Screening Of Effective Fraction For Anti Liver Injury Induced By LPS From P.chinense P.and Potential Mechanism

Objective: The liver is not only the main site for endotoxin limination,but also one of the earliest and most obvious target organs injury induced by endotoxemia.Liver injury can lead to a large number of endotoxin overflow into the body circulation and cause multiple organ damage.P.chinense P.has significant curative effect on protecting liver function,but it has not been reported for endotoxin induced liver injury.Therefore,the purpose of this study was to(1)Screen the effective fraction from different solvent rough extractives of PCP on hepatic injury induced by LPS.(2)The effective fraction from different solvent rough extractives of PCP were hierarchical extracted,further screening the effective inhibition part of hepatic injury induced by LPS and studying on its mechanism.Methods:(1)The rough extractives of PCP:(1)The PCP was refluxed with different solvents(water,70% ethanol,95% ethanol)in a water bath by the solid-liquid ratio of 1:10,and the extract liquid of PCP was collected to concentrate,then calculated the extraction rate.(2)The vitro alternative model: By means of MTS and ELISA,we detected the OD value of RAW264.7 cell proliferation and calculate the relative growth rate(RGR),and evaluated the safe concentration of different solvent rough extractives of PCP on the RAW264.7 cell;In inverted microscope,morphological changes were observed by Giemsa staining;The supernatant inflammatory cytokine(IL-1?,TNF-?)content was measured by ELISA;to evaluate the optimum concentration of LPS on RAW264.7 cells,and the optimal position of the rough extract of PCP inhibition activatied RAW264.7 cells induced by LPS.(3)In vivo animal experiment: Kunmin mice were randomly divided into: the control group,hepatic injury induced by LPS group,epatic injury induced by LPS + gansu granule group(positive control),hepatic injury induced by LPS + 70% ethanol extract of PCP group,hepatic injury induced by LPS + 95% ethanol extract of PCP group,epatic injury induced by LPS + water extract of PCP group.In addition to the blank group,each group of mice were caudal vein injected with LPS(4mg·kg-1)to induced hepatic injury in mice.The hepatic injury induced by LPS group without any treatment,the drugs were respectively medicine each group by gavage 12 hours before and 2 hours after LPS injection,12 hours after LPS injection,We collected abdominal aorta blood in mice,isolated the serum and liver tissue were collected for the preparation of homogenate and paraffin section for histopathological detection.Superoxide dismutase(SOD)levels in liver homogenate were measured by superoxide dismutase and TBA colorimetric method was used to detect the level of malondialdehyde(MDA).(2)The hierarchical extraction of PCP: Using Pursh 70% ethanol extract,according to extract: warm water =1:10(W/V)ratio of 65? water bath dissolved into water suspension,then successively with petroleum ether,ethyl acetate,n-butanol extraction for three times in 65? water bath and extraction ratio is extract: Solvent=1:3,the extraction of petroleum ether extract,ethyl acetate extract and n-butanol extract were collected respectively,and the extraction rate was calculated.(2)The vitro screening and vivo animal experiments: The testing indexes were the same as those of vitro model in methods(1).(3)Mechanism study: Immunofluorescence was used to detect LPS induced apoptosis associated specklike protein(apoptosis-associated speck-like protein containing a CARD domain,ASC)and nucleotide binding oligomerization domain like receptors(nucleotide-binding oligomerization domain-like receptors,NLRP3)expression of the optimal concentration and the intervention of PCP on the expression of ASC and NLRP3 induced by LPS.Results:(1)The ratio of PCP crude extract and the hierarchical extraction:(1)PCP crude extract: The successively ratio of 70%,95%,water ethanol extract of PCP was 13.8%,13.4%,15.8%.(2)PCP hierarchical extraction :the successively ratio of petroleum ether,ethyl acetate,n-butanol extract of PCP was 2.75%,30.66%,17.54%.(2)The safety concentration of PCP crude extract and extractant:(1)PCP crude extract: When the safety concentration of 70%,95%,water ethanol extract of PCP is less than 100 mg·L-1,25mg·L-1,400 mg·L-1,the cell toxicity grade level ?1.(2)PCP hierarchical extraction: When the safety concentration of petroleum ether,ethyl acetate,n-butanol extract of PCP is less than 50 mg·L-1,100mg·L-1,400 mg·L-1,the cell toxicity grade level ?1.(3)The optimal concentration of LPS induced activation of RAW264.7 cells: In inverted microscope(×200),we observed RAW264.7cell appearing round,and there was not cell processes,less cell dendritesand in the control group.There was activated sign of RAW264.7 in 0.25mg·L-1-8mg·L-1 LPS group,for example gather phenomenon significantly reduced,pseudopodia increased,cells body appeared swollen,the edge is not clear,irregular shape and so on.Compared with the control group,IL-1?,TNF-? of RAW264.7 cells were increased in 0.25mg·L-1-8mg·L-1 LPS(P<0.05,P<0.01).(4)Optimal point of crude extracts and extracts of P.chinense P.inhibitthe activation of RAW264.7 cell:(1)Under the inverted microscope(×400),compared with other groups,70% ethanol extract andethyl acetate extract could significantly improve RAW264.7 cell morphological changes that induced by LPS.(2)Compared with the LPS group,70% ethanol extract of PCP was significantly decreasing the IL-1?,TNF-? level in cell supernatant(P<0.05,P<0.01)when concentrations were 50 mg·L-1,25 mg·L-1,12.5 mg·L-1,water extract of PCP was significantly decreasing the IL-1? and TNF-? level in cell supernatant(P<0.05,P<0.01)when concentrations were 50 mg·L-1,25 mg·L-1;ethyl acetate extract of PCP was significantly decreasing the IL-1? and TNF-? level in cell supernatant(P<0.05,P<0.01)when concentrations were 50 mg·L-1 and 25 mg·L-1 was significantly decreasing the TNF-? level in cell supernatant(P<0.05);N-butanol extract of PCP was significantly decreasing the IL-1? and TNF-? level in cell supernatant(P<0.05)when concentrations was 50 mg·L-1.(5)optimal point of crude extracts and extracts of PCP against the mice liver injury induced by lipopolysaccharide:(1)The changes of mice's liver tissue were observed under the light microscope.Compared with the LPS group,liver tissue injury has improved and inflammatory exudation has decreased in gansu granule +LPS group,70% ethanol extract +LPS group,ethyl acetate extract +LPS group and n-butanol extract +LPS.(2)Serum: compared with the control group,the serum levels of ALT and AST were significantly higher than other groups(P < 0.01).Compared with the model group,the serum level of AST are decreased significantly in gansu granule group,70% ethanol extract group and n-butanol extract group in(P < 0.01);(3)Liver homogenate: Compared with the controlgroup,the level of SOD in each group were not obvious,the level of MDA in the model group was significantly increased(P < 0.01).Compared with the model group,the levels of MDA were significantly decreased in the positive drug group,70% ethanol extract,petroleum ether extract,ethyl acetate extract and n-butanol extract(P<0.05,P<0.01).(6)immunofluorescence assay:(1)Compared with the control group,the expression of NLRP3 and ASC in the 1mg·L-1-8mg·L-1 group was up-regulated,and the 2mg·L-1,4mg·L-1 LPS group was the most obvious.(2)The expression of ASC and NLRP3 in ethyl acetate and n-butanol group were significantly lower than those in LPS group,and the effect of ethyl acetate was stronger.Compared with LPS group,the ASC and NLRP3 of the petroleum ether group had no obvious change.Conclusions:(1)The effective fraction from different solvent rough extractives of PCP on hepatic injury induced by LPS is 70% ethanol extract of PCP.(2)In fractional extraction of PCP 70% ethanol extraction,the main components of effective mitigate hepatic injury induced by LPS were concentrated in the ethyl acetate fraction.(3)The effectiveness of PCP mitigate hepatic injury induced by LPS may be related to the regulation of ROS/NLRP3/IL-1? pathway.