Phloroglucinol inhibits oxytocin-induced contraction in rat gastric circular muscle and uterine smooth muscle

Abstract

Phloroglucinol is commonly used to alleviate dysmenorrhoea and stomach cramps. However, there is little evidence of phloroglucinol in the mechanism of primary dysmenorrhoea (PD) development. In this study, a PD rat model was established. The effects of phloroglucinol on the contraction of rat gastric circular muscle and uterine smooth muscle induced by oxytocin (OT) were investigated. The writhing response, and levels of oestradiol (E2), prostaglandin e2 (PGE2), and prostaglandin f2α (PGF2α) were determined. The protein and mRNA levels of OT receptor (OTR) were detected. OT showed a significant promoting effect on gastric circular muscle and uterine smooth muscle contraction. However, phloroglucinol strongly inhibited the contraction induced by 10⁻⁶mol/L of OT. We also found that phloroglucinol reduced writhing response and attenuated uterine damage. Compared to the blank group, E2 and PGF2α were significantly increased, but PGE2 was significantly decreased in the PD model group. Phloroglucinol was found to reverse the changes of E2, PGF2α and PGE2. Moreover, phloroglucinol reduced the protein and mRNA levels of OTR. In conclusion, phloroglucinol could attenuate PD and inhibit the contraction of rat gastric circular muscle and uterine smooth muscle induced by OT. The mechanism might be related with the regulation of OTR expression.

IMPACT STATEMENT

• What is already known on this subject? Phloroglucinol is commonly used to alleviate dysmenorrhoea and stomach cramps. However, there is little evidence of phloroglucinol in the mechanism of primary dysmenorrhoea (PD) development.

• What do the results of this study add? Phloroglucinol could attenuate PD and inhibit the contraction of rat gastric circular muscle and uterine smooth muscle induced by OT. The underlying mechanisms of phloroglucinol for PD treatment may be associated with OTR.

• What are the implications of these findings for clinical practice and/or further research? These findings provide novel ideas for the role of phloroglucinol in PD development.

Keywords:

• Primary dysmenorrhoea
• phloroglucinol
• oxytocin
• gastric circular muscle
• uterine smooth muscle

1. Introduction

Dysmenorrhoea is a common clinical disease of gynaecology (Ju et al. 2014). Severe symptoms can affect the normal life and work. Dysmenorrhoea can be divided into primary dysmenorrhoea (PD) and secondary dysmenorrhoea according to the aetiology of disease occurrence (De Sanctis et al. 2016). PD refers to painful menstruation without pelvic disease. Women with PD had enhanced contractions of the uterine muscle, which is associated with the release of prostaglandins and other inflammatory mediators in the uterus (Xiao et al. 2019). Secondary dysmenorrhoea is defined as a pathological pelvic condition with painful menstruation. Currently, it is treated with non-steroidal anti-inflammatory drugs and oral pills, which may had adverse effects on digestive system, liver and kidneys disorders (Rodrigues et al. 2011). Thus, it is necessary to investigate the processes and mechanisms of PD development.

Oxytocin (OT) is a peptide hormone and is secreted by the posterior pituitary gland in the human body. The main role of OT is the regulation of lactation and contraction of the womb during childbirth (Gimpl and Fahrenholz 2001). Recently, numerous studies have shown that it is also involved in the regulation of gastrointestinal motility, mucosal homeostasis, intestinal permeability and immune functions (Wang et al. 2016). Xie et al. (2003) found that OT suppressed the motility of proximal colon in rabbits. OT could regulate negatively the spontaneous contraction in rat distal colon (Wang et al. 2017). Evidence showed that a number of the observed effects of OT are realised by its action on smooth-muscle cells (Garriz et al. 2021).

Phloroglucinol is a pure smooth muscle antispasmodic drug with no atropine and no papaverine (Wu et al. 2021). Phloroglucinol was discovered in the nineteenth century (Annahazi et al. 2014). It has been explored as a therapeutic method for various diseases and was commercialised in the European countries (Blanchard et al. 2018). It has been used for relieving severe vomiting, dysmenorrhoea and gastrointestinal spasm (Clara et al. 2020). It has no effect on anticholinergic effect and seems to be less toxic than most other antispasmodic drugs (Li et al. 2011). However, it remains unknown whether phloroglucinol can alleviate OT-induced contraction of rat gastric circular muscle and uterine smooth muscle.

This study aimed to investigate the effect of phloroglucinol on the contraction of rat gastric circular muscle and uterine smooth muscle induced by OT and determined whether phloroglucinol exerts a protective effect on PD in vivo.

2. Material and methods

2.1. Animals

SD rats (animal licence number: SCXK (Xiang) 2019-0004) 190 ± 10 g) were kept in a sterile environment at a room temperature of 23 ± 2 °C, and had free access to food and water. All experiments were approved by the Animal Use Ethics Committee of the Third Affiliated Hospital of Zhengzhou University. All efforts were carried out to minimise animal suffering and to minimise the number of animals used.

2.2. Detection of contraction of rat gastric circular muscle

The rats were sacrificed by decapitation, and the stomach was quickly removed and placed in an ice-cold Krebs solution. We cut the gastric cavity along the small curvature of the stomach and rinsed the gastric cavity. The signal was input to the physiological recorder to record the muscle strip’s spontaneous contraction activity. The muscle strips were incubated for 1 h under 1 g of a preload (washed with fresh Krebs solution every 15 min at 37 °C). After the spontaneous contraction of the muscle strips stabilised, 50 μL OT of different concentrations was added to make the OT in the perfusion bath with final concentrations of 10^–9, 10^–8, 10^–7, 10^–6mol/L to change muscle strip tension. Subsequently, phloroglucinol (10⁻⁶mol/L), atosiban (10⁻⁶mol/L) or atropine (10⁻⁶mol/L) was added and recorded 15 min. Before changing the concentration, the muscle strips were washed three times with fresh solution at 37 °C and each interval was 15 min. The previous step was repeated after the spontaneous contraction was restored. The same volume of normal saline was used as the solvent control. Atropine used as positive control in this study. The reason is as follows: M-cholinergic receptor and α, β2 adrenergic receptor exist in gastrointestinal smooth muscle. Excitation of M-cholinergic receptor or inhibition of α and β2 receptor pathway can cause gastrointestinal smooth muscle contraction, tension enhancement and increase of contraction amplitude. Atropine is a cholinoceptor blocking drug, which competitively antagonises M-cholinergic receptor, inhibits gastrointestinal smooth muscle spasm, and reduces the amplitude and frequency of peristalsis. Atropine has been widely used to relieve gastrointestinal colic for a long time. In vitro experiments have also proved that atropine can relieve gastrointestinal smooth muscle tension (Davison et al. 1983, Takeuchi 2012).

2.3. Detection of contraction of rat uterine smooth muscle

The rats were sacrificed by decapitation and cut at the cervix on both sides to obtain unilateral uterine horns. The muscle strips were incubated for 1 h under a 1 g of preload. After the spontaneous contraction was stable, different concentrations of OT were added to investigate the changes of contraction of the muscle strips. We make the final concentration of OT in the perfusion bath with final concentrations of 10⁻⁹, 10⁻⁸, 10⁻⁷, 10⁻⁶mol/L, respectively, and recorded 15 min. Subsequently, phloroglucinol (10⁻⁶mol/L), atosiban (10⁻⁶mol/L) or atropine (10⁻⁶mol/L) was added and recorded 15 min.

2.4. PD rat model establishment

Rats were randomly divided into six groups (n = 6, each group), including blank group, PD model group, low-dose phloroglucinol group (20 mg/kg), high-dose phloroglucinol group (40 mg/kg), aspirin group (250 mg/kg) and atropine (1 mg/kg). The rats in the blank group were injected with saline, and the rats in the other groups were injected with oestradiol benzoate 5 mg·kg⁻¹d⁻¹ (No. 1812081, Tianjin KingYork Pharmaceutical Co. Ltd., Tianjin, China) subcutaneously on days 1–7. Those rats were fast overnight on days 7 and 8. Subsequently, rats in the model group were intraperitoneally injected with 100 U·kg⁻¹(No. 09200802, Shanghai Hefeng Pharmaceutical Co. Ltd., Shanghai, China) of OT on day 8. In addition to intraperitoneal injection of 100 U·kg⁻¹ of OT, rats in the low-dose phloroglucinol group were given 20 mg/kg of phloroglucinol (No. 20190704, Hubei Wushi Pharmaceutical Co. Ltd., Wuhan, China), rats in the high-dose phloroglucinol group were given 40 mg/kg of phloroglucinol, rats in the aspirin group was given 250 mg/kg of aspirin (No. 19071602, Reyoung Pharmaceutical Co. Ltd., Zibo, China) and rats in the atropine group were given 1 mg/kg of atropine (No. 62007271, Suicheng Pharmaceutical Co. Ltd., Zhengzhou, China). The rats in the model group were injected with normal saline and the time of writhing within 40 min was investigated. The rats were sacrificed at 24 h, and the uterine tissues were collected for subsequent study.

2.5. Writhing response

Rats were intraperitoneally injected with OT (2 U) in each group after 30 min administration. The number of writhing in each rat within 30 min were recorded immediately after the injection of OT. At the same time, the incubation period from the injection of OT to the first writhing reaction was also recorded. The rat’s hind limbs and trunk were stretched, the abdomen was contracted and one limb was rotated inside, indicating that the dysmenorrhoea rat model was successfully established.

2.6. Haematoxylin-eosin (HE) staining

The pathological changes of uterus of rats were evaluated by HE staining. Briefly, the whole uterus was fixed with 10% formaldehyde after excision. The thickened myometrium without haemorrhagic focus was selected and cut into 1 cm × 1 cm × 0.5 cm specimens. Then, 3 μm thick sections were prepared for HE staining after routine dehydration and paraffin embedding. The pathological changes in uterus of rats were observed using a light microscope (LEICA DM500, Wetzlar, Germany).

2.7. Enzyme-linked immunosorbent assay (ELISA)

The uterine tissues were homogenised using nine times of grinding forces with the homogenisation medium (Cryomill, Servicebio, Wuhan, China) followed by centrifuging at 3000–4000 r for 10 min. We then took the supernatant to prepare a 10% tissue homogenate, and place it in 4 °C for subsequent study. Corresponding ELISA kits were used to detect the levels of oestradiol (E2) (USCN life Science Inc., Wuhan, China), prostaglandin E2 (PGE2) (USCN life Science Inc., Wuhan, China) and prostaglandin F2α (PGF2α) (USCN life Science Inc., Wuhan, China) in uterine tissues according to the manufacture’s instructions.

2.8. Immunohistochemistry

The immunohistochemical method was used to compare the expression of OTR in rats’ uterine tissues of each group. The paraffin-embedded slides were deparaffinized and hydrated. Subsequently, antigen retrieval was conducted with citric acid. The sections were blocked with 5% BSA, incubated overnight at 4 °C with primary antibody against OTR (1:200; Proteintech, USA) followed by incubation with a secondary antibody (1:200; Proteintech, USA) for 1 h at room temperature and exposed to DAB for 5 min. In the end, the sections were stained with haematoxylin. Every slice in each group was randomly selected with a field of view (200X) to take pictures. Image-Pro Plus 6.0 software was used to select the same brown-yellow as the unified standard for the positive of all photos. Each photo was analysed to obtain the relative integrated optical density (IOD) value and positive pixel area of the tissues to be tested. Furthermore, we calculated the IOD per stained area (pixels) (IOD/area). The bigger the value, the higher the positive expression level.

2.9. Quantitative reverse transcription PCR (qRT-PCR)

Total RNA was extracted from uterine tissues using TRIzol (TaKaRa, Dalian, China) in accordance with the manufacturer’s instructions. The cDNA synthesis kit (Thermo Fisher Scientific) was used to synthesise cDNA. qRT-PCR was performed on an ABI Q6 (Applied Biosystems Inc., USA) using SYBR Green Real-Time PCR Master Mix (Toyobo, number QPK-201). The PCR mixtures consisted of 1 μL of cDNA, 12.5 μL of 2 × Master Mix, 0.5 μL of 10 μmol/L forward primer, 0.5 μL of 10 μmol/L reverse primer and 10.5 μL of ddH₂O. The qRT-PCR procedure had the following stages: 95°C for 10 min, 40 cycles of 95°C for 30 s, 60°C for 60 s. The 2^–ΔΔCT method was used to calculate the relative expression of OTR. GAPDH was used as a control gene. Each experiment was carried out in six copies. The primer sequence of OTR and GAPDH was as follows: OTR forward: 5′-GCACGGGTCAGTAGTGTCAAGC-3′, OTR reverse: 5′-GGCAATGATGAAGGCAGAAGC-3′; GAPDH forward: 5′-CTGGAGAAACCTGCCAAGTATG-3′, GAPDH reverse: 5′-GGTGGAAGAATGGGAGTTGCT-3′.

2.10. Data analysis

The average tension of 3 min before the OT addition was set up as the baseline. The average tension at 1, 3, 5, 10 and 15 min after the addition of OT or drug was the effective value of different periods. The baseline and effective value were obtained by calculating the integral value of the muscle strip of tension curve (the area under the curve) in the corresponding period. The ratio R of the effective value to the baseline was used as an indicator of the effect of OT on the muscle strip movement (the value is 1 before addition). GraphPad Prism 7.0 software was used for statistical analysis. The data were expressed as mean ± standard deviation (SD). The difference was determined by one-way ANOVA or t-test, and p < 0.05 was accepted as a significant difference.

3. Results

3.1. OT promotes the contraction of gastric circular muscle and uterine smooth muscle

We investigated the effects of different concentration (10⁻⁹, 10⁻⁸, 10⁻⁷, 10⁻⁶mol/L) of OT on the contraction of gastric circular muscle and uterine smooth muscle. Gastric circular muscle strips start to contract after OT treatment, and the contraction value reached a peek at the 3 min (Figure 1(A)). Except for the slight relaxation in the first few minutes after adding 10⁻⁹ moL/L of OT, the other three OT concentrations made the muscle strips tension rise as a concentration-dependent manner. OT also had a significant positive effect on uterine smooth muscle contraction, and it is dose-dependent (Figure 1(B)). The contraction reached the maximum value at 3 min, and then decreased. These data indicated that 10⁻⁶mol/L of OT had the most significant promoting effect on the contraction of gastric circular muscle and uterine smooth muscle.

Figure 1. The effect of different concentration of oxytocin (OT) on gastric circular muscle strips and uterine smooth muscle. (A) The effect of different concentration of OT on gastric circular muscle strips. (B) The effect of different concentrations of OT on uterine smooth muscle. Data are expressed as mean ± SD. n = 6. **p < 0.01 vs. control.

3.2. Phloroglucinol inhibits the contraction of gastric circular muscle and uterine smooth muscle induced by OT

We evaluated whether phloroglucinol exerts the inhibitory effect on the contraction of gastric circular muscle and uterine smooth muscle induced by OT. It was found that the phloroglucinol, atropine and atosiban have no obvious effect on gastric circular muscle strips when OT was at 10⁻⁹mol/L (Figure 2(A)). However, atropine and atosiban showed significant inhibitory effects when OT was at 10⁻⁸, 10⁻⁷ and 10⁻⁶mol/L. Phloroglucinol remarkably inhibited the contraction of gastric circular muscle when OT was at 10⁻⁶mol/L. In addition, atropine and atosiban significantly inhibited the contraction of uterine smooth muscle induced by OT, while phloroglucinol only dramatically inhibited the contraction of uterine smooth muscle when OT was at 10⁻⁶mol/L (Figure 2(B)). These results suggested that phloroglucinol suppressed the OT-induced contraction of gastric circular muscle and uterine smooth muscle.

Figure 2. The effect of phloroglucinol on contraction of gastric circular muscle strips and uterine smooth muscle induced by OT. (A) The effect of phloroglucinol on the contraction of gastric circular muscle strips induced by OT. (B) The effects of phloroglucinol on uterine smooth muscle contraction induced by OT. Data are expressed as mean ± SD. n = 6. *p < 0.05, **p < 0.01 vs. control.

3.3. Phloroglucinol inhibits writhing response and alleviates uterine damage of PD rats

To investigate the effect of phloroglucinol on PD rats, we carried out writhing test and histopathological examination. The number of twists was significantly increased, while latent period was remarkably decreased in the model group compared with the blank group (Figure 3(A, B)). Phloroglucinol significantly reduced the writhing reaction caused by OT in a dose-dependent manner, and it effectively increased the writhing latency (Figure 3(A, B)). In addition, the effect of phloroglucinol was further investigated by detecting the uterine tissues. As shown in Figure 3(C), in the blank group, the structure of each layer of the tissue is clear, and no edoema was observed. However, in the model group, obvious edoema in the endometrium lamina propria was observed. The severity of edoema in the endometrium lamina propria in the phloroglucinol group was less than that of the model group, and the effect was in agreement with the aspirin group and the atropine group. Overall, phloroglucinol relieved pain and alleviated uterine damage of PD rats.

Figure 3. Phloroglucinol inhibits writhing response and alleviates uterine damage. (A) The effect of phloroglucinol on the number of twists in rat writhing experiment. (B) The effect of phloroglucinol on latent period in rat writhing experiment. (C) Pathological change of the uterine tissues with H&E staining. Scale bars are 100 µm. n = 6. ***p < 0.001, **p < 0.01 vs. blank group. ##p < 0.01, #p < 0.05 vs. model group.

3.4. Phloroglucinol reduces E2 and PGF2α levels, and enhances PGE2 level in uterine tissues

To analyse the impact of phloroglucinol on the uterine-related indexes, the levels of E2, PGF2α and PGE2 were examined in uterine tissues using ELISA. The E2 and PGF2α levels of the model group were significantly increased compared with the blank group, while the PGE2 level was significantly lower than that of the blank group (Figure S1). Phloroglucinol, aspirin and atropine could reverse the changes of PGF2α and PGE2 to a certain extent compared with the model group. Phloroglucinol also reversed the changes of E2. In summary, phloroglucinol reduces E2 and PGF2α levels, and enhances PGE2 level in uterine tissues.

3.5. Phloroglucinol reduces OTR expression in uterine tissues

To analyse the impact of phloroglucinol on the OTR, the protein and mRNA expression of the OTR was examined in uterine tissues using immunohistochemistry and qRT-PCR. The protein expression of OTR in the model group increased significantly compared to the blank group (Figure S2(A)). Aspirin and atropine had no significant effect on the protein expression of OTR. Phloroglucinol significantly reduced the protein expression level of OTR in a dose-dependent manner compared with the model group. Moreover, it was found that the mRNA expression of OTR in the model group increased significantly compared to the blank group (Figure S2(B)). Aspirin and atropine had no significant effect on the mRNA expression of OTR. Phloroglucinol could reduce the expression of OTR mRNA in a dose-dependent manner relative to the model group. Collectively, phloroglucinol inhibited OTR expression in uterine tissues of PD rats.

4. Discussion

Dysmenorrhoea is a common clinical gynecological disease and affects women’s work and quality of life. In this study, we found that phloroglucinol could significantly inhibit the contraction of uterine smooth muscle and gastric circular muscle induced by OT. In addition, phloroglucinol could alleviate PD, as evidence by the decreased number of writhing, and the decreased inflammation and edoema in the uteri. Moreover, phloroglucinol significantly reduced E2, PGF2α and OTR expression and enhanced PGE2 expression compared to the model group. This study provides valuable evidence on the effect and mechanism of phloroglucinol on dysmenorrhoea.

It has been reported that the gastrointestinal function of adult women changes rhythmically with the menstrual cycle, and adult women experience more gastrointestinal discomfort at the beginning of the follicle (Bjornsson et al. 2006). The human gastrointestinal tract also expresses OTR gene and protein, so oxytocin is very likely to be a gastrointestinal hormone that regulates gastrointestinal motility. OT can also inhibit gastric motility, feeding and reduce gastric secretion by changing the excitability of neurons such as nucleus of solitary tract and dorsal nucleus of vagus nerve (Asad et al. 2001). Conventional doses of OTR may not cause abnormal gastrointestinal activity in animals or humans. The author thinks that the first reason is that OTR has a short half-life, only exists in the human body for 1–6 min, so it cannot act for a long time. Second, the density of OTR in the gastrointestinal tract is much smaller than that of the uterus, so the contraction of uterine smooth muscle is more obvious, and the contraction of the gastrointestinal tract is relatively moderate; but most importantly, the activity of the gastrointestinal tract is regulated by a combination of sympathetic nerves, parasympathetic nerves and enteric nervous system. The effect of OT on the gastrointestinal tract may be affected by other nervous systems and hormones, and the gastrointestinal adverse reactions may be related to its effect on the gastrointestinal tract receptors. Therefore, we speculate that after the sympathetic and parasympathetic nervous systems are unconstrained, especially after oestrogen induces the expression of intestinal OTR, OT will cause similar intestinal muscle contractions in humans or animals.

OT plays an important role in the regulation of gastrointestinal activity. A study showed that intravenous injection of OT could increase the stomach and duodenum pressure in rabbits (Li et al. 2007). Another study demonstrated that OT significantly inhibited gastric emptying and gastrointestinal transit in female rats (Wu et al. 2003). Ohlsson et al. (2004) discovered that OT promoted colonic motor activity in healthy women. In this study, phloroglucinol remarkably inhibited the contraction of gastric circular muscle induced by OT, suggesting that phloroglucinol may play a physiological role in regulating gastrointestinal activity.

Phloroglucinol is widely used as a spasmolytic agent for smooth muscle in clinic, and has high application value in the treatment and operation of urinary system and gastrointestinal system (Li et al. 2011). It has been successfully used to treat dysmenorrhoea, gastrointestinal colic and severe vomiting (Park et al. 2011). In this study, we found that phloroglucinol inhibited the contraction of uterine smooth muscle and gastric circular muscle induced by OT and significantly reduced OT-induced writhing response. Our results were in agreement with those previous reports (Wei et al. 2018, Huang et al. 2021). Overall, these results suggest that phloroglucinol may be a potential therapeutic drug for dysmenorrhoea.

Prostaglandin is a kind of unsaturated fatty acid with extensive physiological activity. Prostaglandins have been reported to play a key role in the uterine contraction (Olson 2003, Li et al. 2021). PGF2α and PGE2 are the two most prominent prostaglandins. Studies reported that PD is related with elevated levels of PGF2α (Liu et al. 2017, Wei et al. 2018). Ge-Gen Decoction repressed OT-induced contraction of uterus and obviously attenuated the OT-induced increased PGF2α expression (Yang et al. 2016). Nevertheless, the level of PGE2 in PD is debateable. Decreased levels of PGE2 in PD were observed in serum and uterine of PD mice (Yang et al. 2015). However, Huang et al. (2016) found that the PGE2 level was increased in uterine tissues of PD mice. Thus, the mechanism by which PGE2 are decreased in PD needs further research. In this study, we discovered that OT significantly increased the PGF2α level and decreased the PGE2 level, whereas phloroglucinol remarkably reversed the changes, suggesting that phloroglucinol may suppress OT-induced contraction through modulating PGE2 and PGF2α expression.

OTR takes part in many physiological reactions, including smooth muscle contraction, prostaglandin expression and lactation (Yarmolinskaya et al. 2020). OT was reported to induce myometrium contractions through OTR (Zingg and Laporte 2003). A study discovered that endogenous OT stimulated phasic contraction of gallbladder in rabbits through OTR (Liu et al. 2003). Xi et al. (2019) found that OTR mediated OT-induced contractions of the colon in the 5-hydroxytryptamine knockout mice. Therefore, regulating OTR levels to relieve abnormal contraction of the smooth muscle and gastrointestinal tract may be a new therapeutic strategy. In this study, phloroglucinol could inhibit the expression of OTR, which further confirmed that phloroglucinol’s therapeutical effect on dysmenorrhoea may be related with OTR.

In summary, phloroglucinol can effectively inhibit the contraction of uterine smooth muscle and gastric circular muscle induced by OT, and alleviated PD. The underlying mechanisms of phloroglucinol for PD treatment may be associated with OTR. These findings suggest that phloroglucinol may be a novel strategy for alleviating PD.

Ethical approval

This study was approved by the Ethics Committee of the Third Affiliated Hospital of Zhengzhou University.

Author contributions

QL and NS designed the study. HG, JN and JL contributed to experiment and methodology. RS, YB and SZ collected and analysed the data. WY wrote the manuscript. All authors read and approved the final manuscript.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported by Medical Science and Technology Project of Henan Province (No. 2018020214).