Studying the nature of ascending-descending-floating-sinking of Chinese medicines based on gonadotropin-releasing hormone

FU Yang,YUAN Peipei,ZENG Mengnan,HOU Ying,GAO Liyuan,WEI Yaxin,ZHENG Xiaoke,FENG Weisheng

FU Yang,YUAN Peipei,ZENG Mengnan,HOU Ying,GAO Liyuan,WEI Yaxin,ZHENG Xiaoke,FENG Weisheng,Department of Pharmacy,Henan University of Chinese Medicine;The Engineering and Technology Center for Chinese Medicine Development of Henan Province;Co-construction Collaborative Innovation Center for Chinese Medicine and Respiratory Diseases by Henan &Education Ministry,Zhengzhou 450046,China

Abstract OBJECTIVE:To explore the nature of ascendingdescending-floating-sinking of Traditional Chinese Medicine on normal rats using gonadotropin-releasing hormone (GnRh).METHODS:Normal male Sprague-Dawley rats were orally administered six floating Chinese medicines and seven sinking Chinese medicines for 14 d.Then,GnRh and relevant indicators were detected.Initially,the different effects of floating and sinking drugs on the body were explored.To verify the effects of floating and sinking drugs on the body,normal rats were orally administered Mahuang (Herba Ephedra Sinica) and Tinglizi (Semen Lepidii Apetali) for 14 d.Then,GnRh antagonists were administered.We observed the changes of relevant indicators and clarified the correlation between GnRh and the laws of floating and sinking.RESULTS:Floating Chinese medicines significantly increased the testicular coefficients;GnRh level in the serum;the protein level of GnRh receptor (GnRhR) in the testis;and the mRNA levels of androgen receptor (AR),small C-terminal domain phosphatase (SCP) 3,SCP2,SYCE1,SMC1B,SMC3,and Rec8 (P <0.01 or <0.05). Sinking Chinese medicines did not react similarly,while GnRh antagonists blocked the regulatory effect of Mahuang (Herba Ephedra Sinica) but did not affect Tinglizi (Semen Lepidii Apetali).CONCLUSIONS:GnRh may be closely related to the nature of ascending-descending-floating-sinking of Chinese medicine.Floating Chinese medicines may promote their medicinal properties by regulating GnRH level;however,sinking Chinese medicines did not affect GnRh level.

Keywords:gonadotropin-releasing hormone;receptors,LHRH;Mahuang (Herba Ephedra Sinica);Tinglizi (Semen Lepidii Apetali);testis;ascending-descending-floating-sinking.

1.INTRODUCTION

The concept of ascending-descending-floating-sinking in Traditional Chinese Medicine (TCM) refers to the effect of drugs on the human body,called the ‘drug potential’.This plays an extremely important role in the treatment of diseases.ascending-descending-floating-sinking is an important part of TCM and plays a guiding role in clinical treatment.The ascending-descending-floatingsinking of TCM is relative to the effects of the diseases and summarises the curative effect on these diseases.Currently,the ascending-descending-floating-sinking of most TCMs is unavailable,and the understanding of ascending-descending-floating-sinking itself varies.The research on ascending-descending-floating-sinking is often based on the effect of a certain floating Chinese medicine or a certain sinking Chinese medicine.The lack of comprehensive and systematic methods to comb and explain the theory of ascending-descending-floatingsinking leads to the lack of scientific understanding in the field of TCM.We put forward the Chinese medicine floating drug theory:The ascending-descendingfloating-sinking of TCM is the theory that medicinal properties can be summarised through the special properties of TCM,and the efficacy after combination can be evaluated using animal experimentation and systems biology.

Normal rats were used to study the cold and hot properties of medicines.After repeated experimentation,it was confirmed that the 14-day intragastric administration of TCMs in normal rats is conducive to finding the effects of these drugs on the body.Hot TCM can promote energy metabolism,and cold TCM can inhibit energy metabolism.1This study adopts a similar research model using normal rats with typical floating and sinking Chinese medicines to discuss the concept of ascending-descending-floating-sinking.The following clinically recognised floating and sinking Chinese medicines were selected:Mahuang (Herba Ephedra Sinica,MH),Chaihu (Radix Bupleuri Chinensis,CH),Shengma (Rhizoma Cimicifugae Foetidae,SM),Gegen(Radix Puerariae Lobatae,GG),Bohe (Herba Menthae Haplocalycis,BH),Jiegeng (Radix Platycodi,JG),Dingxiang (Flos Syzygii Aromatici,DX),Kuxingren(Semen Armeniacae Amarum,KXR),Chuanniuxi (Radix Cyathulae,CNX),Fuling (Poria,FL),Daodou(Canavaliae Semen,DD),Xuanfuhua (Flos Inulae Japonicae,XFH),Tinglizi (Semen Lepidii Apetali,TLZ),and two-way regulating medicine Chuanxiong (Rhizoma Chuanxiong,CX) was used as the control.After 14 d of oral administration in normal male rats,the regularity of ascending-descending-floating-sinking was checked by measuring the organ coefficients and the central nervous system,circulation system,digestive system,endocrine system,and energy metabolism-related indicators.

In this study,the level of axial gonadal hormones and meiosis-related indicators were tested.To observe the corresponding changes,MH and TLZ were used with a gonadotropin-releasing hormone (GnRh) antagonist:cetrorelix (CE).Therefore,the aim of this study was to explore the effects of floating and sinking Chinese medicine on GnRh.The similarity and difference between the effects were also explored to provide theoretical basis for subsequent studies on TCM.

2.MATERIALS AND METHODS

2.1.Ethics approval

All the experiments in this study were conducted in accordance with the protocol approved by the Institutional Animal Care and Use Committee of Henan University of Chinese Medicine (Approval No.DWLL201908112).

2.2.Plant material and preparation of plant extracts

All 14 TCMs were purchased from Beijing Tongrentang Pharmacy.Professor Chen Suiqing of Henan University of Chinese Medicine authenticated them.Herbs (1 kg)were boiled twice in distilled water for 1 h each and filtered.

2.3.Experimental animals

Male Sprague-Dawley (SD) rats (190–240 g) were purchased from Vital River Laboratory Animal Technology (Beijing,China).The approval number for animal experiments is SCXK (Beijing) 2016-0006.Animals were kept at room temperature with 12 h light/dark cycles and housed in groups of four rats per cage.The bedding materials were granules of corncob.

2.4.Methods:The experiment groups

2.4.1 Effect of floating or sinking Chinese medicines onormal rats

All rats were fed for 3 d with clean food and drinking water.A total of 150 male SD rats were randomly divided into 15 groups:normal (NC),MH,CH,SM,GG,BH,JG,DX,KXR,CNX,FL,DD,XFH,TLZ,and CX.The dosage of the TCMs were administered to the rats according to the 2020 Pharmacopoeia.The dosage of TCMs were 42 times that of human medicine,except for MH which was 28 times.

2.4.2 Effects of MH and TLZ with GnRh antagonists on normal rats

A total of 60 male SD rats were randomly divided into 6 groups:NC group,NC+CE group,MH+CE group,MH group,TLZ group,and TLZ+CE group.Each antagonist group was injected intraperitoneally:300 µg·kg-1·d-1CE.The MH and MH+CE groups were administered 4.2 g/kg of MH.The TLZ and TLZ+CE groups were administered 4.2 g/kg of TLZ.

2.5.Sample collection

After 14 d of administration,rats were euthanised with pentobarbital sodium.Blood samples from the aorta were centrifuged for 10 min at 3000 rpm.Tissues of rats from each group were stored at－80 ℃.

2.6.Enzyme-linked immunosorbent assay (ELISA)

Contents of serum GnRh,luteinising hormone (LH),and testosterone (T) were detected according to the manufacturer's instructions using GnRh (E-EL-0071c),LH (E-EL-R0026c),and T (E-EL-0155c) ELISA kits(Elabscience Biotechnology Co.,Ltd.,Wuhan,China).

2.7.Immunofluorescence

The levels of testicular tissue-related protein gonadotropin-releasing hormone receptor (GnRhR),androgen receptor (AR),and small C-terminal domain phosphatase 3 (SCP3) were detected using the GnRhR antibody (19950-1-AP,Proteintech,Wuhan,China),the SCP3 antibody (23024-1-AP,Proteintech),and the AR antibody (22089-1-AP,Proteintech).Fluorescent Cy3-coupled secondary antibodies were used for detection.

2.8.Western blotting

Testicular protein was extracted using a total protein extraction kit (BC3710,Solarbio Science &Technology Co.,Ltd.,Beijing,China).Protein quantification was performed using the BCA kit (PC0020,Solarbio Science&Technology Co.,Ltd.,Beijing,China).The SDSPAGE gel preparation kit (64136301,Bio-Rad,Hercules,CA,USA) was used for gel preparation.Each well containing 50 μg of protein was subjected to 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane.After sealing for 1.5 h,the primary antibodies against GnRhR (1∶500) and β-Actin (1∶5000) were added and incubated overnight at 4 ℃.After washing five times with PBST (PBS with 0.2% Tween20),the secondary antibodies (9910T,Cell Signaling Technology,Inc.,Danvers,MA,USA) were added and incubated for 1 h.The blots were then washed three times with PBST before western blot analysis.The Odyssey two-colour infrared fluorescence imaging system was used for imaging.The western blot values were expressed after normalisation to β-actin levels using the Image Studio software (LI-COR Biosciences,Lincoln,NE,USA).

2.9.Real-time Quantitative Polymerase Chain Reaction(RT-qPCR)

Total RNA was extracted from testis tissue using an RNA extraction kit (Solarbio,Beijing,China),according to the manufacturer’s instructions.The cDNA was obtained with a reverse transcription kit (D7168M,Applygen Technologies Inc.,Beijing,China).The cDNA was used as a template for SYBR qPCR Master Mix (Vazyme Biotech,Nanjing,China) for RT-qPCR.The primer sequences for GnRhR,AR,SCP3,SYCP2,Rec8,SYCE1,SMC1B,SMC3,and GAPDHare listed in Table 1.

2.10.Statistical analysis

All data are presented as the mean ± standard deviation().The data were analysed with SPSS 25.0 statistical software (IBM Corp.Released 2017.IBM SPSS Statistics for Windows,Version 25.0.Armonk,NY,USA).The statistically significant differences between groups were determined by one-way analysis of variance.P <0.05 was considered statistically significant.

3.RESULTS

3.1.Effects of typical floating and sinking Chinese medicines on testicular coefficient and sex hormone levels in normal male rats

The main chemicals in typical floating and sinking Chinese medicines are listed in Table 2.Compared with that in the normal group,neither floating nor sinking Chinese medicine treatment had any effect on the rat testis structure (Figure S1).Five floating Chinese medicines significantly increased the testicular coefficient and GnRh level in the serum of rats (P <0.01 or <0.05),compared to those of the NC group.The seven sinking Chinese medicines did not show similar effects(Figure 1).It is suggested that an increase in testicular coefficient may be related to an increase in GnRh levelin vivo.

3.2.Effects of typical floating and sinking Chinese medicines on GnRhR in the testis of normal male rats

The expression of GnRhR in the testis of normal rats was significantly increased by five typical floating Chinese medicines (P <0.01 or <0.05),compared with that of the NC group.However,the seven sinking Chinese medicines had no effect on the expression of GnRhR(Figure 2).

3.3.Effects of typical floating and sinking Chinese medicines on sperm meiosis-related mRNA levels in the testis of normal male rats

The levels of GnRhR,AR,and SCP3 mRNA in the testis were significantly increased by the typical floating Chinese medicines (P <0.01 or <0.05),compared with those of the NC group (Table 3).The typical sinking Chinese medicines significantly reduced the level of SCP3 mRNA in the testis (P <0.01) (Table 3).This suggests that the effects of typical floating and sinking Chinese medicines on sperm meiosis might be different.Therefore,the expression of genes regulating sperm meiosis that are closely related to SCP3 were detected.The levels of SYCE1,SMC1B,SMC3,SYCP2,and Rec8 in the typical floating Chinese medicine-treated group were significantly increased (P <0.01 or <0.05),compared with that of the NC group,as shown in Table 4.The levels of SYCE1,SMC1B,and Rec8 were significantly reduced in the typical sinking Chinese medicine-treated group (P <0.01 or <0.05) (Table 4).

Table 1 Primer sequences

Table 2 Main chemicals in typical floating and sinking Chinese medicines

3.4.Effects of MH and TLZ with CE on testicular coefficient and sex hormone levels in normal male rats

To verify the results of the previous experiments,the GnRh antagonist CE was selected for the experiment.MH and TLZ were selected for subsequent antagonistexperiments.MH significantly increased testicular coefficient and GnRh levels (P<0.01),compared with those of the NC group,and TLZ had no effect on testicular coefficient and sex hormone levels.Following the administration of antagonists,the testicular coefficient and the levels of LH and T in the serum of rats in the NC+CE group were significantly lower than those of the NC group (P<0.01).Testicular coefficient of the MH+CE group significantly decreased (P<0.01),compared with that of the MH group (Figure 3).

Figure 1 Effects of typical floating and sinking Chinese treatments on testicular coefficient and sex hormone levels in normal male rats

3.5.Effects of MH and TLZ with CE on protein levels of GnRhR,AR,and SCP3 in the testis of normal male rats

Immunofluorescence results showed that the levels of GnRhR,AR,and SCP3 were significantly decreased after administration of CE compared with those of the NC group (P<0.01 orP<0.05).MH significantly increased the protein levels of GnRhR,AR,and SCP3 (P<0.01 orP<0.05);however,CE significantly inhibited these effects (P<0.01).TLZ had no effect on the levels of GnRhR,AR,and SCP3 as shown in Figure 4.Western blotting was conducted to examine the level of GnRhR in rat testis,which further verified the results of immunofluorescence analysis (Figure 5).

Figure 2 Effect of typical floating and sinking Chinese medicines on GnRhR expression in the testis of normal male rats

Figure 3 Effects of MH and TLZ with cetrorelix on testicular coefficient and sex hormone levels in normal male rats

Figure 4 Effects of MH and TLZ with cetrorelix on protein levels of GnRhR,AR,and SCP3 in the testis of normal male rats using immunofluorescence assay

Figure 5 Effects of MH and TLZ with cetrorelix on protein levels of GnRhR,AR,and SCP3 in the testis of normal male rats using Western blotting

3.6.Effects of MH and TLZ with CE on sperm meiosisrelated mRNA levels in the testis of normal male rats

The mRNA levels of GnRhR,AR,and SCP3 in the testis of rats were significantly decreased in the NC+CE group,compared with those of the NC group (P <0.01 or <0.05).MH significantly increased the mRNA levels of GnRhR,AR,and SCP3 (P <0.01 or <0.05);however,these effects were abolished by CE.TLZ had no effect on the level of GnRhR;however,the mRNA levels of GnRhR,AR,and SCP3 were significantly decreased after combining CE (P <0.01 or <0.05) (Table 5).

The sperm meiosis-related mRNA levels of SMC1B,SMC3,SYCP2,and Rec8 in the testis of rats were significantly decreased (P <0.01 or <0.05),compared with those of the NC group.MH significantly increased the mRNA levels of SYCE1,SMC1B,SMC3,SYCP2,and Rec8 (P <0.01 or <0.05);however,these effects were blocked by CE.TLZ had no effect on the level of sperm meiosis-related mRNA (Table 5).

Table 3 Effects of typical floating Chinese medicines on sperm meiosis-related mRNA levels in the testis of normal male rats ( )

Table 3 Effects of typical floating Chinese medicines on sperm meiosis-related mRNA levels in the testis of normal male rats ( )

Notes:NC:Normal control group;MH:Mahuang (Herba Ephedra Sinica);CH:Chaihu (Radix Bupleuri Chinensis);SM:Shengma (Rhizoma Cimicifugae Foetidae);GG:Gegen (Radix Puerariae Lobatae);BH:Bohe (Herba Menthae Haplocalycis);JG:Jiegeng (Radix Platycodi).MH(4.2 g/kg),CH (7 g/kg),SM (7 g/kg),GG (10.5 g/kg),BH (4.2 g/kg),JG (7 g/kg) were administered to the rats of different groups by oral gavage.GnRhR:gonadotropin-releasing hormone receptor;AR:androgen receptor;SCP3:Synaptonemal complex protein 3;SYCE1:synaptonemal complex central element 1;SMC1B:Structural maintenance of chromosomes protein 1B;SMC3:Structural maintenance of chromosomes protein 3;SYCP2:Synaptonemal complex protein 2;REC8:meiotic recombination protein.Compared with the normal group,aP <0.01,bP <0.05.

Table 4 Effects of typical sinking Chinese medicines on sperm meiosis-related mRNA levels in the testis of normal male rats ( )

Table 4 Effects of typical sinking Chinese medicines on sperm meiosis-related mRNA levels in the testis of normal male rats ( )

Notes:NC:normal control group;DX:Dingxiang (Flos Syzygii Aromatici);KXR:Kuxingren (Semen Armeniacae Amarum);CNX:Chuanniuxi(Radix Cyathulae);FL:Fuling (Poria);DD:Daodou (Canavaliae Semen);XFH:Xuanfuhua (Flos Inulae Japonicae);TLZ:Tinglizi (Semen Lepidii Apetali);CX:Chuanxiong (Rhizoma Chuanxiong).DX (2.1 g/kg),KXR (7 g/kg),CNX (7 g/kg),FL (10.5 g/kg),DD (6.3 g/kg),XFH(6.3 g/kg),TLZ (7 g/kg),and CX (7 g/kg) were administered to the rats of different groups by oral gavage.Compared with the normal group,aP<0.05,bP <0.01.

Table 5 Effects of MH and TLZ with cetrorelix on sperm meiosis-related mRNA levels in the testis of normal male rats ()

Table 5 Effects of MH and TLZ with cetrorelix on sperm meiosis-related mRNA levels in the testis of normal male rats ()

Notes:MH (4.2 g/kg) and TLZ (4.2 g/kg) were administered by oral gavage for the different groups.Each antagonist group injected intraperitoneally 300 µg·kg-1·d-1 cetrorelix.NC:Normal control group;NC+CE:Normal+cetrorelix group;NC:Normal control group;NC +CE:Normal+cetrorelix group;MH:Mahuang (Herba Ephedra Sinica) group;MH+CE:Mahuang (Herba Ephedra Sinica)+cetrorelix group;TLZ:Tinglizi (Semen Lepidii Apetali) group;TLZ+CE:Tinglizi (Semen Lepidii Apetali)+cetrorelix group.GnRhR:gonadotropin-releasing hormone receptor;AR:androgen receptor;SCP3:Synaptonemal complex protein 3;SYCE1:synaptonemal complex central element 1;SMC1B:structural maintenance of chromosomes protein 1B;SMC3:structural maintenance of chromosomes protein 3;SYCP2:synaptonemal complex protein 2;REC8:meiotic recombination protein.Compared with the normal group,aP <0.01,bP <0.05;compared with the MH group;cP <0.01,dP <0.05.

4.DISCUSSION

Ascending-descending-floating-sinking is an important part of the basic theory of TCM and one of the most important approaches for treating diseases.Huang Di Nei Jing first proposed the theory of ascending-descendingexiting-entering and systematically discussed it from two aspects of physiological function and pathological change.16Su Wen·Ju Tong Lun provides information on the influences of six exogenous evils on the rise and fall ofQi.It also points out that ‘hundreds of diseases are caused byQi’ and emphasises on the relationship betweenQimovement and diseases.‘Febrile Diseases’are regarded as the earliest ones for which the theory of ascending-descending-floating-sinking was applied.The ascending-descending-floating-sinking of TCM is relative to the trend of diseases and is summarised based on the effects of the disease.If diarrhoea and anal prolapse are caused by the lack of spleenQi,the disease trend is Low and Sink.CH,SM,and other floating drugs should be used to tonifyQiand elevate yang.For those with cough due to rebellion of the lung-Qi,KXR should be used to relieve cough and asthma.Therefore,TCM medication selection often requires a detailed analysis of disease tendency to select the corresponding floating or sinking medicines.

Floating Chinese medicines mainly act upward and outward by increasing yang and are used to treat cold and vomiting.Sinking Chinese medicines mainly act downward and inward by suppressing yang,and are used to treat hiccup,toxicity,and renal disorders.CH,SM,and GG have the role of Shubiao.17,18SM and GG decoction,obtained from ‘Yan's Theory of Infant Formula’,cools the muscles,clears rash,detoxifies,and treats measles at early stages.19Bu-Zhong-Yi-Qi decoction is the core component of Li Dong-Yuan,which is used for the treatment of spleen and stomach diseases.SM and CH can lift the depressedQiin the spleen and stomach.The ascendingQiof Shaoyang can warm the heart and lungs.20BH,anti-wind evil stagnation,is good at introducing medicine into Yingwei.Yinqiao Powder,used since the Qing Dynasty,and BH and Jinyinhua(Flos Lonicerae) mentioned in ‘Wenbing Tiaobian’ can be used for the treatment of fever,mild evil wind,cold,headache,and other symptoms.21Huang Gongxiu’s‘Bencao Qiuzhen’ recorded that JG could remove the cold in the lungs and help the medicine act upward.22MH increases sweating and relieves asthma.KXR can relieve asthma and cough.Both are components of MH decoction,used to restore the function of the lungs.FL is sweet,light,and flat,and it removes dampness,promotes diuresis,invigorates the spleen,tranquilizes the heart,and is a key ingredient of the Wuling powder.23TLZ has a bitter and cold nature.TLZ has the effect of purging lung heat,promoting diuresis,and eliminating phlegm.24TLZ Dazao (Fructus Jujubae) Xiefei decoction use has been recorded in ‘Jingui Yaolue’ by Zhang Zhongjing.The bitter and cold nature of TLZ and Dazao (FructusJujubae) are used to remove evil without injuring the right.25As the so-called ‘all flowers are rising,Xuan Fu is down’,XFH can reduceQito remove fullness and cease vomiting.Zhang Zhongjing commonly used in the XFH Daizhe decoction is used to treat typhoid fever.26‘Compendium of materia medica’ has recorded that DD and DX have the effect of warming the spleen and stomach and stopping hiccups.CNX can guide the medicine down.CNX contained in Zhang Xichun's‘Yixue Zhongzhong canxilu’ can replenish theYinof the liver and kidney and induce blood downward.27‘Bencao Huiyan’ has recorded the bidirectional regulation of CX.Combined with ancient literature records and clinical efficacy,in this study,we selected six types of typical floating Chinese medicines—MH,CH,SM,GG,BH,and JG — and seven types of typical sinking Chinese medicine — DX,KXR,CNX,FL,DD,XFH,and TLZ.The two-way regulating medicine CX was used as the control.Normal male rats were tested first.

The heart and lung belong to the Shang Jiao.They cooperate with each other to ensure the normal circulation ofQiand blood.The spleen and stomach belong to Zhong Jiao.They coordinate with each other to complete digestion and absorption of food.The normal entry and exit ofQican maintain metabolism of the body.The movement and promotion ofQiincreases blood generation and circulation.The heart nourishes the whole body by transporting blood and substances that support life.The normal operation of blood is the basis of normal mental activity.Therefore,the functionality of the circulatory system (cardiac function-related indexes,renal blood flow indexes,and blood pressure),central system (autonomic activity),and digestive system(plasma gastrin and somatostatin levels) in rats were detected in this experiment.No difference in the effects of typical floating and sinking Chinese medicines on these systems were observed (see supplementary materials).The most surprising finding was that floating Chinese medicines could promote an increase in the testicular coefficient,except BH.To monitor the side effects of these drugs,the HE sections of the testis were observed to confirm that there was no damage to the testis of male rats under neither floating nor sinking Chinese medicine treatment (see supplementary materials).Chinese medicine theory believes that essence is the fundamental substance that constitutes the human body.The kidney stores essence to maintain human growth,reproduction,and viscera function.‘Jing’is born to parents and stored in the kidney.GnRh is a decapeptide hormone synthesised and secreted by the hypothalamic arcuate nucleus and preoptic medial nucleus neuroendocrine cells.In male animals,GnRh mainly stimulates the synthesis and secretion of testosterone in the gonad and regulates the gonadal axis,promotes the development of secondary sexual characteristics and testicular maturation,and maintains the normal function of the reproductive system.This is similar to the function of kidney essence in TCM theory.The smooth movement ofQiin and out of the body is closely associated with the lungs,spleen,and kidneys.Among them,the kidney is the foundation of the body.The harmony of pneuma is the foundation of gestation,and Tian Gui is the premise of gestation.If kidneyQiis not adjusted,Tian Gui deficiency causes infertility.Tian Gui is mainly related to menstruation,semen production,and maintenance of the second sex syndrome,which is similar to the function of GnRh.Therefore,it is speculated that GnRh may be related to kidneyQi.Combined with the experimental results,it is suggested that the floating Chinese medicines can increase GnRh level.Previously,some scholars showed that the floating Chinese medicines GG can increase the GnRh levels of ovariectomised female rats and can increase the GnRh levels of male mice with reproductive system damage.28,29However,it was noted that the floating Chinese medicines did not increase the levels of LH and T in the serum,which may be related to the extensive effect of floating Chinese medicines on regulating immunity.30-32In vivo,GnRh can induce a cascade of reactions by stimulating GnRhR expression.33It has been found that GnRhR is expressed in many normal and pathological tissues outside the hypothalamus and pituitary.The reactivity and function of GnRh and GnRhR are specific.34The experimental results showed that the floating Chinese medicines significantly increased the levels of GnRh,and the sinking Chinese medicines had no effect.Western blot and reverse transcription-polymerase chain reaction (RT-PCR)analyses were used to detect the level of GnRhR in rat testis.It was found that the floating Chinese medicines significantly increased the expression of GnRhR,and the sinking Chinese medicines had no effect on its expression.RT-PCR showed that floating Chinese medicines could significantly increase the mRNA level of AR,and sinking Chinese herbs showed no effect.

Spermatogenesis is a complex biochemical event,involving the participation of the hypothalamus and the pituitary gland via secretion of the hypothalamic hormone GnRh.35Meiosis is a key continuous process and is an important part of spermatogenesis in the testis.36Data on sperm motility was not obtained;however,the expression of sperm meiosis-related genes were determined in this study.Synaptic complex protein 3(SCP3) is a key factor in meiosis,mainly expressed in primary spermatocytes.SCP3 knockout homozygous male mice were infertile.Therefore,testicular coefficient decreased significantly and caused apoptosis in spermatogenic cells.36The experimental results showed that the floating Chinese medicines significantly increased the level of SCP3,and the sinking Chinese medicines reduced it significantly.Genes closely related to SCP3 were found through the STRING database,including SCP2,SYCE1,SMC1B,SMC3,and Rec8.36-40Using RT-PCR,we found that floating Chinese medicines significantly increased the levels of sperm meiosis-related genes.This may be related to the increase of GnRh and GnRhR levels in the testis,reflecting the nature of Lift-Float.Sinking Chinese medicines did not increase GnRh and GnRhR levels in the testis but decreased the levels of SYCE1,SMC1B,and Rec8,which may be unrelated to GnRh level,reflecting the property of Low-Sink.

Based on the above results,it is suggested that the Lift-Float property may be related to GnRh level.The floating Chinese medicines may increase the expression of GnRhR and AR in rat testis through GnRh,thus promoting sperm meiosis via the Lift-Float property.MH and TLZ were selected as the representative TCMs to test floating and sinking,respectively.

The GnRh antagonism experiment was carried out using CE.GnRh antagonists are analogous structures produced by modifying the amino acids of GnRh.GnRh antagonists have stronger affinity toward GnRhR and can quickly inhibit the gonadal axis.After administration,the levels of follicle stimulating hormone and LH decreased within several hours.41The results showed that after intraperitoneal injection of CE,the testicular coefficient,levels of LH and T in the serum,levels of GnRhR,AR,SCP3,and related meiotic genes were significantly decreased.These findings suggest that CE reduces the levels of sex hormones in rats and inhibits sperm meiosis.After administration of MH,the testicular coefficient of rats,gonadal axis hormone levels,expression levels of GnRhR,AR,SCP3,and mRNA of sperm meiosis-related factors increased significantly.These effects were inhibited by CE,indicating that MH plays a role in the Lift-Float property by regulating the levels of GnRhin vivo.TLZ administration showed no effect.Considering that both MH and TLZ can be used to treat asthma,we will establish an animal model of asthma in our followup studies.This model will be used to test the effects of MH and TLZ on lung injury indicators;the results will be compared with those obtained with the use of a GnRh antagonist,CE.

In this study,the correlation between ascendingdescending-floating-sinking property of drugs and GnRh level was determined,to provide new ideas for better understanding this theory.It also provides a theoretical foundation for identifying other targets of the ascendingdescending-floating-sinking property.In the follow-up study,metabolomics and proteomics technology will be used to study the components of various organs of rats and clarify the material basis for their roles.However,pharmacological studies on other floating Chinese medicines combined with antagonists to explain further the correlation between Lift-Float properties and GnRh level are needed.

In conclusion,GnRh may be closely related to the nature of ascending-descending-floating-sinking of Chinese medicines.Floating Chinese medicines may regulate GnRh level,and sinking Chinese medicines have no effect on GnRh expression.

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