Comparative Study Of Rhubarb Different Extracts On Hypothalamic-Pituitary-Ovarian Axis Of Rats

As a kind of traditional Chinese herb,rhubarb plays an important position in thousands of years of TCM clinic.But in recent years,the incidence of adverse reactions of rhubarb increases yearly due to vast and irrational use,which has attracted attention of researchers.The early study on rhubarb reproductive toxicity indicated that applying RWD over a long period of time could cause atrophy of uterus and ovary,decrease the level of serum P?progesterone?and E₂?estradiol?,elevate the level of serum LH?luteinizing hormone?and FSH?follicle stimulating hormone?,and cause dysfunction of HPOA?hypothalamic-pituitary-ovarian axis?.But its toxic material basis is still unknown.Based on the previous study,the female rats were given corresponding rhubarb different extracts for 60 days,this research aims to observe the efffect of rhubarb different extracts on the mRNA and protein expression of GnRH,GnRH-R and FSH? which plays an important role in HPOA,as well as organization structure of hypothalamic arcuate nucleus and adenohypophysis,and probe into the toxic effect of rhubarb different extract on HPOA from molecular,protein and morphology levels.Then screen the main toxic extract,laying foundation for the study of reproductive toxicity material basis of rhubarb.Objective:As proved by preliminary study on rhubarb reproductive toxicity,applying RWD over a long period of time has obvious damage to every level of HPOA.By observing the effect of rhubarb different extracts on function and structure of hypothalamic arcuate nucleus and adenohypophysis,this research aims to screen the main reproductive toxicity extract of rhubarb,laying the foundation for further screening and analyising the reproductive toxicity part and component of rhubarb,and providing theoretical basis for its safe application in clinic.Methods:The CE?chloroform extract?,EAE?ethyl acetate extract?,NAE?n-butyl alcohol extract?and WSS?water-soluble substance?of rhubarb were prepared by polarity gradient extraction method.Female adult rats were randomly divided into NC?normal control?group,RWD group and different extracts groups.Rats were given corresponding medicines for 60 days.During the period,general state of rats was observed.Calculate the body mass growth rate of rats.The expression of GnRHmRNA in hypothalamus and GnRH-RmRNA and FSH?m RNA in hypophysis was detected by RT-qPCR?Reverse transcription-quantitative polymerase chain reaction?.The protein expression of GnRH in hypothalamic arcuate nucleus and GnRH-R and FSH? in gonadotropic cells were detected by immunohistochemistry method.The pathologic change of hypothalamic arcuate nucleus and adenohypophysis was observed with light microscope.The ultrastructure of hypothalamic arcuate nucleus neurons and adenohypophysial goadotrophic cells was observed by transmission electron microscope.Result:1 General state of ratsEffect on appearance of rats: In NC group,the fur of rats was white and lustrous;But in RWD group,the fur of rats was dim and lacklustre,and the color of tail and local coat was red,and with the extension of drug delivery time,the coat became redder and redder.But in the CE,EAE,NAE and WSS groups,the pigmentation of rats was not obvious.Effect on stools: In NC group,stools of rats was normal.In RWD group,rats had loose stools in an early drug delivery,and returned to normal about 10 days later.But the stools in CE group were dry out,and stools in EAE,NAE and WSS groups were basically normal.2 Effect on mRNA expression of hormones associated with HPOA2.1 Effect on expression of GnRHmRNA in hypothalamusCompared with NC group,the expression of GnRHmRNA in RWD group rose 236.63 percent?P <0.01?,the expression of GnRHmRNA in CE group and EAE group respectively rose 137.19 percent and 135.15 percent?P <0.05?.The expression of GnRHmRNA in NAE group and WSS group respectively fell 23.22 percent and 46.62 percent compared with NC group,but there was no statistical significance?P >0.05?.The results indicated that the influence extent of different samples on expression of GnRHmRNA in hypothalamus was RWD > CE > EAE > WSS > NAE.2.2 Effect on expression of Gn RH-RmRNA in hypophysisThe expression of GnRH-RmRNA in RWD group rose 137.13 percent compared with normal control group?P <0.05?.Compared with NC group,the expression of GnRH-RmRNA in CE group,EAE group,NAE group and WSS group respectively fell 48.44 percent,39.68 percent,41.56 percent and 47.79 percent,but there was no statistical significance?P >0.05?.The results indicated that the influence extent of different samples on expression of GnRH-RmRNA in hypophysis was RWD > CE > WSS > NAE > EAE.2.3 Effect on expression of FSH?mRNA in hypophysisCompared with NC group,the expression of FSH?mRNA in RWD group rose 194.31 percent?P <0.01?,the expression of FSH?mRNA in CE group and EAE group respectively rose 131.15 percent and 125.94 percent?P <0.05?.The expression of FSH?mRNA in NAE group and WSS group respectively fell 35.97 percent and 36.64 percent compared with NC group,but there was no statistical significance?P >0.05?.The results indicated that the influence extent of different samples on expression of FSH?mRNA in hypophysis was RWD > CE > EAE > WSS > NAE.3 Effect on protein expression of hormones associated with HPOA3.1 Effect on protein expression of GnRH in hypothalamic arcuate nucleusCompared with NC group,the protein expression of GnRH in RWD group rose 136.84 percent?P <0.01?,the protein expression of GnRH in CE group and EAE group respectively rose 31.58 percent and 36.84 percent?P <0.05?.The protein expression of GnRH in NAE group and WSS group respectively rose 10.53 percent and 21.05 percent compared with NC group,but there was no statistical significance?P >0.05?.The results indicated that the influence extent of different samples on protein expression of GnRH in hypothalamic arcuate nucleus was RWD > EAE > CE > WSS > NAE.3.2 Effect on protein expression of GnRH-R in adenohypophysis cellsThe protein expression of GnRH-R in RWD group fell 22.67 percent compared with NC group?P <0.05?.Compared with NC group,the protein expression of GnRH-R in CE group,EAE group,NAE group and WSS group respectively fell 14.67 percent,17.33 percent,14.67 percent and 10.67 percent,but there was no statistical significance?P >0.05?.The results indicated that the influence extent of different samples on protein expression of GnRH-R in adenohypophysis cells was RWD > EAE> CE ? NAE > WSS.3.3 Effect on protein expression of FSH? in adenohypophysis cellsCompared with NC group,the protein expression of FSH? in RWD group rose 70.18 percent?P <0.01?,the protein expression of FSH? in CE group and EAE group respectively rose 22.81 percent and 26.32 percent?P <0.05?.The protein expression of FSH? in NAE group and WSS group respectively rose 1.75 percent and 15.79 percent compared with NC group,but there was no statistical significance?P >0.05?.The results indicated that the influence extent of different samples on protein expression of FSH? in adenohypophysis cells was RWD > EAE > CE > WSS > NAE.4 Organization structure changes of hypothalamic arcuate nucleus and pituitary gland under light microscope4.1 Effect on organization structure of hypothalamic arcuate nucleusUnder the light microscope,hypothalamic arcuate nucleus neurons of NC group distributed evenly,density was big,cytoplasm stained deep,and nucleus is clear.The hypothalamic arcuate nucleus neurons of RWD group showed little neurons,fuzzy boundary of nuclear membrane,chromatin marginalization,nissl substance dissolving,as well as hell cells.However,there were no apparent pathologic changes in different extracts groups.4.2 Effect on organization structure of pituitary glandUnder light microscope,the pituitarsy gland of NC group had many adenohypophysis cells arranged regularly and abundant sinusoid capillary.Compared with NC group,the total number of adenohypophysis cells in RWD group reduced and the cells arranged in irregular,the number of sinusoid capillary increased.However,there were no apparent pathologic changes in different extracts groups.5 Ultrastructure changes of hypothalamic arcuate nucleus neurons and adenohypophysial goadotrophic cells under electron microscopy5.1 Effect on ultrastructure of hypothalamic arcuate nucleus neuronsUnder electron microscopy,the ultrastructure of hypothalamic arcuate nucleus neurons of rats in RWD group,CE group and EAE group all appeared different degrees of pathological changes,compared with NC group.In RWD group,the hypothalamic arcuate nucleus neurons showed extremely irregular karyomorphismen,increased nuclear bag,mitochondria and lysosome,as well as rough endoplasmic reticulum aggregations and chromation condensed in margin.In CE group,the hypothalamic arcuate nucleus neurons showed broaden nucleus cisternal space and increased mitochondria.In EAE group,the hypothalamic arcuate nucleus neurons showed enlarged nucleolus,mitochondria swelling and crista fragmentation,as well as increased mitochondria,rough endoplasmic reticulum and lysosome.However,there were no apparent ultrastructural pathologic changes in NAE group and WSS group,compared with NC group.5.2 Effect on ultrastructure of adenohypophysial goadotrophic cellsUnder electron microscopy,the ultrastructure of adenohypophysial goadotrophic cells of rats in RWD group,CE group and EAE group all appeared different degrees of pathological changes,compared with NC group.In RWD group,the adenohypophysial goadotrophic cells showed extremely irregular karyomorphismen,broken nuclear envelope,expanded Golgi apparatus,aggregated heterochromatin increased.In CE group and EAE group,the adenohypophysial goadotrophic cells showed hypertrophied mitochondria and slightly dilated endoplasmic reticulum and Golgi apparatus.In addition,the concentric round lamellar structures consisted of rough endoplasmic reticulum could be seen in RWD group,CE group and EAE group.However,there were no apparent ultrastructural pathologic changes in NAE group and WSS group,compared with NC group.Conclusion:CE,EAE and RWD could elevate the expression of GnRHmRNA in hypothalamus and FSH?mRNA in adenohypophysis,increase the protein expression of GnRH in hypothalamic arcuate nucleus and FSH? in adenohypophysis cells,and cause protein synthesis of hypothalamic arcuate nucleus neurons and adenohypophysial goadotrophic cells enhanced.Effect of CE and EAE on function and structure associated with HPOA is similar to RWD,which indicates that CE and EAE both play a commanding role in the reproductive toxicity of rhubarb.