Analysis of the relationship between liver-qi stagnation and premature ovarian failure based on exosomes

Chen Yu,Jing-Yan Song,Hai-Cui Wu,Fang Lian＊

1Traditional Chinese Medicine College of Shandong University of Traditional Chinese Medicine,Jinan Shandong,250011,China.

2The First Clinical Medical College of Shandong University of Traditional Chinese Medicine,Jinan Shandong,250011,China.

3Reproductive and Genetic Center of Integrated Chinese and Western Medicine affiliated Hospital of Shandong University of Traditional Chinese Medicine,Jinan Shandong,250014,China.

Abstract The incidence of premature ovarian failure,which is related to women’s excessive pressure and mental tension,has increased in recent years.Premature ovarian failure has become one of the diseases that seriously disturb women’s physical and mental health,and its incidence can be regarded as liver depression and qi stagnation.Patients have an increasing demand for treatment,not only for fertility with the improvement of social living standards and the development of healthcare technology.Exosomes,as small membrane vesicles containing complex RNA and proteins,have the function of mediating cell communication and transmitting information in the pathological state caused by liver-qi stagnation.Exosomes in the hypoxic microenvironment can protect cells from damage and promote the pathological process.Moreover,exosomes in an inflammatory environment can play an active anti-inflammatory role.Exosomes can reduce the apoptosis of granulosa cells by expressing miRNA and so on to restore ovarian function.The purpose of treating premature ovarian failure can be achieved in this way.This paper introduced the relationship between the three,discussed the guiding significance of Chinese medicine theories in the treatment of exosomes and premature ovarian failure,and provided new research ideas for the treatment of integrated traditional Chinese medicine and western medicine.

Key words:Liver-qi stagnation,Premature ovarian failure,Exosomes,Hypoxic microenvironment,Inflammatory reaction

Background

Premature ovarian failure refers to amenorrhea occurrence,elevated gonadotropin levels,and reduced estrogen levels in women before 40 years old,accompanied by varying degrees of perimenopausal symptoms.Patients may have hot flashes,night sweats,dryness,and burning sensation of the reproductive tract,decreased sexual desire,cardiovascular disease,changes in mood and cognitive function,osteoporosis,and other manifestations.The incidence of premature ovarian failure is on the rise.Premature ovarian failure is not recorded in ancient Chinese medicine books,and most of its description is“amenorrhea”“infertility”“blood dry menstrual closure”and so on.Premature ovarian failure is more common in women of childbearing age.These patients are prone to anxiety,depression,and other negative emotions due to long-term mental stress and the greater pressure.The liver mainly discharges the generation of bad mood that is most likely to lead to liver-qi stagnation.Thus,clinical liver-qi stagnation type premature ovarian failure occupies a certain proportion.As specifically secreted vesicles,exosomes can be secreted under both physiological and pathological conditions.Studies have shown that bone marrow mesenchymal stem cell-derived exosomes can improve ovarian function in chemotherapy-induced premature ovarian failure rats by inhibiting granulocyte apoptosis[1,2].Mesenchymal stem cells can also restore premature aging ovarian function through antioxidant effects[3].Simultaneously,the pathological changes caused by liver-qi stagnation(e.g.,hypoxia and inflammation)are closely related to oxidative stress and apoptosis in the microenvironment where exosomes exist.Therefore,this paper explores the relationship between liver-qi stagnation and premature ovarian failure from the perspective of exosomes to provide new diagnosis and treatment ideas for the study of traditional Chinese medicine and western medicine on premature ovarian failure.

Liver-qi stagnation and premature ovarian failure

Premature ovarian failure is accompanied by amenorrhea,follicular dysplasia,infertility,premature failure,osteoporosis,and other manifestations classified in traditional Chinese medicine as“amenorrhea”“blood dry”“infertility”and so on.In traditional Chinese medicine,the kidney is referred to as a functional system with the kidney as the center,which is closely related to the bladder,bone,pulp,brain,hair,ears,and the two Yin(excretion system for reproduction,feces and urine),and is related to human reproduction,growth and development,digestion,endocrine,intelligence,and physical ability.Similarly,in traditional Chinese medicine,the liver is regarded as a functional system centered on the liver,closely related to the eyes,fingernails,muscles,gallbladder,intestines,and stomach,and related to the reproductive,blood,endocrine,nervous,mental,and emotional systems.The kidney dominates reproduction,conceals essence,refines blood,and blood is the material menstruation base.Therefore,menstruation is closely related to the kidneys.The liver controls dispersion and blood storage in addition to nourishing the whole body.The blood stored in the liver is injected into blood.Menstruation and oocyte secretion cannot affect liver drainage.The liver can adjust qi and promote the movement of blood and body fluid,which affects emotions.If the physiological function of the liver is abnormal,qi and blood cannot normally run.Blood movement needs qi promotion.However,qi cannot promote blood if stagnant,leading blood stagnation and blood stasis which eventually leads to cellular vein blockage.Liver meridian encircles the female external genitalia for circulation,stagnates liver qi,then the discharge function is abnormal.The liver stores blood.If the liver qi is depressed,blood cannot flow on time.Liver-qi stagnation,drainage function disorders,and the water and grain in the stomach are difficult to transform into subtle substances.Therefore,liver-qi stagnation affect women’s menstruation,reproductive,and other physiological activities.Thus,the pathogenesis of premature ovarian failure has a certain connection with liver-qi stagnation.Congenital kidney storage essence leads to growth,development,and reproduction.Therefore,traditional Chinese medicine believes that kidney deficiency is based on the liver,spleen,and kidney mutual disease in terms of the etiology and pathogenesis of premature ovarian failure.Ye recorded in the Medical Case of Clinical Guidelines that“women are born with liver”and believed that the treatment of gynecological diseases should be based on the liver.

Patients with premature ovarian failure clinically often have liver-qi stagnation.About 60%of premature ovarian failure patients have an onset of emotional factors[4].If the female is studying or working,long-term pressure and mental tension falls and can produce a negative mood,liver-qi stagnation,and affect ovarian function.Wang[5]believes that patients with premature ovarian failure should focus more attention on life rules according to the time of the diurnal flow of the 12 meridians.Sleeping before 11 o’clock is conducive to the liver’s operation and catharsis,thus relaxing the whole body.Moreover,Teng[6]believed that premature ovarian failure caused by liver-qi stagnation could be treated from four aspects:soothing liver and regulating qi,clearing liver and reducing fire,nourishing blood and softening liver,and psychological counseling.Furthermore,Xu[7]believed that liver depression was the superficial pathogenesis for the etiology and pathogenesis of premature ovarian failure,and Xiangfu was a necessary product in the treatment.In premature ovarian failure treatment,Chai[8]believed that liver depression can lead to early depletion of menstrual fluid if liver depression is often accompanied by a syndrome.Moreover,the common drugs are Chaihu(Radix Bupleuri),Yujin(Radix Curcumae),Xiakucao(Spica Prunellae),Xiangfuzi(Rhizoma Cyperi),Hehuanpi(Cortex Albiziae)and so on to relieve liver depression by their virulence and dispersion.Thus,no lack of premature ovarian failure caused by liver-qi stagnation was noted during clinical observation.Careful syndrome differentiation should be carried out in the treatment of premature ovarian failure,and the products to soothe the liver and relieve depression can be applied to help patients feel comfortable and achieve a better curative effect.

Relationshipbetweenexosomesand premature ovarian failure

Premature ovarian failure is a syndrome with multiple etiologies.The specific etiology is currently unclear,and it is believed that its pathogenesis involves genetic factors,autoimmune factors,enzymological disorders,iatrogenic factors,few original follicle reserve,follicle atresia,quick depletion,and so on[9].Moreover,exosomes are small vesicles with a diameter of 30–150 nm containing RNA and proteins,which exist in natural body fluids and are widely distributed in the blood,urine,follicular fluid,cerebrospinal fluid,milk,and so on[10].Exosomes have stable biological properties and a lipid bilayer that prevents the content degradation and enables them to directly enter the target cells.This“carrier”signal transduction mode is efficient and stable.In addition,studies have shown that exosomes derived from bone marrow mesenchymal stem cells can also participate in tissue repair and are expected to replace stem cells as a new therapeutic tool for tissue repair[11].The pathogenesis of premature ovarian failure is mainly related to granulosa cell apoptosis,genetic factors,immune factors,and mitochondrial dysfunction.The apoptosis of granulosa cells induces apoptosis of oocytes.Excessive oocyte atresia is the cause of premature ovarian failure.Genetic factors are mainly due to genetic mutations that cause follicular atresia or aging.Abnormal function and the number of T lymphocyte subsets can lead to excessive ovarian immune response and decreased ovarian reserve function.The abnormal expression of mitochondrial DNA in oocytes can affect ovarian function and cause premature ovarian failure[12].Most of the studies on exosomes and premature ovarian failure are modeled through iatrogenic factors to explore the relationship between exosomes and ovarian function[2,13,14].Iatrogenic factors besides pelvic surgery,radiation,and chemotherapy drug use represent a significant share.Antitumor drugs(e.g.,cyclophosphamide and nitrogen mustard)have obvious adenotoxicity and can damage ovarian function from three aspects:inducing apoptosis of mother cells,inhibiting the function of membrane cells and granulosa cells,and affecting the level of reproductive hormones[15].

Granulosa cell apoptosis and exosomes

Premature ovarian failure is caused by excessive follicular atresia,and the apoptosis of granulosa cells undoubtedly induces this process.Zhang et al.[13]showed that in chemotherapy-induced premature ovarian failure(POF)mice,exosomes derived from humanamnioticepithelialcellsinhibited chemotherapy-induced granulosa cell apoptosis by transmitting functional miRNAs such as miR-1246,protected ovarian blood vessels from damage,and were involved in maintaining the number of damaged ovarian primordial follicles.Sun et al.[1]found that miR-644-5p carried by bone marrow mesenchymal stem cell derived exosomes inhibits the apoptosis of ovarian granulocytes by targeting p53,suggesting that miR-644-5p can potentially treat POF and restore ovarian function.Yang et al.[2]found that bone marrow mesenchymal stem cell derived exosomes can prevent follicle atresia in cyclophosphamide-treated rats by miR-144-5p transmission,and miR-144-5p can be transferred to the coculture of cyclophosphamide damaged granulosa cells to reduce the apoptosis of granulosa cells.This study provides new information on the potential of exosome miR-144-5p in the treatment of ovarian failure.Xiao et al.[14]found that amniotic fluid stem cell-derived exosomes can prevent follicle atresia in cyclophosphamide-treated mice through the delivery of miR-146a and miR-10a,and their potential target genes are critical to cell apoptosis.Downregulation of these two miRNAs in amniotic stemcell-derivedexosomesattenuatedthe antiapoptotic effect of mouse-injured granulosa cells in vitro in which miR-10a played a dominant role.In the case of ovarian damage,exosomes can transmit information to inhibit granulosa cell apoptosis to achieve the repair.

Oxidative stress and exosomes

Oxidative stress can damage mitochondrial DNA,leading to mitochondrial dysfunction and induce ovarian failure[16].Seok et al.[3]transplanted human placenta-derived mesenchymal stem cells into ovariectomized rat model,analyzed serum exosomes,and found that they contain antioxidant factors,which can restore ovarian function by upregulating antioxidant factors,providing a new idea for the treatment of POF.

In conclusion,exosomes play a certain role in the treatment of POF and can be used as the direction of diagnosis and treatment for research.For POF caused by granulosa cell apoptosis,exosomes can inhibit granulosa cell apoptosis through information expression.Exosomes can also restore ovarian function by resisting oxidative stress.The etiology of POF is relatively complex,and the relationship between the pathogenesis of genetic factors,immune factors,and exosomes is still to be explored.

The relationship between exosomes and liver-qi stagnation

According to traditional Chinese medicine theory,the liver can promote the development of qi and promote the movement of the qi and blood.The liver plays a leading role in draining and promoting body fluid circulation.Contrarily,liver-qi stagnation is not free.Qi and blood stasis are noted and became turbid.Moreover,liver loss and discharge,body fluid metabolism disorder,and sputum gathering were noted.The liver controls dispersion,adjusts the qi,and regulates the qi and blood.If liver qi is stagnant,qi and blood operation are not easily smoothed to damage the liver collaterals.Consequently,qi liver collaterals and blood deficiency easily cause hypoxia[17].In the experimental study of rats,hypoxia exists in liver stagnation and spleen deficiency[18].Hypoxia is a factor that cannot be ignored to cause cell damage.Simultaneously,the discovery of the production and induction mechanism of exosomes in the hypoxia environment is of practical significance for the prevention and treatment of diseases.Thus,hypoxia is a factor causing cell damage that cannot be ignored.Yang qi cannot be separated from the liver.If the liver qi is stasis,then the function of the liver cannot be normally played.The yang qi is crabbed.Thus,depression and heat change.Inflammation can be considered as the yang-heat range of traditional Chinese medicine[17].

Qimovementstagnation,hypoxic microenvironment and exosomes

You et al.[19]found that hypoxic conditions led to increased production of exosomes in liver cancer cells,and the miR-1273f level in exosomes was higher.In addition to activating the Wnt/β-catenin signaling pathway,the miRNA was also responsible for the direct replication of hypoxic exosomes in hepatoma carcinoma cells.Chen et al.[20]found that exosomes from MSCs mainly protect cells from hypoxia-induced apoptosis by carrying miR-21,alleviating endoplasmic reticulum stress,and inhibiting p38 MAPK signals.These results suggest that mesenchymal stem cell exosomes can improve the survival rate of microencapsulated islets and benefit diabetic patients.Cheng et al.[21]found that in vitro mesenchymal stem cell exosomes enhance the survival rate of cardiomyocytes under hypoxia.Hannafon et al.[22]suggested that breast cancer exosomes contain MTA1 and can transfer it to other cells,resulting in hypoxia and changes in estrogen receptor signal transduction in the tumor microenvironment.Moreover,Chen et al.[23]found that under hypoxia conditions,ovarian cancer cell-derived exosomes deliver miRNAs to induce M2 macrophages to polarize,thereby promoting the proliferation and migration of ovarian cancer cells.Thus,exosomes can physiologically adapt to the environment under hypoxic conditions and promote pathological processes.

Exosomes transmit information under the pathological state of hypoxic microenvironment caused by liver-qi stagnation,showing different roles.They can actively replicate,promote the disease process,or protect cells from hypoxic damage.Simultaneously,exosomes were found to restore ovarian function by expressing antioxidant factors during the treatment of POF.These findings can provide a basis for the treatment of POF caused by liver-qi stagnation by exosomes.

Turbid accumulation,inflammatory response,and exosomes

Studies have shown that the inflammatory factor expression in liver depression patients is elevated[24].Informationcanbetransmittedthrough exosome-mediated cellular communication in the presence of inflammatory infection.Singla et al.[25]found that embryonic stem cell-derived exosomes or embryonic stem cells can increase M2 macrophages and anti-inflammatory cytokine IL-10,inhibit inflammation-induced cell pyroptosis,and improve cardiac remodeling for cardiotoxicity induced by doxorubicin.Li et al.[26]found that exosomes derived from mesenchymal stem cells can inhibit apoptosis and inflammation by reducing the expression of inflammatory factors(e.g.,interleukin-6,tumor necrosis factor-α,nuclear factor κB,and interferon-gamma).Moreover,it has a protective effect on renal ischemia-reperfusion injury.Zhao et al.[27]found that exosomes of adipose stem cells from patients with osteoarthritis were found to express higher microRNA-145 and microRNA-221 levels,which are associated with increased proliferation and chondrogenic potential of periosteum cells,suggesting that they have the potential to serve as a therapeutic tool to reduce chronic inflammation and promote cartilage regeneration.Patel et al.[28]studied the exosomes of adipo-derived stem cells for traumatic brain injury treatment in rats,analyzed the brain and spleen transcriptome using RNA Seq,and showed that theexosomeMALAT1generegulates inflammation-related pathways,cell cycle,cell death,and regeneration molecular pathways.In the study of pancreatitis treatment,Wang et al.[29]found that the overexpression of the KLOTHO gene using the exosomes of mesenchymal stem cells can reverse apoptosis and reduce the levels of interleukin-6 and tumor necrosis factor-α,suggesting that it has an anti-inflammatory effect.In the presence of an inflammatory environment in the body,exosomes can promote the expression of related genes and inhibit the expression of inflammatory factors to carry out a positive anti-inflammatory effect,thereby inhibiting the inflammatory response.Moreover,in the inflammatory response caused by liver-qi stagnation,exosomes can inhibit the inflammatory response and cell apoptosis by reducing the expression of inflammatory factors.Furthermore,exosomes can protect ovaries from damage by inhibiting the apoptosis of granulosa cells in the treatment of POF.This coincides with POF caused by liver-qi stagnation.

Exosomes can be released by almost all cell types as tiny vesicles are secreted by cells.Exosomes can protect cells against hypoxia,inhibit inflammatory factors,and prevent cell apoptosis in the hypoxic microenvironment,inflammatory reaction,and other pathological environments similar to liver-qi stagnation.In the POF treatment process,it can inhibit the apoptosis of granulosa cells by expressing RNA and restoring ovarian function by upregulating antioxidant factors to achieve the purpose of treating POF.From the perspective of the pathogenesis of POF,exosomes can intervene and treat POF from the two aspects of inhibiting granulosa cell apoptosis and resisting oxidative stress.From the perspective of integrated traditional Chinese and western medicine,exosomescanalsobeantioxidationand anti-inflammatory,which may be the pathological factors of POF in the type of liver depression syndrome.The specific diagnosis and treatment methods of exosomes in POF still need more exploration.

Conclusion

To sum up,the author believes that exosomes are associated with liver-qi stagnation as well as POF.The incidence of POF has been continuously increasing annually in recent years.Most of the disease is based on kidney deficiency,and the disorder of the liver,spleen,and kidney is the common disease.Negative emotions(e.g.,anxiety or depression)have become factors that cannot be ignored with the development of time and the progress of society.A long-term negative emotional state can easily lead to liver qi discomfort,causing liver depression and qi stagnation.POF accompanied by liver and qi stagnation has become increasingly common.The traditional Chinese medicine for relaxing the liver and relieving depression has a certain effect on POF,but the specific mechanism of liver-qi stagnation causing POF is still unclear,which needs to be further studied.Under the guidance of traditional Chinese medicine theory,the study of the treatment of exosomes in POF can provide a new idea for the treatment of POF by combining traditional Chinese and western medicine.