Injury Mechanism Of Natural PM2.5 Exposure On Ovarian Function And Protective Effect Of Polydatin From PM2.5-Induced Ovarian Damage

Objective: To explore the influence of natural PM2.5 exposure on ovarian function and the injury mechanisms involved.Accordingly,potential protective agent polydatin was screened out and verified.Methods: Sixty-four 8-week-old female mice were randomly divided into two groups: one group in filtered air(FA)and the other exposed to PM2.5-bearing air(PM).1 and 4 months later,estrus cycle monitoring and mating test were conducted to assess the ovarian endocrine and reproductive function.After 4-month exposure,blood and ovaries of mice were collected and experimented to evaluated the ovarian reserve function and explore the injury mechanisms.Then,KGNs(human granule cell lines)were treated with water-and DMSO-soluble components of PM2.5 to verify the molecular mechanisms.On the basis of abovementioned results and literature review,polydatin was considered as a good candidate for preventing ovarian damages from PM2.5 exposure and verified next.Forty-eight8-week-old female mice were randomly divided into four groups: control(CONT),PM2.5(PM),PM2.5 + low-dose polydatin(PM+PD(L)),PM2.5 + high-dose polydatin(PM+PD(H)).Weight and estrus cycle were monitored.Ovarian reserve function was assessed by follicle count,and molecular mechanisms were detected with the methods of TUNEL,electron microscope,oil red O staining,IHC,WB et.al.Furthermore,human ovarian cortex tissues cultured in vitro were treated with DMSO-soluble component of PM2.5 and polydatin to verify the molecular mechanisms.Results: Compared with the FA group:(1)After 4-month exposure,the incidence of irregular estrus cycle,serum E2 and FSH were significantly increased(P<0.05)in PM group.(2)After both 1-and 4-month exposure,the pregnancy index and average litter size decreased while time for mating prolonged in PM group.After 4-month exposure,the average litter size of post-delivery mice and weight of PND3 pups in PM group also decreased significantly(P<0.05).(3)Histomorphologically,the number of primordial follicles,antral follicles and total healthy follicles decreased significantly(P<0.05)in PM group.(4)TUNEL results showed promoted granulosa cell apoptosis in PM group.The protein level of pro-apoptotic factors ?-H2 AX,BAX,and the terminal apoptosis marker Cleaved Caspase3 increased(P<0.05)in PM group.(5)RNA-Seq showed that differentially expressed genes were largely enriched in cholesterol metabolism and ovarian steroid hormone synthesis pathway,which were closely related to ovarian endocrine function changes.(6)The oxidative damage markers NTY and 4-HNE increased,while antioxidant enzymes SOD2,NQO1 and CYT-C were downregulated(P<0.05)in ovaries of PM group.(7)The expression of inflammation-related factors IL6 and Caspase1 increased markedly(P<0.05),and NF?B pathway was activated in PM group.Soluble components of PM2.5induced oxidative stress and inflammation of human granule cell lines KGN,inhibited cell proliferation and promoted apoptosis.In the second part,compared with the PM group:(1)Polydatin improved the ovarian endocrine function of PM2.5 exposed mice.The incidence of irregular estrus cycle decreased,The level of serum E2(P<0.05)and P4 increased.(2)The number of primordial follicles,antral follicles and the proportion of primordial follicles increased in the PM+PD(L)/(H)group(P<0.05).(3)Polydatin inhibited granulosa cell apoptosis and reduced follicular atresia.Compared with the PM group,the protein level of ?-H2 AX,BAX and Cleaved Caspase3 decreased in the PM+PD(L)/(H)group(P<0.05).(4)Polydatin promoted autophagy in ovaries of PM2.5 exposed mice,reducing damaged mitochondrial and abnormal lipid accumulation.(5)Polydatin alleviated the oxidative stress and inflammation damage in ovaries of PM2.5 exposed mice.Compared with the PM group,the level of 8-OHd G,NTY and 4-HNE decreased in the PM+PD(L)/(H)group(P<0.05);spleen weight and serum IL6 in the PM+PD(L)/(H)group decreased,the ovarian protein level of Caspase1 and IL6 were downregulated(P<0.05),and the NF?B pathway was inhibited.Additionally,polydatin inhibited oxidative stress and inflammation of human ovarian cortex that were caused by DMSO-soluble component of PM2.5.Conclusions: Long-term natural PM2.5 exposure impaired ovarian reproduction and endocrine function,and decreased ovarian reserve function as well.Oxidative stress,inflammation and steroid hormone metabolism changes were involved in the damage mechanisms.Polydatin can reduce PM2.5 exposure-induced ovarian damages and protect ovarian function,not only by inhibiting oxidative stress and inflammatory response,but also by enhancing cell autophagy to improve intracellular environment.