Lycium barbarum Polysaccharides Suppress Radiation-Induced Microglial Inflammation by Inhibiting the IKKβ/IκBα/NF-κB Pathway

Current research examined Lycium barbarum polysaccharides (LBP) for combating LPS-induced inflammatory responses in primary bovine mammary epithelial cells. We uncovered a preventive role of LBP in reducing detrimental effects induced by LPS including inhibition of NF-κB and MAPK along with peroxisome proliferator-activated receptor-γ (PPARγ) activation. The decrease in cell proliferation due to LPS was curtailed by pretreatment with LBP. Moreover, the effect of LBP on regulation of inflammatory responses in bovine mammary epithelial cell was PPARγ dependent. Collectively, data suggest that LBP reverses LPS-induced inflammatory response via MAPK/NF-κB signaling in a PPARγ-activation-dependent manner. Thus, the study provides new insights into therapeutic strategies for combating mastitis using LBP and highlighted the link between PPARγ and regulation of mammary cell inflammation.

Lycium Barbarum Polysaccharides (LBP) are the active components of Wolfberry (a traditional Chinese medicine) which has long been used for improving visual function. This study aims to investigate localized changes of retinal function in a partial optic nerve transection (PONT) model, and effects of LBP on visual function. The multifocal electroretinograms (mfERG) were obtained from 30 eyes of 30 Sprague-Dawley rats. The rats were divided into 6 groups (five treatment groups and one control group). Starting from the first day of the experiment, the rats in the (PONT+LBP) group and the (LBP) group were dosed with LBP; rats in the (PONT+PBS (phosphate buffered saline)) group and the (PBS) group were dosed with PBS via nasogastric tube every day until euthanized. The dorsal part of the optic nerve was transected in the (PONT), (PONT+LBP) and (PONT+PBS) groups at the end of week 1 (day 7 after LBP or PBS feeding began). The mfERG was measured at three time points: week 2, week 3 and week 5. Significant reduction of P1 and PhNR amplitudes of the mfERG were observed in all retinal regions a week after PONT. Feeding with LBP prior to PONT preserved retinal function. All mfERG responses returned to the normal range in the superior retina, which corresponds to the transected dorsal region of the optic nerve, while most of the inferior retinal responses were significantly increased at week 4 after PONT. The ventral part of the retina had secondary degeneration which was not only limited to the ganglion cell layer, but is a widespread effect affecting the outer retina. LBP altered the functional reduction caused by PONT by regulating the signal from the outer retina.

Cell injury in the cardiovascular endothelia caused by oxidative stress is among the major inducers of endothelium dysfunction and serves an important role in initiating cardiovascular diseases (CVDs). Therefore, protecting and improving the normal function of endothelial cells are considered key measures against CVDs. As a traditional Chinese medicinal component, Lycium barbarum is regarded to have high medicinal value. The present study aimed to investigate the potential anti-apoptosis and anti-oxidation effects of Lycium barbarum polysaccharides (LBPs) on injured rat artery endothelial cells, to demonstrate the experimental and medicinal values of LBPs. In the present study, the aortic endothelial cells of rats were cultivated and randomly divided into five groups: A control group, H2O2-injured group (H2O2 group), H2O2+LBPs (110 µg/ml) group (low-dose group, LT), H2O2+LBPs (220 µg/ml) group (medium-dose group, MT) and H2O2+LBPs (440 µg/ml) group (high-dose group, HT). Among these, the activity of superoxide dismutase (SOD), and the levels of malondialdehyde (MDA) and nitric oxide (NO) were detected by colorimetry. Additionally, the expression of B-cell lymphoma-2 (Bcl-2) and Bcl-2-associated X protein (Bax) were detected by western blotting. It was observed that SOD activity and NO content decreased while MDA content increased significantly in the H2O2 group (P<0.05 vs. control); that SOD activity in the MT and HT group, and NO content in all three LBP groups were increased, while MDA content in the three LBP groups was decreased, compared with the H2O2 group (all P<0.05); that Bcl-2 expression decreased significantly in the H2O2 group while the expression of Bax increased significantly compared with the control group (both P<0.05); and that Bcl-2 expression in all three LBP groups increased, while Bax expression in the MT and HT groups decreased compared with the H2O2 group (all P<0.05), with these altered Bax levels being statistically similar to those in the control group (P>0.05). On light microscopy, the cells in the control group exhibited spindle-shaped morphology, consistent sizes, defined boundaries, and distinct nuclei of equivalent sizes with round or oval morphology. Additionally, the chromatin in the nuclei was evenly distributed, and all cells were adhered in a paving-stone arrangement. Notably, only few cells died. Conversely, the cells in the H2O2 group exhibited signs of damage and enlarged gaps, and focal cells died. In the HT group, the cells once again appeared adherent and exhibited similar morphological status to the normal cells. Overall, these results indicate that LBPs serve a protective role in oxidative-injured vascular endothelial cells through anti-apoptosis and anti-oxidation effects.

The effect of Lycium barbarum polysaccharides on the male rats׳ reproductive system and spermatogenic cell apoptosis exposed to low-dose ionizing irradiation

Background The primary pathological basis of diabetic retinopathy (DR) is new blood formation due to anoxia and inflammation,which results in breakdown of blood-retinal barrier (BRB).Vascular endothelial growth factor (VEGF) is a key factor promoting neovascularization.Researches determined that lycium barbarum polysaccharides (LBP) can protect cells against oxidative damage.However,the study of LBP in ophthalmology is lack.Objective This study was to investigate the effects of LBP on the dynamic pathological change of retinal vessels and expression of VEGF in retina of diabetic rats.Methods One hundred and seventeen SPF male SD rats were randomly divided into the normal control group,the diabetic mellitus (DM) group and the LBP group according to random number table.Type 1 DM models were induced by intraperitoneal injection of streptozotocin (STZ,55 mg/kg) in the rats of the DM group and the LBP group,and then 250 mg/kg LBP was intragastically administered in the rats of the LBP group.The morphological change of retinal vessels was dynamically observed by retinal stretched preparation with Evans blue (EB) in 4,10 and 16 weeks after modeling.E B (45 mg/kg) was slowly injection via jugular vein,and 1％ polyoxymethylene was infused into the left ventricule.The eyeballs were extracted and retina were isolated.EB content in the retinas (mg/g) was calculated using retinal stretched preparation method at the time points mentioned above.Expressions of VEGF protein and mRNA in the retinas were detected by immunohistochemistry and real-time quantitative PCR at various time points,respectively.Results Retinal stretched preparation with EB exhibited that the abnormal degree in the shape,diameter of vessels and leakage of the retinal blood vessels were significantly slighter in the LBP group than those of the DM group in 4,10,16 weeks after modeling.At 4,10,16 weeks,EB content in the retinas was (12.17±1.55),(16.46±1.60) and (19.55±1.49) mg/g,which was significantly lower than (15.76± 1.90),(21.61 ±2.05) and (26.30±2.28) mg/g of the DM group (P＜0.05).Immunochemistry showed that the expression of VEGF protein primarily located at retinal ganglion cells (RGCs) layer.The staining intensity for VEGF protein was weaker in the LBP group than that of the DM group.The expression levels of VEGF protein (A value) in the LBP group were 0.234±0.011,0.331±0.023 and 0.536±0.031at various time points,with significant decline in comparison with 0.281±0.018,0.533±0.055 and 0.765±0.075 of the DM group (all at P＜0.05).Real-time quantitative PCR revealed that the expression levels of VEGF mRNA were 0.157±0.013,0.505 ±0.114 and 1.577±0.074 in the LBP group at various time points,which were significantly lower than 0.235±0.209,1.043±0.084 and 2.446±0.061 of the DM group (all at P＜0.05).Conclusions LBP can alleviate the DM-induced retinal vasculopathy,lessen the leakage of vessels well,and further protect the BRB. Key words: Diabetes mellitus/complication; Retina/diabetic retinopathy; Lycium barbarum polysaccharide; Vascular endothelial growth factor

Objective To investigate the effect of lycium barbarum polysaccharides (LBP) on oxidative stress in renal tissue of rats with renal interstitial fibrosis (RIF). Methods The RIF rat model was established by unilateral ureteral obstruction (UUO). A total of 108 specified pathogen free (SPF) class healthy adult male Sprague-Dawley (SD) rats were randomly divided into sham operation group, UUO model group and treatment group. The treatment group was further divided into low, medium and high dose of LBP groups and benazapril group. From the next day of the operation, the rats were given continuous intragastric administration for 3 weeks. The LBP low, medium and high dose groups were given 400, 600, 800 mg·kg-1·d-1 LBP, respectively. The benazapril group was administered with 1.05 mg·kg-1·d-1 benazepril hydrochloride. The sham operation group and UUO model group were daily fed normal saline solution by gavage. Six rats were sacrificed randomly at 7, 14 and 21 days after operation. Their blood samples were collected to detect the serum creatinine (Scr) and the kidney organ index was calculated. The pathological changes on the surgical side were observed by both HE staining and Masson staining. Meanwhile, the levels of malondialdehyde (MDA) and superoxide dismutase (SOD) in the renal tissue were detected by colorimetry detection. The expression of transforming growth factor-β1 (TGF-β1) protein was detected by immunohistochemical staining and the expression of TGF-β1 mRNA was detected by real time PCR. Results (1) Compared with the sham group, the Scr and kidney organ index of the UUO model group and treatment groups increased at each time point (all P<0.05). Compared with the UUO model group, the kidney organ index of LBP low dose group in the 7th days, the LBP medium and high dose group in the 21st days as well as benazapril group in the 7th and 21st days were significantly lower (all P<0.05). (2) Renal pathological change: compared with the sham operation group, both the renal tubular interstitial injury index and collagen positive area of the else groups were higher at each time point (all P<0.05). Compared with the UUO group, the tubulointerstitial injury index and collagen staining positive area of LBP dose groups and benazapril group significantly decreased at different time points (all P<0.05). (3) Compared with the sham group, in renal tissue of the other groups the level of MDA increased, SOD level decreased, while the expressions of TGF-1 mRNA and protein increased (all P<0.05). Compared with the UUO model group, LBP low, medium and high dose group as well as benazapril group had lower MDA level, higher SOD level as well as lower expressions of TGF-1 mRNA and protein at each time point (all P<0.05). Conclusions The pathological injury in UUO rats can be improved by the LBP. The LBP can alleviate the oxidative stress status of the kidney tissue by decreasing MDA and increasing SOD. The further study on the LBP delaying the progression of RIF is to be conducted. Key words: Fibrosis; Ureteral obstruction; Oxidative stress; Lycium barbarum polysaccharides

Background The pathogenesiof age-related maculadegeneration (AMD) iassociated with the senility of human retinal pigmenepithelium (RPE) cells.Seeking drug to arresRPE cell senility iof significance fothe prevention and treatmenof AMD.Research showed thathe lycium barbarum polysaccharide (LBP) can delay senility,buitinfluence on RPE cell aging iunclear.Objective Thistudy wato discusthe protective effecand mechanism of LBP on RPE cell aging.MethodPorcine retinal neural epithelial layewaisolated,and photoreceptooutesegmen(POS) waextracted by density gradiencentrifugation and marked by FITC.The POwathen co-cultured with RPE cellin the medium containing 0.01,0.10 and 1.00 g/L LBP fo24 hours.The areof fluorescence,representing the amounof POphagocytosed by RPE cells,wameasured undethe fluorescenmicroscope to evaluate the influence of LBP on the phagocytifunction of RPE cells.The POS-induced RPE lipofuscin-uptake cell model waestablished by co-culturing human RPE cellwith porcine POfo3 weeks.The RPE-POco-culture cell model waincubated in medium containing 0.01,0.10 o1.00 g/L LBP,and the autofluorescence caused by lipofuscin up-taken into RPE cellwadetected with flow cytometry.cell counting kiwaused to assescell proliferation and viability (value) 24,48 and 72 hourafteculturing.ResultPorcine POpresented athin rodundethe lighmicroscope and appeared abilayedisc-like structureundethe transmission electron microscope,and itFITC-labeled yellow-green autofluorescence waobserved undethe fluorescenmicroscope.No POwaup-taken into the RPE cellin the normal control group,buthe areof POphagocytosed by RPE cellwagradually enlarged with increasing doseof LBP,showing significandifference among the group(F =21.425,P =0.006).Compared with the POcontrol group,the phagocytosed areincreased avariouconcentrationof LBP+POgroup(P＜0.01).Flow cytometry showed thathe autofluorescence value in the POcontrol group wamore highethan thaof the normal control group.Athe LBP dose increased,the autofluorescence value in the RPE celldeclined gradually and iwaneathe normal value in the 1.00 g/L LBP+ POgroup.The rate of proliferation of the lipofuscin RPE cellvaried with the increase of doseof LBP with the maximal value in the normal RPE group and minimal value in the lipofuscin RPE group,and the rate of proliferation of the lipofuscin RPE cellascended with increasing doseof LBP until neathe normal value in the 1.00 g/L LBP + lipofuscin RPE cellgroup (P＞0.05).ConclusionLBP enhance the anti-aging effecof human RPE cellby strengthening the phagocytiability to POand the ability to remove lipofuscin and by heightening the proliferation of human RPE cells. Key words: Retinal pigment epithelial cell; Lycium barbarum polysaccharide; Senility; Photoreceptor

Objective To study the effects of different concentration lycium barbarum polysaccharide (LBP) on mouse islet β cell line MIN6 cells viability and apoptosis.Methods MIN6 cells were cultured and divided into control group and different concentration LBP intervene group,treated separately with 0,100,200,400 mg/L LBP for 24 h.Cells viability and apoptosis were separately detected by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and flow cytometry.The expression of phosphorylated extracellular regulated protein kinase (ERK) and B-cell lymphoma 2 (Bcl-2) were analyzed by Western blot.Results Compared with control group,MIN6 cells viability was promoted (all P ＜ 0.01),apoptosis was repressed (all P ＜0.01) in 100,200 mg/L LBP intervene group,while MIN6 cells viability was decreased (P ＜ 0.05) and apoptosis was increased (P ＜ 0.05) in 400 mg/L LBP intervene group.Compared with control group,protein levels of Bcl-2 and phosphorylated ERK were increased in 100,200,400 mg/L LBP intervene group (F =65.26,P ＜0.01 ; F =14.85,P ＜0.01),and decreased in 400 mg/L LBP intervene group.The difference of levels of Bcl-2 and phosphorylated ERK were statistically significant between 200 mg/L LBP intervene group and 400 mg/L LBP intervene group (all P ＜ 0.01).Conclusions Lower concentration LBP could promote the proliferation and inhibit the apoptosis of MIN6 cells,while the effects of higher concentration LBP were on the contrary.The mechanisms were associated with ERK signaling pathway. Key words: Lycium barbarum polysaccharide; MIN6 cells ; Cells viability ; Apoptosis

The impacts of natural plant extracts, including Lycium barbarum polysaccharides (LBP), mulberry leaf flavonoids (MLF), and ginkgo leaf flavonoids (GLF), on high glucose (HG)-induced injury in HBZY-1 mesangial cells were analyzed. LBP, MLF, and GLF were individually extracted, and their purities were determined. HBZY-1 cells cultured in low glucose (LG) served as the Normal group (NG), while HG culture represented the HG group. Subsequently, HBZY-1 cells in the HG group were treated with 300 μg/mL of LBP, MLF, and GLF, resulting in the HG+LBP, HG+MLF, and HG+GLF groups, respectively. Cell viability (CV) was assessed using the MTT assay, and cellular morphological changes were observed through HE staining. Meanwhile, lactate dehydrogenase (LDH) and nitric oxide (NO) levels were examined using the microplate method. Furthermore, malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) levels were detected by ELISA. mRNA levels in IL-6, IL-1β, and TNF-α were assessed using real-time quantitative polymerase chain reaction (rt-qPCR), and Collagen IV was examined using immunofluorescence. The results revealed that the polysaccharide content in the LBP extract was 63.57%, the flavonoid contents in the MLF and GLF extracts were 55.40% and 48.62%, respectively. Based on the NG group, HG group exhibited decreased CV, increased LDH and MDA, decreased NO, SOD, and GSH-Px, elevated mRNA levels in IL-6, IL-1β, and TNF-α, and reduced Collagen IV, showing great differences (P &lt; 0.05). Based on the HG group, the HG+LBP, HG+MLF, and HG+GLF groups demonstrated increased CV, decreased LDH and MDA levels, increased NO, SOD, and GSH-Px levels, decreased mRNA levels in IL-6, IL-1β, and TNF-α, and increased Collagen IV, presenting obvious differences (P &lt; 0.05). Comparing to the HG+LBP group, the HG+MLF and HG+GLF groups exhibited enhanced CV, downshifted LDH and MDA, elevated NO, SOD, and GSH-Px, downregulated mRNA in IL-6, IL-1β, and TNF-α, and increased Collagen IV (P &lt; 0.05). No significant changes in CV, LDH, MDA, NO, SOD, GSH-Px, IL-6, IL-1β, TNF-α, or Collagen IV were observed in the HG+MLF and HG+GLF groups to the NG group (P &gt;0.05). In conclusion, natural plant extracts such as LBP, MLF, and GLF exerted protective effects against injury in HBZY-1 mesangial cells caused by HG and possessed significant anti-inflammatory, antioxidant stress, and antifibrotic damage properties.

Disruption of colonic homeostasis caused by aberrant M1/M2 macrophage polarization contributes to the development of inflammatory bowel disease (IBD). Lycium barbarum polysaccharide (LBP) is the primary active constituent of traditional Chinese herbal Lycium barbarum L., which has been widely demonstrated to have important functions in regulating immune activity and anti-inflammatory. Thus, LBP may protect against IBD. To test this hypothesis, the DSS-induced colitis model was established in mice, then the mice were treated with LBP. The results indicated that LBP attenuated the weight loss, colon shortening, disease activity index (DAI), and histopathological scores of colon tissues in colitis mice, suggesting that LBP could protect against IBD. Besides, LBP decreased the number of M1 macrophages and the protein level of Nitric oxide synthase 2(NOS2) as a marker of M1 macrophages and enhanced the number of M2 macrophages and the protein level of Arginase 1(Arg-1) as a marker of M2 macrophages in colon tissues from mice with colitis, suggesting that LBP may protect against IBD by regulating macrophage polarization. Next, the mechanistic studies in RAW264.7 cells showed that LBP inhibited M1-like phenotype by inhibiting the phosphorylation of STAT1, and promoted M2-like phenotype by promoting the phosphorylation of STAT6. Finally, immunofluorescence double-staining results of colon tissues showed that LBP regulated STAT1 and STAT6 pathways in vivo. The results in the study demonstrated that LBP could protect against IBD by regulating macrophage polarization through the STAT1 and STAT6 pathways.

Lipopolysaccharide (LPS)-induced inflammation is the leading cause of multiple organ failure in sepsis. Pyruvate kinase 2 (PKM2) is a protein kinase and transcriptional coactivator that plays an important role in glycolysis. Recent studies have confirmed that glycolysis maintains the M1 differentiation and induces immune activation in macrophages. Lycium barbarum polysaccharide (LBP), the main bioactive component of Chinese wolfberry, suppresses glycolysis and inflammation. Here, RAW264.7 macrophages were treated with LBP for evaluating its effects against LPS-induced inflammation. The differentiation of M1/M2 macrophages was assessed by flow cytometry for assessing the cell surface markers, CD86 and CD206. The enrichment of hypoxia inducible factor (HIF)-1α and ubiquitin in the PKM2 protein complex was determined by co-immunoprecipitation. LBP suppressed LPS-induced glycolysis, differentiation of M1 macrophages, and the production of interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and high mobility group (HMG) 1 proteins. The suppressive effects of LBP were similar to those of PKM2 knockdown, but were abolished by the overexpression of PKM2. LPS elevated the mRNA and protein levels of PKM2. LBP reduced the LPS-induced expression of PKM2 protein, but had no effects on the expression of PKM2 mRNA. LPS inhibited the ubiquitination of PKM2, probably by downregulating the expression of ubiquitin ligases, including Nedd4L, Nedd4, and Gnb2. LBP interfered with the inhibition of PKM2 ubiquitination by upregulating the expression of Nedd4L, Nedd4, and Gnb2. In conclusion, LBP suppressed the LPS-induced inflammation by altering glycolysis and the M1 differentiation of macrophages. The effects of LBP were mediated by the downregulation of PKM2 via enhanced ubiquitination.

Objective To evaluate effects of Lycium barbarum polysaccharide (LBP) on expression of vascular endothelial growth factor (VEGF) and hypoxia inducible factor-1α (HIF-1α) in ultraviolet B (UVB) -radiated HaCaT cells. Methods Conventionally cultured HaCaT cells were divided into control group and LBP groups, which were firstly treated with DMEM, 12.5, 25.0, 50.0 and 100 μg/ml LBP solution respectively for 4 hours, and then were irradiated by UVB at different intensity of 0, 20, 40, 60 mJ/cm2 separately. After 24-hour continuing culture, CCK-8 assay was performed to determine the cell survival rate, and an enzymatic-biochemical method to estimate the activity of superoxide dismutase (SOD) . RT-PCR and Western blot analysis were conducted to measure the mRNA and protein expression of HIF-1α and VEGF respectively. Results Compared with the control group at the same UVB radiation dose, the 12.5-, 25.0-and 100.0-μg/ml LBP groups showed different extents of increase in survival rates of UVB-radiated cells (P < 0.05) , and the 50.0-μg/ml LBP group showed the highest cell survival rate (P < 0.01) . Among all the LBP groups, SOD activity was highest in the 50.0-μg/ml LBP group (P < 0.01) . Along with the increase of UVB radiation dose, the mRNA and protein expression of HIF-1α and VEGF all gradually increased. Compared with the control group, the 50.0-μg/ml LBP group could effectively reduce the mRNA and protein expression of HIF-1α and VEGF in HaCaT cells (all P < 0.05) . Conclusion LBP may play a role in protecting cells from UVB radiation-mediated damage, likely by influencing the mRNA and protein expression of HIF-1α and VEGF in HaCaT cells. Key words: Ultraviolet rays; Lycium barbarum; Polysaccharides; Endothelial growth factors; Hypoxia inducible factor 1α; HaCaT cells

Objective To explore the effect of lycium barbarum polysaccharide (LBP) and decellularized nerve xenograft combination on nerve regeneration and recovery in repairing rat sciatic nerve defects. Methods Xenogeneic acellular nerve scaffolds were prepared by chemical extraction of tibial nerves from rabbits. A 10 mm left sciatic nerve defect model was created in 90 healthy adult Sprague-Dawley rats who were randomly divided into 5 groups: blank group (n=18), model group (n=18), LBP group (n=18), scaffold group (n=18), LBP and scaffold combination group (n=18). Two weeks after model creation. The sciatic nerve defect in LBP, scaffold and combination groups was repaired either by LBP, by scaffold, or by the LBP xenograft combination. After the second surgery, the rats survived for 4, 8 and 12 weeks, respectively. Gross observation of the scaffold, nerve conduction study to measure sciatic nerve motor nerve conduction velocity (MNCV), and neuromorphometry analysis of the graft were carried out at these three time points. Results Neuroma formation at the proximal nerve end was observed in the model group and LBP group. Adhesion of nerve ends to the surrounding tissue was more severe in the model group than in the LBP group. The sizes of the nerve grafts in the scaffold group and combination group were comparable. Proficient vascularization on the surface of these grafts was seen. There was only slight adhesion to the surrounding tissue. The regenerated nerve fibers in the combination group were denser and better oriented. Motor nerve conduction velocity recovery and the number of myelinated nerve fibers in the grafts were measured at 12 weeks postoperatively for blank, model, LBP, scaffold and combination groups, respectively. The differences among the groups were significant (P<0.05). Conclusion The combination of LBP and xenogeneic acellular nerve scaffold enhances peripheral nerve regeneration and recovery when used to repair nerve defects. Key words: Sciatic nerve; Nerve regeneration; Xenogeneic acellular nerve scaffold; Lycium barbarum polysaccharide

Objective: Here, we aimed to explore the effect of LBP in combination with Oxaliplatin (OXA) on reversing drug resistance in colon cancer cells through in vitro and in vivo experiments. We also aimed to explore the possible mechanism underlying this effect. Finally, we aimed to determine potential targets of Lycium barbarum polysaccharide (LBP) in colon cancer (CC) through network pharmacology and molecular docking. Methods: The invasion ability of colon cancer cells was assessed using the invasion assay. The migration ability of these cells was assessed using the migration assay and wound healing assay. Cell cycle analysis was carried out using flow cytometry. The expression levels of phosphomannose isomerase (PMI) and ATP-binding cassette transport protein of G2 (ABCG2) proteins were determined using immunofluorescence and western blotting. The expression levels of phosphatidylinositol3-kinase (PI3K), protein kinase B (AKT), B-cell lymphoma 2 (Bcl-2), and BCL2-Associated X (Bax) were determined using western blotting. Forty BALB/c nude mice purchased from Weitong Lihua, Beijing, for the in vivo analyses. The mice were randomly divided into eight groups. They were administered HCT116 and HCT116-OXR cells to prepare colon cancer xenograft models and then treated with PBS, LBP (50mg/kg), OXA (10mg/kg), or LBP + OXA (50mg/kg + 10mg/kg). The tumor weight and volume of treated model mice were measured, and organ toxicity was evaluated using hematoxylin and eosin staining. The expression levels of PMI, ABCG2, PI3K, and AKT proteins were then assessed using immunohistochemistry. Moreover, PMI and ABCG2 expression levels were analyzed using immunofluorescence and western blotting. The active components and possible targets of LBP in colon cancer were explored using in silico analysis. GeneCards was used to identify CC targets, and an online Venn analysis tool was used to determine intersection targets between these and LBP active components. The PPI network for intersection target protein interactions and the PPI network for interactions between the intersection target proteins and PMI was built using STRING and Cytoscape. To obtain putative targets of LBP in CC, we performed GO function enrichment and KEGG pathway enrichment analyses. Results: Compared with the HCT116-OXR blank treatment group, both invasion and migration abilities of HCT116-OXR cells were inhibited in the LBP + OXA (2.5mg/mL LBP, 10μΜ OXA) group (p < 0.05). Cells in the LBP + OXA (2.5mg/mL LBP, 10μΜ OXA) group were found to arrest in the G1 phase of the cell cycle. Knockdown of PMI was found to downregulate PI3K, AKT, and Bcl-2 (p < 0.05), while it was found to upregulate Bax (p < 0.05). After treatment with L. barbarum polysaccharide, 40 colon cancer subcutaneous tumor models showed a decrease in tumor size. There was no difference in the liver index after LBP treatment (p > 0.05). However, the spleen index decreased in the OXA and LBP + OXA groups (p < 0.05), possibly as a side effect of oxaliplatin. Immunohistochemistry, immunofluorescence, and western blotting showed that LBP + OXA treatment decreased PMI and ABCG2 expression levels (p < 0.05). Moreover, immunohistochemistry showed that LBP + OXA treatment decreased the expression levels of PI3K and AKT (p < 0.05). Network pharmacology analysis revealed 45 active LBP components, including carotenoids, phenylpropanoids, quercetin, xanthophylls, and other polyphenols. It also revealed 146 therapeutic targets of LBP, including AKT, SRC, EGFR, HRAS, STAT3, and MAPK3. KEGG pathway enrichment analysis showed that the LBP target proteins were enriched in pathways, including cancer-related signaling pathways, PI3K/AKT signaling pathway, and IL-17 signaling pathways. Finally, molecular docking experiments revealed that the active LBP components bind well with ABCG2 and PMI. conclusion: Our in vitro experiments showed that PMI knockdown downregulated PI3K, AKT, and Bcl-2 and upregulated Bax. This finding confirms that PMI plays a role in drug resistance by regulating the PI3K/AKT pathway and lays a foundation to study the mechanism underlying the reversal of colon cancer cell drug resistance by the combination of LBP and OXA. Our in vivo experiments showed that LBP combined with oxaliplatin could inhibit tumor growth. LBP showed no hepatic or splenic toxicity. LBP combined with oxaliplatin could downregulate the expression levels of PMI, ABCG2, PI3K, and AKT; it may thus have positive significance for the treatment of advanced metastatic colon cancer. Our network pharmacology analysis revealed the core targets of LBP in the treatment of CC as well as the pathways they are enriched in. It further verified the results of our in vitro and in vivo experiments, showing the involvement of multi-component, multi-target, and multi-pathway synergism in the drug-reversing effect of LBP in CC. Overall, the findings of the present study provide new avenues for the future clinical treatment of CC.

Vitiligo is a skin disease characterized by lack of functional melanocytes. Lycium barbarum polysaccharide (LBP) has been demonstrated to preserve keratinocytes and fibroblasts against oxidative stress. This study aimed to explore the efficacy and underlying mechanisms of LBP on autophagy in H2 O2 -damaged human melanocytes. Cellular viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and annexin V-fluorescein isothiocyanate/propidium iodide double staining. Reverse transcription-polymerase chain reaction, western blotting and electron microscopy were performed to detect autophagy. The protein expression level of Nrf2 and p62 were assessed by western blotting. Plasmid transfection and lentiviral infection were used to overexpress and silence Nrf2 in PIG1 cells. LBP promoted the proliferation and inhibited apoptosis of H2 O2 -damaged PIG1 cells. LBP increased the proliferation of H2 O2 -damaged PIG1 cells via induction of autophagy, and Nrf2 shRNA experiment confirmed that LBP activated the Nrf2/p62 signal pathway. These results suggest that LBP may be used for the treatment of vitiligo. PRACTICAL APPLICATIONS: Goji berry is the mature and dried fruit of Lycium barbarum L., which is a common food with a long history in China, as well as a Traditional Chinese Medicine. Our previous research found that LBP could activated the Nrf2/ARE pathway in an ultraviolet (UV)-induced photodamage model of keratinocytes, and increase the levels of phase II detoxification and antioxidant enzymes. We firstly confirmed the anti-vitiligo effects of L. barbarum polysaccharide (LBP) by inducing autophagy and promoted proliferation of human melanocytes, and LBP induced autophagy via activating the Nrf2/p62 signaling pathway in this study. These results proved that LBP can be an effective therapy for vitiligo treatment.