ZHANG Tiantian,LI Guowen,CHU Zhifeng,LI Jie,YU Lijun,GAO Yuan,WANG Huifeng

ZHANG Tiantian,CHU Zhifeng,YU Lijun,GAO Yuan,Department of Pharmacy,Shanghai Songjiang Fangta Hospital of TCM,Shanghai 201600,China

LI Guowen,Department of Pharmacy,Shanghai TCM-Integrated Hospital,Shanghai 200080,China

LI Jie,Department of Traditional Chinese Medicine,Shanghai Songjiang Fangta Hospital of TCM,Shanghai 201600,China

Abstract OBJECTIVE: To investigate the most effective weight ratio of Qiancao (Radix Rubiae Cordifoliae) and Haipiaoxiao (Endoconcha Sepiellae) used to produce decoction for the treatment of abnormal uterine bleeding(AUB) in rats with incomplete abortion,and to study the possible mechanism.METHODS: The models of AUB were established by incomplete drug abortion with mifepristone and misoprostol in pregnant rats.The therapeutic effects of decoctions made by eight different weight ratios of Qiancao (Radix Rubiae Cordifoliae) and Haipiaoxiao(Endoconcha Sepiellae) were observed.RESULTS: Statistical analysis revealed that the most effective weight ratio of Qiancao (Radix Rubiae Cordifoliae) and Haipiaoxiao (Endoconcha Sepiellae) to treat AUB in this study was 2∶1.CONCLUSION: The insights gained from this study would improve understanding of the mechanisms involved in the effect of Qiancao (Radix Rubiae Cordifoliae) and Haipiaoxiao (Endoconcha Sepiellae) on AUB.

Keywords: uterine hemorrhage;Qiancao (Radix Rubiae Cordifoliae);Haipiaoxiao (Endoconcha Sepiellae);herb pair;weight ratio

1.INTRODUCTION

Multi-herb prescription is one of the leading characteristics of Traditional Chinese Medicine (TCM).1The number of herbs in TCM prescription varies from two to even ＞ 50.Because of the complexity of multiherb prescription,the modernization and generalization of TCM has several obstacles.Herb pairs are used as the basic units for a prescription and the simplest form of multi-herb therapy.Thus,the study of herb pairs may act as the foundation in the study of the multi-herb therapy.2Patients with clinically diagnosed abnormal uterine bleeding (AUB) were treated by medicines or operations.Surgical treatments may cause severe anemia and infertility.The medical therapies for AUB could be hormonal or non-hormonal.Non-hormonal therapy,including non-steroidal anti-inflammatory drugs and antifibrinolytics are not sufficiently effective.3The use of hormonal therapy is limited due to various side effects.4Thus,Traditional Chinese Medicine has provided an alternative option to treat AUB patients.5Incomplete abortion induced by mifepristone and misoprostol causes abnormal uterine bleeding,and hence,could be applied to establish the AUB model.6

Qiancao(Radix Rubiae Cordifoliae) and Haipiaoxiao(Endoconcha Sepiellae) is a famous herb pair used to treat AUB in both ancient and modern clinical practice.In the pioneering Chinese medicinal monograph,Huang Di Nei Jing,Siwucegu-luruwan(consisting of four weights of Haipiaoxiao and one weight of Qiancao) was used to treat AUB.7In the integrating Chinese and Western Medicine in 1909 by Zhang Xichun,five herb prescriptions,mainly for bleeding,contain Qiancao(RadixRubiaeCordifoliae) and Haipiaoxiao(Endoconcha Sepiellae) herb pair.Surprisingly,the weight ratio of Qiancao (Radix Rubiae Cordifoliae) and Haipiaoxiao (Endoconcha Sepiellae) varies in the classic prescriptions mentioned above.The most common weight ratios [Qiancao(Radix Rubiae Cordifoliae) to Haipiaoxiao (Endoconcha Sepiellae)] clinically used in ancient prescriptions are 1∶4 (Siwucegu-luruwan),1∶2 (prescription Lixuetang),3 ∶ 4 (prescription Anchongtang and Guchongtang),and 1∶1 (prescription Qingshentang).Hitherto,there is no consensus on the most effective weight ratio of Qiancao (Radix Rubiae Cordifoliae) and Haipiaoxiao (Endoconcha Sepiellae) to treat AUB.Therefore,the study on the weight ratio of Qiancao (Radix Rubiae Cordifoliae) and Haipiaoxiao(Endoconcha Sepiellae) for the treatment of AUB could provide a basis for clinical application of these herbs.

In order to find the optimal weight ratio of Qiancao(RadixRubiaeCordifoliae) and Haipiaoxiao(Endoconcha Sepiellae) to treat AUB,we observed the effects of eight different weight ratios of this herb pair decoction on uterine bleeding volume,hemostasis[including prothrombin time (PT) and activated partial thromboplastin time (APTT)],and the uterine pathological changes due to incomplete abortion in rats.To understand the mechanisms of this herb pair,the serum levels of matrix metalloproteinase 1 (MMP1),prostaglandin I-2 (PGI2),and thromboxane A2 (TXA2)were detected.

2.MATERIALS AND METHODS

2.1.Animals and reagents

A total of 100 healthy female Sprague–Dawley (SD) rats and 50 male SD rats [(200 ± 20) g] were purchased from the Experimental Animal Center of Huazhong University of Science and Technology (certificate number 1100111911010829 for male rats and 1100111911010838for the female rats) and housed under a controlled environment (at temperature of (22 ± 2) ℃and relative humidity of 55% ± 15% with a 12 h light/dark cycle with free access to food and water) in the Animal Laboratory Center of Wuhan Servicebio Technology Co.Ltd.(Wuhan,China).

The decoction pieces of Qiancao (Radix Rubiae Cordifoliae) and Haipiaoxiao (Endoconcha Sepiellae)were purchased from Shanghai Tongjitang Pharmaceutical Co.Ltd.(Shanghai,China) and were identified by Professor Li Guowen of Shanghai TCMIntegrated Hospital.The voucher specimens were deposited in the Shanghai Songjiang Fangta Hospital of TCM (the voucher specimen numbers are 2018YP00401 for Qiancao and 2018YP00402 for Haipiaoxiao).

Rat MMP1,PGI2,and TXA2enzyme-linked immunosorbent assay (ELISA) kits were obtained from Cloud-Clone Corp.(Wuhan,China).PT and APTT kits were purchased from Rayto Life and Analytical Sciences Co.Ltd.(Shenzhen,China).

2.2.Preparation of decoctions

The most common weight ratios of Qiancao (Radix Rubiae Cordifoliae) to Haipiaoxiao (Endoconcha Sepiellae) clinically used in ancient prescriptions are 1∶1,3∶4,1∶2,and 1∶4.Hence,we prepared eight decoctions of different weight ratios [Qiancao (Radix Rubiae Cordifoliae) to Haipiaoxiao (Endoconcha Sepiellae);1∶0,2∶1,1∶1,3∶4,1∶2,1∶4,1∶8,0∶1].The chosen experimental weight ratios contain the most common weight ratios traditionally used in ancient prescriptions.

As the traditional dosage for human patients is 30 g decoction pieces/day,the dosage for rats should be 540 mg/200 g body weight according to the Meeh-Rubner equation.Then,54 g proportionally mixed decoction pieces of different weight ratios were added to 1080 mL purified water,respectively.After immersed for 30 min,the pieces were decocted in the water for 1 h.The decoctions were filtered and collected.The filtrate was concentrated to 200 mL for animal experiments.The volume of gavage was 1 mL/100 g body weight.This process was repeated daily during gavage.

2.3.Animal experiments

All animal experiments were approved by the Experimental Animal Ethics Committee of Wuhan Servicebio Technology Co.,Ltd.(Approval No.2019018)and performed in accordance with China’s Guidelines for Care and Use of Laboratory Animals.

Female SD rats were mated with males of proven fertility in the evening.If sperm was found in the vaginal smears the next morning,it was considered as day 1 of gestation.8On day 7 of pregnancy,female rats were randomly divided into a negative control group (GN),a positive control group (GP),and eight experimental groups (G1–G8).Incomplete abortions were induced with 8.3 mg/kg of mifepristone (8:00) and 100 μg/kg of misoprostol (18:00) in all pregnant rats.9,10For the following 7 d after incomplete abortion,rats in GN were treated with 1 mL distilled water/100 g body weight.GP rats were treated with Gongxuening capsule for 7 d.It is a patent Chinese botanical drug,widely used in the clinic to treat bleeding,such as excess catamenia,postpartum flooding,and uterine bleeding.11The clinical dosage of Gongxuening capsule for humans was 0.78 g/d.According to the Meeh-Rubner equation,the dose for rats was 7 mg/100 g body weight.G1-G8 rats were treated with decoctions made by proportionally mixed decoction pieces [Qiancao (Radix Rubiae Cordifoliae) to Haipiaoxiao (Endoconcha Sepiellae);1∶0,2∶1,1∶1,3∶4,1∶2,1∶4,1∶8,0∶1] at 270 mg/100 g body weight.All medicine and placebo were administered by gavage and the volume of gavage was 1 mL/100 g body weight.On day 14 of gestation,blood was withdrawn from the inferior vena cava of all rats 2 h after the final treatment.All rats were sacrificed by cervical dislocation,and laparotomy was performed.

2.4.Determination of uterine bleeding volume

After misoprostol administration,a cotton ball (90 mg)was inserted into the rat vagina.The cotton ball was replaced at 8:00 and 18:00 every day until day 14 of gestation.Cotton balls were stored at -20 ℃ until analysis.12Then,0.02 mL tail venous blood sample was withdrawn with syringe and mixed with 4 mL NaOH(50 g/L,V1).The blood-stained cotton balls were washed with an appropriate amount of NaOH two times,and the volume of NaOH (V2) was recorded.The absorbance (A)was measured at 546 nm.The formula of volume uterine bleeding was as follows:13

Uterine bleeding volume (mL)=tail vein blood (0.02 mL)×V2× the washed sample (A)/ tail vein blood (A) ×V1

2.5.Measurement of PT and APTT

Blood samples collected were anticoagulated with 3.8%sodium citrate solution,and plasma was obtained by centrifugation at 3500 rpm 4 ℃ for 15 min.14The plasma was used to assay PT and APTT using commercial kits purchased from Rayto Life and Analytical Sciences Co.,Ltd.(Shenzhen,China),according to the manufacturer’s instructions.

2.6.Histopathological examination

Uterine tissue was fixed in 4% paraformaldehyde solution,dehydrated at 4 ℃ for 24-48 h,and embedded in paraffin.Subsequently,the tissue was sliced in to 4-μm sections and stained with hematoxylin-eosin (HE).15The pathological histology was observed under a microscope.

2.7.ELISA

Serum was separated from the blood samples by centrifugation at 3500 rpm,4 ℃ for 5 min.ELISA was conducted to assess the serum level of MMP1,TXA2,and PGI2content,according to the manufacturer’s protocols (Cloud-Clone Corp.,Wuhan,China).TheAvalues were obtained at 450 nm,and the concentrations were calculated by converting the optical density against a standard curve.

2.8.Statistical analysis

All statistical data were analyzedviaSPSS GraphPad Prism software version 8.0 (GraphPad Software,Inc.La Jolla,CA,USA) statistical software.Data are presented as mean ± standard deviation (±s).The significance of difference between the groups was analyzed by oneway analysis of variance,followed by multiple comparison test for various parameters.Pvalue ＜ 0.05 was considered statistically significant.

3.RESULTS

3.1.Effects on bleeding volume

As shown in Table 1,the bleeding volume decreased significantly in GP and G1-G8 compared to GN (P＜0.001).Also,the bleeding volume decreased at the same in G2,G3,and G8 rate compared to GP.

3.2.Effects of PT and APTT on thrombin system

Experimental data are shown in Table 1.PT and APTT are significantly shortened in GP and G1-G8 compared to GN (P＜ 0.001).PT in G1,G2,G3,and G8 is significantly shortened compared to GP (P＜ 0.05,P＜0.001,P＜ 0.001,andP＜ 0.001,respectively),and compared to GP,APTT in G1-G8 was also shortened (P＜ 0.001).

3.3.Immunohistochemistry of uterine

After drug-induced incomplete abortion,hormone withdrawal triggered the influx of inflammatory cells into the endometrium and led to the degradation and necrosis of decidua and chorion.16

The immunohistochemical staining of the uterus in all the groups is shown in Figure 1.

Figure 1 Immunohistochemistry of uterus

Table 2 shows the patho-scores of uterine tissues in each group.The lower the patho-score,the less dilated interstitium vascular,the less infiltration of inflammatory,and the fewer necrotic decidual cells.Compared to GN,the patho-score in GP,G2,to G8 had reduced significantly (P＜ 0.001),and the patho-score in G2 reduced significantly compared to GP (P＜ 0.01).

3.4.Effects on serum levels of MMP1,TXA2,and PGI2

As shown in Table 3,serum MMP1 levels in GP and G1-G7 decreased significantly compared to GN (P＜ 0.001).Moreover,serum MMP1 levels in G2 decreased significantly than those in GP (P＜ 0.01),while the TXA2levels elevated significantly in GP and G2 compared to GN (P＜ 0.01 and ＜ 0.001).No difference was detected in serum PGI2levels in all groups.

Table 1 Effects on bleeding volume,PT and APTT on thrombin system ( ± s)

Table 1 Effects on bleeding volume,PT and APTT on thrombin system ( ± s)

Notes: GN: negative control group treated only with distilled water;GP: positive control group treated with Gongxuening capsule;G1-G8:treated with decoctions made by proportionally mixed decoction pieces [Qiancao (Radix Rubiae Cordifoliae) to Haipiaoxiao (Endoconcha Sepiellae);1∶0,2∶1,1∶1,3∶4,1∶2,1∶4,1∶8,0∶1].PT: prothrombin time;APTT: activated partial thromboplastin time.Compared to the positive control group (GP),aP ＜ 0.001,cP ＜ 0.05;compared to the negative control group (GN),bP ＜ 0.001.

Table 2 Patho-grade of uterine tissues in rats ( ± s)

Table 2 Patho-grade of uterine tissues in rats ( ± s)

Notes: GN: negative control group treated only with distilled water;GP: positive control group treated with Gongxuening capsule;G1-G8: treated with decoctions made by proportionally mixed decoction pieces [Qiancao (Radix Rubiae Cordifoliae) to Haipiaoxiao (Endoconcha Sepiellae);1∶0,2∶1,1∶1,3∶4,1∶2,1∶4,1∶8,0∶1].Compared to the positive control group (GP),aP ＜ 0.001,cP ＜ 0.01;compared to the negative control group (GN),bP ＜ 0.001.

Table 3 Effects on serum levels of MMP1,TXA2,and PGI2 (pg/mL, ± s)

Table 3 Effects on serum levels of MMP1,TXA2,and PGI2 (pg/mL, ± s)

Notes: GN: negative control group treated only with distilled water;GP: positive control group treated with Gongxuening capsule;G1-G8:treated with decoctions made by proportionally mixed decoction pieces [Qiancao (Radix Rubiae Cordifoliae) to Haipiaoxiao (Endoconcha Sepiellae);1∶0,2∶1,1∶1,3∶4,1∶2,1∶4,1∶8,0∶1].MMP1: matrix metalloproteinase 1;TXA2: thromboxane A2;PGI2: prostaglandin I-2.Compared to the positive control group (GP),aP ＜ 0.001,cP ＜ 0.01,dP ＜ 0.05;compared to the negative control group (GN),eP ＜ 0.01,bP＜ 0.001.

4.DISCUSSION

As a traditional herb pair,Qiancao (Radix Rubiae Cordifoliae) and Haipiaoxiao (Endoconcha Sepiellae)have been applied in the clinical treatment on AUB for thousands of years.Its therapeutic effect has been identified in numerous cases.However,the applied weight ratio of this herb pair varies in different prescriptions.Thus,it is crucial to study the most effective weight ratio of Qiancao (Radix Rubiae Cordifoliae) and Haipiaoxiao (Endoconcha Sepiellae)for clinical application.

The bleeding volume of AUB rats reduced notably after the treatment of Qiancao (Radix Rubiae Cordifoliae) and Haipiaoxiao (Endoconcha Sepiellae).The bleeding volume in G2,G3,and G8 decreased notably compared to GN.The results showed that the weight ratio of 2∶1,1∶1,and 0∶1 [Qiancao (Radix Rubiae Cordifoliae) to Haipiaoxiao (Endoconcha Sepiellae)] reduced the bleeding volume effectively among all the studied weight ratios.

PT reflects the activity of extrinsic coagulation,and APTT reflects the endogenous clotting activity.17The activation of clotting factors within the extrinsic and final common pathways (factors I,II,V,VII,and X) shortenthe PT,elevate or stimulate the clotting factors within the intrinsic and final common pathways (PK,HMWK,and factors VIII,IX,XI,and XII),resulting in shortened APTT.18In this study,PT and APTT were shortened after treatment of Qiancao (Radix Rubiae Cordifoliae) and Haipiaoxiao (Endoconcha Sepiellae).The weight ratio of 2 ∶1,1 ∶1,and 0 ∶1 [Qiancao (Radix Rubiae Cordifoliae) to Haipiaoxiao (Endoconcha Sepiellae)]shortened the PT more effectively compared to GP (P＜0.001).These findings were consistent with the reduced bleeding volume,and may suggest that the weight ratio of 2 ∶1,1 ∶1,and 0 ∶1 [Qiancao (Radix Rubiae Cordifoliae) to Haipiaoxiao (Endoconcha Sepiellae)]can activate the extrinsic pathway.

Drug-induced incomplete abortion triggers the influx of inflammatory cells into the endometrium and the degradation and necrosis of decidua and chorion.16The effect of Qiancao (Radix Rubiae Cordifoliae) and Haipiaoxiao (Endoconcha Sepiellae) on reducing uterine bleeding volume has been studied widely.However,only a few studies have conducted immunohistochemistry of the uterus.Herein,Qiancao (Radix Rubiae Cordifoliae)and Haipiaoxiao (Endoconcha Sepiellae) (2∶1) significantly reduced the interstitium vascular dilation,inflammatory cells infiltration,and necrotic decidual cells.MMPs are a group of matrix-degrading proteins that include ＞ 22 human zinc-dependent proteolytic enzymes.19Among these MMPs,MMP1 is the most ubiquitously expressed interstitial collagenase.In nonfertile cycles,progesterone withdrawal results in increased MMP activity and promotes uterine bleeding and tissue sloughing.20As shown in this study,Qiancao(RadixRubiaeCordifoliae) and Haipiaoxiao(Endoconcha Sepiellae) treatment decreased the serum MMP1 levels significantly compared to the negative group.Moreover,serum MMP1 levels in G2 decreased significantly than in GP and (P＜ 0.01).This result illustrated that the weight ratio of 2∶1 [Qiancao (Radix Rubiae Cordifoliae) to Haipiaoxiao (Endoconcha Sepiellae)] may be the most effective treatment to reduce serum MMP1 in AUB rats.TXA2and PGI2have the opposite effectsin vivo.In activated platelets,arachidonate is converted into TXA2,which stimulates the platelet activation process.21In endothelial cells from the vessel wall,arachidonate is converted into PGI2,which inhibits platelet activation.22Thus,regulating the balance between TXA2and PGI2may be a key approach to treat AUB.Interestingly,serum TXA2levels elevated significantly in GP and G2 compared to GN (P＜ 0.01 and ＜ 0.001),and no difference was detected in serum PGI2levels in all groups.These results illustrated that weight ratio of 2 ∶1[Qiancao (Radix Rubiae Cordifoliae) to Haipiaoxiao(Endoconcha Sepiellae)] may be the most effective treatment via significantly raised serum TXA2level,and that the regulation of serum PGI2level is not the underlying therapeutic mechanism of Qiancao (Radix Rubiae Cordifoliae) and Haipiaoxiao (Endoconcha Sepiellae).

Strikingly,more than 100 components have been detected from Qiancao.These are anthraquinones,naphthoquinones,cyclic hexapeptides,and terpenoids.23Some are involved in the pharmacological activities of many studies.Purpurin and mollugin are the chemical markers that evaluate the quality of herbal medicine in theChinese Pharmacopeia.24Purpurin has antiangiogenic and anti-oxidant functions,25,26while Mollugin has anti-cancer,27anti-inflammatory,and neuroprotective activities.28Haipiaoxiao (Endoconcha Sepiellae) consists of trace elements,polysaccharides,nucleosides,and other components.According to theChinese Pharmacopeia,24the content of calcium carbonate in Haipiaoxiao (Endoconcha Sepiellae) must at least 86%.A recent study identified proteins and peptides,such as hemocyanin,enolase,myosin,actin,calmodulin,histone and tubulin,in Haipiaoxiao(Endoconcha Sepiellae).29The current study showed that Qiancao (Radix Rubiae Cordifoliae) or Haipiaoxiao(Endoconcha Sepiellae) could not achieve the same effect of the herb pair on treatment of AUB.The active ingredients in the herb pair may enhance each other by regulating its absorption,distribution,metabolism,and excretion.Moreover,many ingredients are inactive but become active in combination.30In conclusion,this study showed that Chinese herbs Qiancao (Radix Rubiae Cordifoliae) and Haipiaoxiao(Endoconcha Sepiellae) exert a therapeutic effect on AUB by shortened PT and APTT and regulated serum level of MMP1 and TXA2.Moreover,rats treated by Qiancao (Radix Rubiae Cordifoliae) and Haipiaoxiao(Endoconcha Sepiellae) have fewer inflammatory cells,necrotic decidual cells,and less interstitium vascular dilation in uterine sections.The comparison among the decoctions of eight different weight ratios illustrated that the weight ratios of 2∶1,1∶1,and 0∶1 [Qiancao(Radix Rubiae Cordifoliae) to Haipiaoxiao (Endoconcha Sepiellae)] reduce the uterine bleeding volume.However,the weight ratio of 2∶1 showed the best effect on reducing serum MMP1 level and increasing serum TXA2level.The drug-induced abortion in rats after treatment of Qiancao (Radix Rubiae Cordifoliae) and Haipiaoxiao(Endoconcha Sepiellae) (2 ∶ 1) improved the immunohistochemical characteristics.Qiancao (Radix Rubiae Cordifoliae) and Haipiaoxiao (Endoconcha Sepiellae) (2∶1) may be the most effective treatment for AUB.Furthermore,it is necessary to study the underlying mechanism of this herb pair and why the weight ratio of 2∶1 is preferred for clinical application.