Co.(Wuhan, China). Goat anti rabbit IgG labeled uorescent antibody was purchased from Santa cruz (Shanghai, China). DMEM medium, fetal bovine serum (FBS), Penicillin and Streptomycin Solution were purchased from HyClone (Logan, UT, USA). LPS were purchased from Sigma (St. Louis, MO, USA). All other chemicals and reagents used in this study were of analytical grade.
Cell culture RAW 264.7 cells were cultured in DMEM medium, supplemented with 10% FBS, 100 U/mL Penicillin, 100 µg/mL Streptomycin Solution and maintained at 37 °C in a humidi ed atmosphere with 5% CO 2 .

Total and nuclear protein extraction
For I κ B, P-I κ B, NF -κ B etc. analysis, protein expression by western blotting, total and nuclear protein extracts were prepared from pure macrophage using commercial kits (Biosynthesis Biotechnology Co., LTD, Beijing, China). The protein concentration was determined by bicinchoninic acid (BCA) assay and stored at − 80 °C until analyzed.

Western blotting
Sixty (60) µg of cell extract were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene di uoride (PVDF) membrane (Millipore, Bedford, MA, USA). The membrane was blocked for 1 h with 5% non fat milk in TBST and then incubated with a rabbit monoyclonal antibody against I κ B (AbcamCompany, UK) at 4 °C over night. After washing with TBST, the membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary antirabbit antibodies (1:5000; Boster Co., Wuhan, China) for 60 min at room temperature. After additional washing, bound conjugates were detected by ECL superSignalTM West Pico substrate (Pierce, Rockford, IL, USA). Proteins were visualized by exposing the blot to X-ray lm, 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 I κ B protein expression was normalized to Actin.
The WB process of P-I κ B, NF -κ B are the same as that of I κ B.

Statistical analysis
All data are expressed as means ± standard error (SEM) and represented at least three independent experiments. ANOVA and Student's t-test were used to perform multiple comparisons for statistical signi cance, and p < 0.05 was considered statistically signi cant.

Time dependent regulation of I κ B phosphorylation by LBP
When the cells were stimulated by LPS, I κ B phosphorylated rapidly, the level of P-I κ B increased and I κ B decreased (see Fig. 1). The phosphorylated I κ B is then ubiquitinated and nally recognized and degraded by proteasome. After adding LBP 5 min alone, I κ B in the cells increased, I κ B level decreased after 10 min, and I κ B increased after 20 min to 30 min, indicating that LBP can also activate I κ B, but its effect is more moderate compared with LPS model. After LPS (1 µ g / ml) combined with LBP (25 µ g / ml), the level of I κ B increased at each time point compared with LPS model, which indicated that LBP inhibited the phosphorylation of I κ B caused by LPS stimulation. Figure 2 After LPS stimulation, compared with the blank group, the expression of I κ B decreased and the expression of phosphorylated I κ B increased (P < 0.05), indicating that LPS has a certain stimulating effect on I κ B. This is consistent with the results of Peng Qiang et al.

Dose effect regulation of I κ B phosphorylation by LBP
The level of I κ B in RAW264.7 cells stimulated by LBP alone (25, 50, 100 µ g / ml) decreased (P < 0.05), but there was no signi cant difference compared with LPS model group (P > 0.05), indicating that the stimulation of I κ B by LBP alone was similar to LPS. Previous studies have shown that LBP can increase the number of peritoneal macrophages and pseudopods in mice, enhance the phagocytic ability, and improve the immune ability of the body [, , ] . In this study, compared with the blank group, the I κ B decreased, the NF -κ B distribution and expression in the cytoplasm decreased, and the NF -κ B distribution in the nucleus increased after the addition of LBP alone, indicating that LBP also has the activation effect on the NF -κ B pathway in RAW264.7 cells, which may be related to the fact that both When LBP (25, 50, 100 µ g / ml) was used alone, the level of P-I κ B in the cells was lower than that in LPS group (P < 0.05), suggesting that the stimulation effect of LBP on I κ B was weaker than that of LPS. After LPS (L µ g / ml) + LBP (25,50 µ g / ml) treatment, the level of I κ B in the cells increased (P < 0.05), which indicated that LBP could weaken the phosphorylation of I κ B by LPS.
Zhang Xiaorui et al. [, ] showed that lbpf4-ol puri ed from LBP could signi cantly up regulate TLR4 / MD2 expression in peritoneal macrophages and RAW264.7 cells. Combined with the results of this study, LBP could activate TLR4 / NF -κ B pathway in macrophages. For the cells only added with LBP, compared with the blank group, the expression level of P-I κ B did not increase, it is possible that ubiquitination of I κ B occurs rapidly after phosphorylation, and is nally recognized and degraded by proteasome.

Regulation of nuclear translocation of NF -κ B by LBP
In Fig. 3, green uorescence represents NF -κ B. It can be seen that there are a lot of green uorescence in the cytoplasm area of normal cells, and the color of the nuclear area is relatively dark. After stimulated by LPS, NF -κ B was activated into the nucleus, so there was a lot of green uorescence in the nucleus. When macrophages were treated with 25 µ g / ml LBP alone for 30 minutes, the green uorescence was distributed in both nucleus and cytoplasm. This indicated that the activation of NF -κ B by LBP was milder than that of LPS. When the cells were treated with LPS (L µ g / ml) + LBP (25 µ g / ml), compared with the LPS model group, the distribution of green uorescence in the cytoplasm increased, which indicated that LBP had a certain inhibitory effect on LPS induced NF -κ B intense nuclear translocation.
As can be seen from Fig. 4, there is a large amount of NF -κ B in the normal growth cell cytoplasm, and the expression level of NF -κ B in the nucleus is low. When LPS stimulated the cells, compared with the blank group, the relative expression of NF -κ B in the cytoplasm decreased (P < 0.05), and the level of NFκ B in the nucleus increased signi cantly (P < 0.05), indicating that LPS can activate NF -κ B.
When LBP (25, 50, 100 µ g / ml) alone was used to stimulate the cells, the level of NF -κ B in the cytoplasm decreased compared with that of the normal control group (P < 0.05), but the decrease was signi cantly smaller than that of LPS model, which indicated that LBP alone could stimulate NF -κ B activation, but it was milder than LPS.
After LPS (1 µ g / ml) + LBP (25, 50, 100 µ g / ml) was used, the NF -κ B level in the cytoplasm was signi cantly higher than that in the LPS model group (P < 0.05), while the NF -κ B level in the nucleus was signi cantly lower than that in the LPS model group (P < 0.05), which indicated that LBP could effectively inhibit the in ammatory stimulation of LPS on cell.
Wang Yalin et al. [, ] found that Dictyophora polysaccharide can inhibit LPS induced NF -κ B in ammatory signal pathway activation by regulating TLR4 expression, I κ B α phosphorylation and NF -κ B nuclear translocation, Peng Qiang et al [, , ] used L. rutenicum polysaccharide (LRGP3) to interfere with LPS stimulated RAW264.7 cells, the results showed that LRGP3 signi cantly inhibited the production of TNF -α, IL-6, NO, the expression of iNOS, TLR4 and the degradation of I κ B α, and reduced the expression of p65nf -κ B protein. The above studies showed that plant polysaccharides had a certain inhibitory effect on the in ammatory activation of RAW264.7 cells induced by LPS.
In this study, compared with LPS group, the level of I κ B in RAW264.7 cells treated with LBP & LPS increased, the distribution and expression of NF -κ B in the cytoplasm increased, and the distribution and expression of NF -κ B in the nucleus decreased, indicating that LBP has a certain inhibitory effect on I κ B phosphorylation and NF -κ B nuclear translocation downstream of TLR4 / MyD88 dependent pathway in LPS stimulated macrophages. Note: compared with LPS group, Pa<0.05.

Figure 2
Dose effect regulation of I κ B phosphorylation by LBP Note: compared with control, Pa<0.05; compared with LPS group, Pb<0.05 .

Figure 3
Immuno uorescence analysis: Regulation of nuclear translocation of NF -κ B by LBP