| Part 1 Effect of hesperetin on lipopolysaccharide-inducedcytokine levels in vitroObjective:To investigate the effect of hesperetin(HES)on lipopolysaccharide(LPS)-induced inflammatory cytokines in vitro.Methods: RAW 264.7,BEAS-2B cells were cultured in RPMI-1640,and inflammatory reaction in vitro was induced by lipopolysaccharide(LPS).The two cells were divided into 4 groups: blank control group(CON group),LPS group,LPS+HES(10?M)pretreatment group and LPS+HES(40 ?M)pretreatment group.RAW 264.7 and BEAS-2B cells were pretreated 2h with HES at a concentration of 10 and 40 ?M in LPS+HES(10 ?M)pretreatment group and LPS+HES(40 ?M)pretreatment group respectively.In addition,the cells were stimulated by LPS for 6h and 24 h respectively.The protein levels of TNF-? and IL-6 induced by LPS in vitro at different concentrations of HES were determined by ELISA stimulated by LPS for 24h;Real-time quantitative reverse transcriptase-polymerase chain reaction(RT-q PCR)was used to analyze the m RNA expression levels of inflammatory cytokines including TNF-? and IL-6 induced by LPS for 6h in vitro in different concentrations of HES.Results: Compared with the CON group,the m RNA expressions and protein levels of TNF-? and IL-6 in RAW264.7 cell were significantly up-regulated when exposed to LPS(P<0.01),while the m RNA levels were down-regulated both in 10 ?M and 40 ?M HES pretreatment groups(P<0.01).The similar results were observed in BEAS-2B cells:compared with the CON group,TNF-? and IL-6 m RNA expressions and protein levelswere reduced significantly with the pretreatment of HES with both 10 ?M and 40 ?M concentrations(P<0.05).Conclusion: HES inhibits LPS-induced inflammatory responses in vitro by attenuating the expression levels of the pro-inflammatory cytokines including TNF-? and IL-6,which is related to the concentration of HES.Part 2 Efect of hesperetin on lipopolysaccharide-induced acute lunginjury in vivoObjective: To investigate the protective effects of hesperetin(HES)on lipopolysaccharide(LPS)-induced acute lung injury(ALI)in vivo.Methods: The ALI mice model was induced by LPS after adaptive feeding in twenty-eight C57BL/6 mice(weighting 22±2g),which were randomly divided into 4groups(seven mice in each group): blank control group(CON group),LPS group,LPS+HES(25 mg/kg)group and LPS+HES(50 mg/kg)group.Mice in LPS+HES(25mg/kg)group and LPS+HES(50 mg/kg)group received difffferent concentrations HES(dissolved in 0.5% CMC-Na)at the volume of 200 ?l·20 g body weight for seven consecutive days prior to LPS challenge by gavage,respectively.Mice were killed 6 h after intratracheal instillation of LPS(5 mg/kg).Serum,bronchoalveolar lavage fluid(BAL fluid)and lung tissue samples were collected.The pathological changes of lung tissue were observed by HE staining;the total protein concentration in the supernatant of bronchoalveolar lavage fluid(BALF)was detected by Bradford protein detection kit;the total cell count in BALF was determined by standard blood cell count;The activity of myeloperoxidase(MPO)in homogenized lung tissue was determined by the enzyme detection kit;the protein concentrations of TNF-? and IL-6 in BALF and serum were determined by enzyme-linked immunosorbent assay(ELISA).The m RNA expression levels of TNF-? and IL-6 in lung tissue were analyzed by real-time quantitative reverse transcriptase-polymerase chain reaction(RT-q PCR)Results: Compared with the CON group,mice in LPS group had interstitial and alveolar interal inflammatory cell infiltration,alveolar integrity was destroyed,alveolar wall was thickening and had alveolar cavity hyperemia;compared with the LPS group,the histological changes induced by ALI were significantly reduced with the pretreatment of HES(25 mg/kg and 50 mg/kg)(P<0.01);HES reduced the degree of pulmonary edema in mice with ALI,and the effect was more obvious in the group with the higher concentration(P<0.01);LPS induced an increase in the total protein concentration in BALF,while HES reduced total protein concentration in BALF(P<0.05);HES significantly reduced the total cell number and neutrophil count in BALF of ALI mice,thereby helping to protect immune function(P<0.05);HES significantly reduced the high levels of MPO caused by LPS(P<0.05);LPS induced TNF-? protein overexpression in lung tissue;however,the HES group(25 mg/kg or 50 mg/kg)was shown to reduce the TNF-? protein expression in lung tissue induced by LPS;LPS increased the protein expression levels of TNF-? and IL-6 in BALF significantly(P<0.01),while HES could effectively reduce the protein expression of TNF-? and IL-6 in BALF(P<0.01).LPS could induce the increase of TNF-?and IL-6 in serum(P<0.01)and HES could decrease the TNF-? protein expression in serum.IL-6 protein expression was reduced in serum with HES(50 mg/kg)(P<0.01),there was no significant difference between HES(25 mg/kg)and LPS group(P>0.05);LPS could induce an increase in m RNA levels of TNF-? and IL-6(P<0.01)in lung tissue of mice.However,HES can reduce the m RNA levels of TNF-? and IL-6(P<0.05).Conclusion: HES could protect LPS-induced ALI in mice by reducing the degree of pulmonary edema,reducing total protein concentration,total cell number and neutrophil counts,decreasing MPO expression,and inhibiting the release of pro-inflammatory cytokines.Part 3 Protective mechanism of hesperetin on lipopolysaccharide-inducedacute lung injury in miceObjective: To investigate whether hesperetin(HES)inhibits the activation of mitogen activated protein kinase(MAPK)and NF-?B signaling pathway by binding to myeloid differentiation 2(MD2),thereby inhibiting lipopolysaccharide(LPS)-induced acute lung injury(ALI).Methods: The Le Dock molecular docking software was used to predict and explore the binding mode between HES and MD2.The expression of the protein related to MAPK and NF-?B signaling pathway was detected by western blot,MD2 and toll-like receptor 4(TLR4)were detected by immunoprecipitation.Results: HES overlapped with the LPS binding sites on MD2,and HES could cut off the complex of MD2 and TLR4 under LPS stimulated.After the induction of LPS,the expression ratios of p-ERK/ERK,p-JNK/JNK,and p-P38/P38 in the LPS group were increased compared with the blank control group,and there was a significant difference(P<0.05).Compared with the LPS group,after pretreatment with HES at the concentrations of 25 mg/kg and 50 mg/kg,the expression ratios of p-ERK/ERK,p-JNK/JNK,and p-P38/P38 were significantly decreased(P<0.01),but the level of protein I?B was increased(P<0.01).Conclusion: HES binds directly to MD2,further inhibiting the activation of MAPKs,regulating the inhibitory effect of the inhibitory protein I?B,preventing the interaction between MD2 and TLR4 to protect LPS-induced ALI mice. |
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