LHCGR-Mediated Improvement Of The Effect Of Lupeol On The Estrous Cycle Of Female Mice By Corn Oil And The Extraction Process Of Lupeol

Objective:Lupeol,as a monomeric compound derived from traditional Chinese medicine,has a variety of pharmacological activities and has been reported in the literature to have anti-tumour,anti-inflammatory,anti-oxidative stress and hypoglycaemic effects.Lupeol has been used as an androgen receptor inhibitor for the treatment of androgen-sensitive/insensitive prostate cancer,and also has an inhibitory effect on breast cancer cells;a few reports have found estrogen-like effects of lupeol,but the exact action and mechanism remain unclear.Therefore,this experiment will investigate the pharmacological effects and molecular mechanisms of lupeol on the female reproductive system such as ovaries,uterus and mammary glands,and optimize the process of extracting lupeol from the local herb Indigofera pseudotinctoria Matsum in Guizhou and compare the LC-MS and GC-MS assays.Methods:This experiment is divided into two parts:（1）Luteinizing hormone receptor（LHCGR,LHR）mediates the mechanism of lupeol-saved corn oil effect on the oestrus cycle of female mice.（2）orthogonal test to optimize the lupeol extraction process and comparison of LC-MS and GC-MS detection.Pharmacological part:1)Female Kunming mice were studied by subcutaneous injection in the dorsal renal region,divided into control,corn oil group,low-dose lupeol,medium-dose lupeol and high-dose lupeol groups for 14 consecutive days.2)Recording changes in body weight of mice.3)Compared with the control group,estradiol was significantly decreased in the corn oil group（P<0.05）,and estradiol was significantly increased in the three groups injected with lupeol.However,progesterone content increased in a dose-dependent manner with lupoel.4)Serum was collected for the determination of blood lipid and the contents of estradiol and progesterone were determined by enzyme-linked immunosorbent assay（ELISA）.5)Weighing the ovaries and uterus to calculate the organ coefficients and observing changes in organ weights.6)H&E staining of mouse ovaries,fallopian tubes,uterus and breasts and observation of histomorphological changes and counting of corpus luteum in the ovaries.7)Detection of ovarian estradiol receptor alpha（ERα）,progesterone receptor（PR）and oocyte marker MVH by protein immunoblotting,together with immunohistochemistry to observe ERαexpression levels and subcellular localization in the ovary.8)RNA-seq analysis of changes in transcriptome levels in the corn oil group versus the low-dose lupeol group;KEGG,GO and GSEA analysis of differential genes using R language and other software,combined with experimental validation to elucidate the key downstream signalling pathways through which lupeol acts.9)Molecular docking of LHCGR,ER,PRGR,etc.using molecular docking tools to identify key targets for lupeol.10)Detection of Lhcgr and Lhb by q RT-PCR.The molecular mechanism of the pharmacological effects of lupeol on female reproductive organs was finally revealed.Extraction part:1)A single-factor test for the extraction of horse-buckthorn seeds was carried out by selecting two factors,solvent and time,according to the available literature,and the resulting infusion was assayed by LC-MS to calculate the lupeol content.2)Based on the results of the single-factor experiment,a three-factor,three-level orthogonal test was designed using L₉（3⁴）orthogonal table to select the best process for the extraction of lupeol from horse buckthorn,and the peak area of the resulting extract was detected by GC-MS.3)The content of lupeol in the single-factor experiment was determined by LC-MS in three solvents（95%ethanol,ethyl acetate and petroleum ether）at 10 times the liquid ratio for 5 and 15 days of extraction,compared with the peak area of the orthogonal experiment under the same process and deduced from the orthogonal experiment.4)Conduct statistical analysis to determine whether the peak area and content of lupeol under the same process of LC-MS and GC-MS have the feasibility of equivalent substitution.Results:Pharmacological component:1)No significant changes in body weight and no significant abnormal changes in lipid quadruplet in the five groups of mice after 14days of continuous injection.2)Different changes in the estrous cycle,with the solvent group all in interestrus and the medium and high dose lupeol groups all in estrus;the low dose lupeol group improved the disturbance brought about by the corn oil and returned to a largely normal state.3)Compared to the control group,estradiol decreased significantly（P<0.05）in the solvent group and increased significantly in the lupeol injection three group,while progesterone levels showed a dose-dependent increase with lupeol.4)Protein immunoblotting and immunohistochemistry showed no significant changes in ERαin the five groups,but PR expression was significantly increased in the low and medium dose lupeol groups（P<0.05）and decreased in the high dose lupeol group;MVH was significantly increased only in the medium dose lupeol group（P<0.05）.5)Increased number of ovarian corpus luteum,thickened endometrium,proliferation of cervical epithelial cells and increased folding of the mucosal layer of the fallopian tubes in the lupeol three group compared to the control and solvent groups.6)Breast duct epithelial cells were not only proliferating in the solvent group and were accompanied by secretory fluid,but in the lupeol three group the epithelial cells returned to normal with no obvious secretory fluid and were inhibited in a dose-dependent manner with lupeol.7)RNA-seq results in the solvent and low-dose lupeol groups showed 47 genes down-regulated and 140 genes up-regulated,and KEGG and GSEA analysis revealed simultaneous enrichment to steroid hormone anabolic process events.8)variable shear differential gene enrichment analysis likewise yielded steroid biosynthetic process events occurring while Lhgcr underwent SE and A3SS variable shear.9)Molecular docking results show that lupeol is more stable in forming three chemical bonds with two amino acid residues of LHCGR（B:ARG150:HE;B:ARG150:HH21;BLEU190:O）and possesses a lower binding energy（-9.0 kcal/mol）;binding to LHB（B:GLU19:OE2）,with a binding energy（-9.84 kcal/mol）.10)The q RT-PCR results for Lhcgr and Lhb showed a significant increase（P<0.05）in low doses of lupeol and a dose-dependent decrease in medium and high doses.Extraction component:1)The samples obtained from the 26 orthogonal experiments were analyzed by GC-MS and converted to a median value of 4503.66μg.The highest lupeol content in the extract was in petroleum ether,5 days of extraction,10 times the stock ratio,and the lowest in 95%ethanol,5 days of extraction,5 times the stock ratio;after excluding petroleum ether as a solvent,95%ethanol,5 days,10 times the stock ratio was the highest.After excluding petroleum ether as a solvent,95%ethanol,5 days,10 times the amount of material ratio was the highest.2)Samples obtained by the same process were converted into peak area values by GC-MS,and the corresponding LC-MS contents were:95%ethanol,5 days,LC content 4750.46μg vs.LC 1766.06μg vs GC peak area 5419;Ethyl acetate,15days,LC 1851.66μg vs GC peak area 5486;Petroleum ether,5 days,LC 21159.37μg vs GC peak area 640;Petroleum ether,15 days,LC 15558.42μg vs GC peak area1088.3)The content was deduced under the same process,the rank sum test results P>0.05 and the corresponding ratios for 95%ethanol,ethyl acetate and petroleum ether were 315.91,208.25 and 1305.83 respectively.Conclusions:Pharmacological component.（1）Lupeol effectively improves the effect of corn oil on the oestrous cycle of female mice.（2）Lupeol increases the number of corpus luteum and increases progesterone and progesterone receptors.（3）Lupeol may affect the synthesis and metabolic pathways of steroid hormones to exert pharmacological effects.（4）Lupeol may exert luteinizing hormone-like effects by binding to LHCGR and regulating Lhcgr variable shear.Extraction component.（1）Based on the safety,economic and industrial production perspectives,horse chestnut seeds were extracted from lupeol using 95%ethanol,maceration for 15 days and 10-fold liquid to material ratio method.（2）The GC-MS peak area values could initially be extrapolated to the LC-MS for broad content,which provided a convenient method for determining the GC-MS peak area values.