Mechanism Of IL-4 Inhibiting The Progression Of Polycystic Ovary Syndrome By Promoting The Polarization Of M2 Type Macrophages

Objective: Polycystic Ovary Syndrome(PCOS)represents one of the most prevalent reproductive endocrinopathies among women of childbearing age,with an incidence rate of 5-10%.It is frequently implicated in menstrual irregularities and infertility cases in females.However,currently there are no drugs that can target the treatment of PCOS,and its pathogenesis is still unclear.In recent years,research has found that immune factors may be involved in the occurrence and development of PCOS disease,but their specific mechanisms of action still need to be elucidated.In response to inflammatory events within the body,macrophages differentiate into two primary activation states: classically activated M1 phenotypes(pro-inflammatory),and alternatively activated M2 phenotypes(anti-inflammatory).M1 macrophages secrete a plethora of pro-inflammatory cytokines including IL-6,IL-18,and TNF-α,exacerbating the inflammatory cascade.On the contrary,M2 macrophages produce anti-inflammatory mediators such as IL-4 and IL-10,mitigating inflammation and facilitating trophoblast proliferation.IL-4 is an anti-inflammatory cytokine that possesses a multitude of physiological effects,including the modulation of metabolic functions and the promotion of macrophage polarization toward the M2 phenotype.The NF-κB/IκB signaling axis represents a crucial pathway for the polarization of macrophages.This study utilizes serum samples from patients with PCOS and employs the construction of a mouse model of PCOS to conduct an in-depth investigation into whether IL-4 facilitates M2 macrophage polarization via the NF-κB signaling pathway.Further,this research examines the regulatory impact of M2-polarized macrophages on ovarian granulosa cell apoptosis and the secretion of the ovulatory marker ADAMTS1.The ultimate goal is to enhance the understanding of the potential pathogenesis of PCOS and to provide a scientific foundation as well as prospective targets for clinical treatment strategies and the innovation of new pharmacological therapies.Research methods:Part 1:According to relevant ethical standards,serum specimens were responsibly procured from individuals with PCOS and a control group of normal,healthy volunteers.After verifying that the baseline conditions between the two groups exhibited no statistical significance,alterations in the protein concentrations and transcriptional levels of serum inflammatory mediators,including IL-1β,IL-4,IL-6,IL-10,TNF-α,and IFN-γ were assessed using ELISA/q RT-PCR method in both the normal control and the PCOS patient groups.Part 2:1.A PCOS-like murine model was established by subcutaneously injecting DHEA into eight-week-old female C57BL/6 mice.IL-4 was utilized as the therapeutic agent for the treatment group,and alterations in ovarian pathological damage were observed using H&E staining.2.Test Model Construction: Body weights of various mouse groups were compared,and ELISA method was used to measure the expression levels of testosterone(T),estradiol(E2),luteinizing hormone(LH),and follicle-stimulating hormone(FSH)in serum,assessing the fluctuations in mouse hormonal profiles.The expression levels of inflammatory mediators,such as IL-1β,IL-4,IL-6,IL-10,TNF-α,and IFN-γ were assessed using ELISA/q RT-PCR method.3.Tissue immunofluorescence was applied to assess alterations in the expression of the M1 macrophage marker i NOS and the M2 macrophage marker CD163 across different mouse cohorts.4.TUNEL assay,Ki-67 immunostaining,and caspase-3 immunohistochemistry were adopted to examine alterations in apoptotic and proliferative activity within ovarian tissues among different groups of mice.5.Immunohistochemistry was used to analyze changes in the expression level of the ovulation marker ADAMST1 in various groups of mice.Part 3:5.To explore the impact of PCOS-like cells on the polarization of macrophages,M0 macrophages were incubated with conditioned media from KGN cells that had been treated under diverse conditions for a duration of 24 hours.The q RT-PCR technique was utilized to monitor alterations in the gene expression levels of M1 macrophage markers,including CD86 and CD80,as well as i NOS,and M2 macrophage markers such as CD163,Arg-1,and CD206,across the different experimental groups.Moreover,ELISA/q RT-PCR method was applied to assess the variations in the protein concentrations and m RNA expression levels of proinflammatory cytokines TNF-α,IL-1β,IL-6,and the anti-inflammatory cytokine IL-10 among the cell groups.6.The Western blot method was used to detect changes in protein levels of p-p65,p65,and IκB in various groups of cells.7.To further simulate the effects of the immune environment within the body on the apoptosis and proliferation of ovarian granulosa cells,we once again employed an in vitro co-culture system to investigate how IL-4 regulates the apoptosis and proliferation of PCOS-like cells by modulating different macrophage polarization states.KGN cells were exposed to macrophage-conditioned media processed under varying conditions for24 hours to induce their activity.Subsequently,we observed and measured the expression levels of apoptosis-related proteins Bax/Bcl-2 as well as cleaved caspase-3and caspase-9 in the various groups of KGN cells.8.The Western blot method was used to detect changes in the expression level of the ovulation marker ADAMTS1 in different cell groups.Results:Part 1:Compared to the control group,the protein concentrations and transcriptional levels of the pro-inflammatory cytokines IL-1β,IL-6,and TNF-α in the serum of patients with PCOS were significantly elevated,while the protein concentrations and transcriptional levels of the anti-inflammatory cytokines IL-4 and IL-10 were markedly decreased.This suggests that there may be an increased expression of M1 macrophages and a reduced expression of M2 macrophages in the serum of PCOS patients.Part 2:6.Histological analysis via H&E staining revealed that,relative to the control group,there was a marked increase in the incidence of fluid-filled cystic follicles and the thickness of the granulosa cell layer in mice treated with DHEA.Additionally,a noteworthy decline in the number of corpora luteum and dominant follicles was observed,corroborating the induction of PCOS in the DHEA-administered mice.Post IL-4 therapy,a significant decrease in cystic follicles was noted,along with an escalation in the count of mature follicles and corpora luteum.The ovarian architecture in mice receiving IL-4monotherapy was also found to be analogous to that of the control cohort.Conclusively,IL-4 treatment has been validated to efficaciously ameliorate ovarian pathology and reinstate ovulation in mice with DHEA-induced PCOS.7.In comparison to the control group,mice in the DHEA cohort experienced weight gain,with concurrent elevations in levels of T,E2,and LH in serum,as well as an augmented ratio of LH to FSH.Subsequent treatment with IL-4 efficiently reduced the levels of T,E2,and LH in serum,enhanced serum FSH concentrations,diminished the LH/FSH ratio,and alleviated the hormonal dysregulation associated with PCOS-like conditions in mice.8.Relative to the control group,the DHEA-treated mice showed a significant increase in serum concentrations of TNF-α,IL-1β,and IL-6,with markedly higher expression levels in ovarian tissues.Conversely,serum levels of IL-10 were decreased,along with significantly lower expression in ovarian tissue.Compared to the DHEA group,the DHEA+IL-4 group demonstrated a reduction in the expression levels of IL-1β,TNF-α,and IL-6 in both serum and ovarian tissues of mice,while the expression level of IL-10 was increased.9.Immunofluorescence analysis revealed an upregulation of pro-inflammatory(M1)macrophages and a downregulation of anti-inflammatory(M2)macrophages in the ovarian tissue of PCOS-like mice.Treatment with IL-4 was found to decrease M1 macrophage expression and promote an increase in M2 macrophage expression.10.In comparison with the control group,the DHEA-treated group exhibited a significant increase in the number of cells positive for TUNEL staining and caspase-3expression,alongside a marked decrease in the expression levels of the ovulation marker ADAMTS1.In contrast,the DHEA+IL-4 treatment group demonstrated significantly reduced numbers of TUNEL-positive cells and lower caspase-3 expression compared to the DHEA group alone,suggesting that IL-4 treatment can notably inhibit ovarian apoptosis,enhance cellular proliferation,and stimulate the expression of the ovulation marker ADAMTS1.Part 3:6.Following exposure of M0 macrophages to KGN cell-conditioned media treated with DHEA,there was a great upregulation in the transcription levels of M1 macrophage markers including CD86,CD80,and i NOS.Concurrently,a substantial downregulation was observed in the transcriptional expression levels of M2 macrophage markers such as CD163,Arg-1,and CD206.Post-treatment with IL-4,a notable reversal of the downward trend in the gene expression of M2 macrophage markers was evident.7.After M0 macrophages were exposed to KGN cell-conditioned media treated with DHEA,there was a notable increase in the expression levels of proinflammatory cytokines TNF-α,IL-1β,and IL-6,which are typically released by M1 macrophages.In contrast,the expression level of the anti-inflammatory cytokine IL-10,commonly associated with M2 macrophages,was significantly reduced.Following treatment with IL-4,this expression pattern was clearly reversed,indicating a shift towards an anti-inflammatory profile.8.Compared to macrophages exposed to conditioning media from isolated KGN cells,those exposed to the conditioned media of PCOS-like cells induced by DHEA exhibited increased levels of phosphorylated p65.This upward trend in phosphorylated p65 levels could be significantly attenuated following IL-4 treatment.Similarly,Western blot analysis revealed an inverse expression pattern for IκB protein relative to that of phosphorylated p65 protein,suggesting that the conditioned medium from PCOS-like cells can prompt polarization of M0 macrophages towards an M1 phenotype.The addition of IL-4 was able to reverse this polarization from M1 to M2 macrophages.This reversal effect is mediated through the p65/IκB signaling pathway proteins.9.Upon a 24-hour incubation of KGN cells with conditioned media from macrophages treated under different conditions,the expression levels of apoptosis-related proteins were examined across the various KGN cell groups.According to the results,KGN cells treated with DHEA and subsequently exposed to conditioned media from M1 macrophages showed significantly increased expression levels of apoptosis-related proteins Bax/Bcl-2 as well as cleaved caspase-3 and caspase-9.While,treatment with IL-4 led to a significant downregulation in the expression levels of Bax/Bcl-2 and cleaved caspase-3 and caspase-9,suggesting that IL-4 treatment can mitigate apoptosis in KGN cells through modulating the Bax/Bcl-2 pathway.10.After DHEA treatment,KGN cells exposed to the conditioned medium of M1 macrophages exhibit significantly lower expression levels of ADAMTS1.However,treatment with IL-4 significantly upregulates the expression of ADAMTS1.This indicates that M1 macrophages suppress ADAMTS1 secretion in KGN cells,while IL-4treatment enhances ADAMTS1 expression.Conclusions:4.Patients with PCOS have high expression levels of M1 macrophages and associated pro-inflammatory cytokines,along with low expression levels of M2 macrophages and associated anti-inflammatory cytokines.5.Treatment with IL-4 can reduce the expression of M1 macrophages and upregulate the expression of M2 macrophages.6.IL-4 facilitates the conversion of M1 macrophages,which are induced by PCOS-like cells,to an M2 phenotype by inhibiting the activation of the NF-κB/IκB signaling pathway.7.IL-4 can reduce apoptosis of PCOS-like cells and promote their ovulatory function,thereby inhibiting the occurrence and development of PCOS.