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. 2025 Nov 10;10(46):55307–55315. doi: 10.1021/acsomega.5c03615

Kaempferol Inhibits Colonic Contractions via L‑Type Voltage-Dependent Calcium Channels in Murine Smooth Muscle Cells

Meng Yang †, Rui Xiao ‡, Wen Yu ∥, Ying Wang ‡, Sijia Chen ‡, Song Zhao ∥, Ziyu Liu ∥, Jiayu Gu ∥, Zibo Zhao ∥, Xuanying Lyu †, Nina Song ‡,§,*
PMCID: PMC12658692  PMID: 41322575

Abstract

Kaempferol, a natural flavonoid in various herbs, exhibits many therapeutical effects of anti-inflammation, antitumor, antioxidation, and so on. While its therapeutic potential has been established in many diseases, Kaempferol’s side effect on colonic motility and the underlying mechanism remains ambiguous. The colonic muscle strips from ICR mice were used in contractile experiments to explore the effects of Kaempferol on colonic contractions. Besides, a whole-cell patch technique was performed on the lysed cells from the colonic muscles to investigate the underlying mechanism of Kaempferol’s inhibitory effects. It was found that Kaempferol could inhibit the contractions of colonic smooth muscle significantly. However, blocking the effect of the enteric nervous system or interstitial cells could not block the inhibitory effect of Kaempferol on colonic contractions. Besides, the agonist of the L-type calcium channel Bay K8644 could suppress the inhibiting effect of Kaempferol on colonic contractions. The whole-cell patch of smooth muscle cells shows that the Kaempferol inhibited the Ba currents via the L-type calcium channel. In conclusion, Kaempferol suppresses colonic contractions by inhibiting the L-type calcium channel in a smooth muscle cell, independent of enteric neurons, chloride channels, or potassium channels, which indicates its potential effects on colonic motility and underlying mechanisms.

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Introduction

Kaempferol (Kae) is a natural flavonoid that exists in a wide range of fruits, vegetables, herbal medicines, and other plants. It has aroused widespread attention from scientists that this natural compound Kae shows various advantages of anti-inflammation, antitumor, antioxidation, antianxiety, and immune regulation. − Through these mechanisms, Kae has therapeutic effects on many diseases, such as cancer, diabetic mellitus, pain, and so on. , However, Kae or other flavonoids could result in acute gastrointestinal symptoms, such as gastritis and constipation. − So, if we use Kae or other flavonoids as therapeutic trials, these side effects on colonic motility should be taken into consideration. Recent studies found that Kae could relax the smooth muscles of blood vessels, gastrointestinal tract, bronchi, uterus, and some other organs via different mechanisms. ,− However, the underlying mechanism of the effect of Kae on colonic motility remains ambiguous.

Colonic motility is modulated by the SIP syncytium, which consists of the interstitial cells of Cajal (ICCs), the platelet derived growth factor receptor alpha positive cells (PDGFRα+ cells) and smooth muscle cells (SMCs). Among these, the smooth muscle cells produce the force of contractions, but the interstitial cells, including ICCs and PDGFRα+ cells which are electrically coupled with SMCs, determine the frequency and amplitude of contractions. The enteric nervous system (ENS) innervates the ICCs and PDGFRα+ cells directly, instead of smooth muscle cells, which forms the two axes in colonic muscular layerthe ENS-ICC-SMC axis and the ENS-PDGFRα+ cell-SMC axis. ICCs transduce both excitatory (acetylcholine) and inhibitory (NO) nervous inputs. , Besides, ICCs are pacemaker cells in gastrointestinal (GI) tracts, which generate slow waves and original force of contractions. Meanwhile, PDGFRα+ cells could transduce inhibitory inputs from the ENS, like purines, vasoactive intestinal peptide (VIP), β-nicotinamide adenine dinucleotide hydrate (β-NAD), and so on. , These neurotransmitters released from the ENS act on the interstitial cells and change the membrane potentials. The changing potentials then propagate to adjacent SMCs and affect the frequency and amplitude of the contractions. Normal colonic motility results from the perfect coordination and balance of the two axes.

Many diseases could disturb the balance of the two axes in the GI tracts and lead to GI motility dysfunction. In previous studies, the ICC network was damaged in diabetes mellitus due to reduced stem cell factor, which led to gastroparesis in mice. , However, the function of the ENS-PDGFRα+ cell-SMC axis was enhanced in colons of diabetic mice, due to the upregulations of PDGFRα+ cells and the small conductance calcium-activated potassium channels specifically expressed on them. , These alterations disturb the balance of the axes in the GI tract and then lead to the motility dysfunctions of the GI tract. As more and more scientists pay attention to herbal drugs with therapeutic effects, low toxicity, and possible economic benefit, it is necessary for us to demonstrate whether Kae has effects on the GI motility.

In this study, the experiments are designed to explore the effect of Kae on colonic motility and its mechanism, according to the theory of the ENS-ICC-SMC axis and the ENS-PDGFRα+ cell-SMC axis. We show that Kae inhibits the colonic contractions via acting on the smooth muscle cells instead of the ENS, ICCs, and PDGFRα+ cells. The results illustrate that Kae could inhibit the L-type calcium channel in SMCs and then inhibit colonic contractions. This study may enable us to have a more comprehensive understanding of the role of Kae and its mechanism.

Methods

Animals and Ethical Approval

The ICR mice were male and 5 weeks old and were obtained from the Shanghai Experimental Animal Center of Chinese Academy of Science. The rearing conditions for mice are constant room temperature, a light–dark cycle of 12 h–12 h, and free access to water and food. All the study procedures were approved by the Committee on the Ethics of Animal Experiment of Shanghai Jiao Tong University School of Medicine (Permit No. Hu 686-2009).

Isometric Force Measurement of Colonic Contractions

The ICR mice were sacrificed for cervical dislocation after isoflurane anesthesia, and colons were removed from the abdomens. Colons, removed from abdomens, were cut along the long axis. The mucous layers were fixed upward on the silicone plate. After peeling away the mucosa layer, the colonic smooth muscles were prepared into muscle strips of 2 × 8 mm in size by cutting along the circular axis and attaching silk thread to both ends of the strips. The muscle strips were hung in organ baths and submerged in 37 °C and oxygenated Krebs solution. The muscle strips were equilibrated for 1 h after setting the muscles at optimum length with a force of 3 mN. The spontaneous contractions of muscle strips were recorded by the isometric force transducer, which was connected to an amplifier (RM6240C, Chengdu, China).

Isolation of Colonic Smooth Muscle Cells

The colonic muscular layers were sheared into small strips of size 1 mm × 8 mm and equilibrated in the 37 °C digestive solution for 20–30 min. The content of digestive solution and the method were listed by Song et al. The freshly isolated cells were cultured in SMGM culture medium and settled in a 5% CO2 incubator for 30 min.

Whole-Cell Patch Clamp Experiments

Freshly isolated smooth muscle cells were plated in the chamber of the stage of an Olympus IX-70 microscope and perfused in external solution. The membrane currents in SMCs were recorded by whole-cell patch techniques of the EPC-10 HEKA amplifier. The pipet tips with the resistance of 2–4 MΩ were used in the patch experiments. All of the procedures of the whole-cell patch were implemented at room temperature in 20 min when the cells were ruptured.

Solutions and Drugs

The Krebs solution contained the following: glucose 11.5 mM, CaCl2 2.5 mM, NaCl 121.9 mM, NaHCO3 15.5 mM, KCl 5.9 mM, MgSO4 1.2 mM, and KH2PO4 1.2 mM. The external perfusing solution contained NaCl 134.8 mM, KCl 4.5 mM, glucose 10 mM, HEPES 10 mM, MgCl2 1 mM, and BaCl2 10 mM and was adjusted to pH 7.4 with Tris. The pipet solution contained CsCl mM, TEA 20 mM, EGTA 10 mM, HEPES 10 mM, Na2ATP 2 mM, MgCl2 4 mM, and was adjusted to pH 7.35 with Tris.

Statistical Methods

The data were showed as means ± SEM of n cells or n mice. Only when the P value was less than 0.05, did we recognize it as statistical significance. The statistical analyses of the experimental data were performed by GraphPad Prism. The t test and one-way ANOVA were used to compare groups at the appropriate time.

Results

The Inhibitory Effect of Kae on Colonic Contractions

We recorded the colonic contractions and the effect of Kae on contractions with isometric force measurement. We found that Kae of 10, 20, and 30 μM exerted different amplitudes of suppressions on the colonic contractions. 10 μM Kae significantly decreased the amplitude of contractions to 74.25 ± 3.18%. Moreover, the 20 μM and 30 μM suppressed the colonic contractions to 60.62 ± 3.24% and 41.44 ± 5.06% (*P < 0.05; n = 8; Figure A,B).

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Contractile responses to Kaempferol in murine colonic muscles. (A) Representative traces of responses of spontaneous contractions to Kaempferol 10, 20, and 30 μM in colonic muscles. (B) Summary of the contractile responses of Kaempferol, as indicated by the relative amplitude at 100 s in the colonic muscles. The data were normalized to the control value (before the application of Kaempferol) (n = 8; *P < 0.05 vs control).

The Role of the ENS in the Inhibitory Effect of Kae on Colonic Contractions

To explore whether the ENS play a role in Kae’s inhibition of colonic contractions, we used the contractile experiments to measure the contractions. First, the Kae (20 μM) decreased the colonic contractions to 60.02 ± 2.79% (*P < 0.05; n = 3; Figure A,C). However, after administration of tetrodotoxin (TTX, 0.4 μM) to block the sodium channel expressed in the ENS, the Kae (20 μM) decreased the colonic contractions to 64.57 ± 4.58% (*P < 0.05; n = 3; Figure A,C), which had no significant differences in comparison to the contractions with no TTX. In addition, without nitric oxide production inhibitor, Nω-nitro-l-arginine methyl ester hydrochloride (l-NAME, 200 μM), the Kae suppressed the contraction amplitude to 62.00 ± 6.71% (*P < 0.05; n = 4; Figure B,D). Meanwhile, with l-NAME, the amplitude of Kae’s suppression on colonic contractions reached 62.20 ± 7.51% (*P < 0.05; n = 4; Figure B,D), which was still not significantly different from the contractions with no l-NAME. These data elucidated that the ENS played no significant role in the inhibitory effect of Kae on colonic contractions.

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Effects of the sodium channel blocker (TTX) and nitric oxide production inhibitor (l-NAME) on Kaempferol’s suppression in murine colonic spontaneous contraction: (A) Representative traces of responses of spontaneous contractions to Kaempferol 20 μM in colonic muscles in the presence or absence of TTX. (B) Representative traces of responses of spontaneous contractions to Kaempferol 20 μM in colonic muscles in the presence or absence of l-NAME. (C) Summary of the effects of TTX on Kaempferol’s suppression of colonic contractions in the form of relative amplitude. The data were normalized to the control value (before the application of Kaempferol) (n = 3; *P < 0.05 vs control). (D) Summary of the effects of l-NAME on Kaempferol’s suppression of colonic contractions in the form of relative amplitude. The data were normalized to the control value (before the application of Kaempferol) (n = 4; *P < 0.05 vs control).

The Role of Interstitial Cells in the Inhibitory Effect of Kae on Colonic Contractions

The interstitial cells, including ICCs and PDGFRα+ cells, transduce inputs from the ENS and modulate the amplitude and frequency of colonic contractions. The calcium-activated chloride channels (ANO1) were specifically expressed in ICCs and contributed to the pacemaker activity and some neural responses in the muscles of colons. Thus, we used the antagonist of ANO1 (Ani9) to study whether ANO1 participated in Kae’s effect on colonic contractions or not and the responses to Kae were measured in muscle strips treated with or without Ani9. Before administration of Ani9, the Kae suppressed the colonic contractions to 51.17 ± 5.40%. Additionally, with Ani9 (1 μM), the Kae decreased the colonic contractions to 48.22 ± 3.23% (*P < 0.05; n = 3; Figure A,B). However, the suppression effects of Kae on contractions had no significant differences with or without Ani9, which indicated that ANO1 in ICCs was not related to the colonic responses to Kae.

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Effects of ANO antagonist (Ani9) on Kaempferol’s suppression in murine colonic spontaneous contraction: (A) Representative traces of responses of spontaneous contractions to Kaempferol 20 μM in colonic muscles in the presence or absence of Ani9. (B) Summary of the effects of Ani9 on Kaempferol’s suppression of colonic contractions in the form of relative amplitude. The data were normalized to the control value (before the application of Kaempferol) (n = 3; *P < 0.05 vs control).

As a part of the SIP syncytium, PDGFRα+ cells also showed great significance in regulating colonic motility. Moreover, the low-conductance calcium-activated potassium channels (SK3 channel) were specifically expressed in PDGFRα+ cells and fundamental to transducing inputs from neurons. So, we recorded the contractions in response to Kae with Apamin (the antagonist of SK3 channels) or no Apamin to find out the role of SK3 channels in Kae’s effects. Without Apamin or with Apamin (300 nM), Kae decreased the colonic contractions to 64.76 ± 3.61% and 59.45 ± 4.82%, respectively (*P < 0.05; n = 5; Figure A,B). It is obvious that there was no significant difference between them, which suggested that the SK3 channels in PDGFRα+ cells did not play an important role in Kae’s effect. Taken together, these data showed that the functional channels in interstitial cells did not participate in Kae’s suppression on colonic contractions.

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Effects of the SK3 channel inhibitor (Apamin) on Kaempferol’s suppression in murine colonic spontaneous contraction: (A) Representative traces of responses of spontaneous contractions to Kaempferol 20 μM in colonic muscles in the presence or absence of Apamin. (B) Summary of the effects of Apamin on Kaempferol’s suppression of colonic contractions in the form of relative amplitude. The data were normalized to the control value (before the application of Kaempferol) (n = 5; *P < 0.05 vs control).

The Role of Potassium Channels in Smooth Muscle Cells in the Inhibitory Effect of Kae on Colonic Contractions

As SMCs generated the force of the colonic contraction and were the effectors of inputs transduced by ICCs and PDGFRα+ cells from the ENS, SMCs also had an impact on colonic motility. Therefore, it was of great significance to explore whether the SMCs take part in Kae’s effect. The data showed that the administration of the antagonist of the ATP-sensitive potassium (KATP) channel (Glibenclamide) could not significantly change Kae’s inhibitory effect on colonic contraction (Figure ). Without Glibenclamide (10 μM), Kae inhibited the amplitude of colonic contractions to 56.58 ± 3.50% (*P < 0.05; n = 3; Figure A,C). With Glibenclamide (10 μM), the amplitude of Kae’s suppression was 54.37 ± 3.34% (*P < 0.05; n = 3; Figure A,C). To find out the role of potassium channels in Kae’s effect, we used the nonselective potassium blocker tetraethylammonium (TEA) in contractile experiments. Without or with TEA (10 mM), Kae decreased the contraction amplitude to 70.34 ± 4.46% and 73.25 ± 3.98%, respectively (*P < 0.05; n = 3; Figure B,D), which had no significant differences between them. These results suggested that the potassium channels, including KATP channels, played no important role in Kae’s effect.

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Effects of the KATP inhibitor (Glibenclamide) and nonselective potassium channel blocker (TEA) on Kaempferol’s suppression in murine colonic spontaneous contraction: (A) Representative traces of responses of spontaneous contractions to Kaempferol 20 μM in colonic muscles with the presence or absence of Glibenclamide. (B) Representative traces of responses of spontaneous contractions to Kaempferol 20 μM in colonic muscles with the presence or absence of TEA. (C) Summary of the effects of Glibenclamide on Kaempferol’s suppression of colonic contractions in the form of relative amplitude. The data were normalized to the control value (before the application of Kaempferol) (n = 3; *P < 0.05 vs control). (D) Summary of the effects of TEA on Kaempferol’s suppression of colonic contractions in the form of relative amplitude. The data were normalized to the control value (before the application of Kaempferol) (n = 9; *P < 0.05 vs control).

The Role of L-Type Voltage-Dependent Calcium Channels in Smooth Muscle Cells in the Inhibitory Effect of Kae on Colonic Contractions

L-type voltage-dependent calcium channels (L-VDCC) were expressed in SMCs and responsible for the contractility of smooth muscles. When we used Bay K8644 (1 μM) to activate the VDCC, the Kae only decreased the contractions to 83.09 ± 3.65% (*P < 0.05; n = 5; Figure A–D), which were significantly higher than that without Bay K8644 (*P < 0.05; # P < 0.05; n = 5; Figure A–D). The data indicated that the VDCC might participate in Kae’s effect on colonic contractions.

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Effects of L-type voltage-dependent Ca2+ channels in Kaempferol’s suppression in murine colonic spontaneous contraction: (A) Representative traces of responses of spontaneous contractions to Kaempferol 20 μM in colonic muscles in the presence or absence of Bay K8644 (activator of VDCCs). (B) Representative traces of Ba currents in smooth muscle cells with the presence or absence of Kaempferol. (C) The summary of currents evoked by step depolarizations from −40 mV to +70 mV in smooth muscle cells with the presence or absence of Kaempferol. (n = 5; *P < 0.05 vs control). (D) Summary of the effects of Bay K8644 on Kaempferol’s suppression of colonic contractions in the form of relative amplitude. The data were normalized to the control value (before the application of Kaempferol) (n = 5; *P < 0.05 vs control). (E) The summarized data of currents at 0 mV in the smooth muscle cells with the presence or absence of Kaempferol. (n = 5; *P < 0.05 vs control).

The whole-cell patch technique was used to investigate the mechanism of VDCCs participating in Kae’s effect. In freshly isolated SMCs, the step depolarizations from −40 mV to +70 mV evoked large amplitude of Ba currents (Figure B). However, the administration of Kae significantly suppressed the Ba currents in the SMCs. The result suggested that Kae could decrease the VDCC currents and exert its inhibitory effect on colonic contractions.

Discussion

As a kind of plant-derived compound, Kaempferol exhibits various advantages on health. Recent studies show that Kaempferol had effects of anti-immune response, ferroptosis inhibition, antioxidant activity, anti-inflammation, and so on, which indicates that Kaempferol owns great potentials as clinical treatments for many diseases. ,− Su et al. found that Kaempferol hydrogel exhibited antipsoriasis properties by its cellular antioxidant and antiproliferative abilities and inhibition of ROS generation. , However, previous studies suggested that Kaempferol could induce the relaxation of smooth muscles in uteri, aortic vessels, and coronary artery. ,, In the GI tract, the force of motility is generated by smooth muscles. We found that Kaempferol suppressed the contractions of colonic smooth muscles (Figure ). It is supposed that when patients take Kaempferol orally, it may affect the GI motility, which may reduce the drug absorption. Therefore, we are curious about whether Kaempferol influences contractions of smooth muscles of the GI tract and its underlying mechanism.

The present study showed that Kaempferol could suppress the contractions of colonic smooth muscles. The inhibitory effect was significant at 10, 20, and 30 μM (Figure ). According to the functional structure of the SIP syncytium in the GI tract, normal GI motility results from coordination of ICCs, PDGFRα+ cells, and smooth muscle cells. The interstitial cells, ICCs and PDGFRα+ cells, are innervated by the ENS and transduce the neural inputs to adjacent smooth muscle cells. There are gap junctions between the interstitial cells and smooth muscle cells, which allows electrical changes to propagate from the interstitial cells to coupled smooth muscle cells and then affects the excitability and contractility of smooth muscle cells. Thus, anything that can affect the ENS and the SIP syncytium could have an impact on GI motility.

First, contractile experiments were carried out to explore whether the ENS participates in Kaempferol’s suppression of contractions in colonic smooth muscles. We used TTX to eliminate the enteric neural inputs by blocking the voltage-gated sodium channels on nerve fibers. The result suggests Kaempferol’s inhibitory effect was not TTX-sensitive because there were no significant differences between without TTX and with TTX (Figure ). Thus, we think that the ENS did not participate in Kaempferol’s effect. Besides, in proximal colons, the dominant enteric inhibitory neural regulation is nitric oxide (NO). NO activates the soluble guanylate cyclase (sGC), which is highly expressed in ICCs, then activates potassium channels, decreases calcium sensitivity, and eventually leads to the relaxation of smooth muscles. Moreover, it has been reported that in porcine coronary artery, the relaxation effect of Kaempferol was related to NO. , Given that, l-NAME, nitric oxide synthase inhibitor, was used to abolish the nitrergic regulation by inhibiting the production of NO to investigate the role of nitrergic regulation in Kaempferol’s effect. In the present study, the data showed that inhibiting the production of NO could not change the amplitude of Kaempferol’s suppression, which indicates that the nitrergic regulation of the ENS did not play a role in Kaempferol’s inhibitory effect of colonic contractions (Figure ). Taken together, Kaempferol exerts the inhibitory effects on colonic contractions not via the enteric nervous system in the GI tract.

Apart from the ENS, the interstitial cells, ICCs and PDGFRα+ cells, also have leading roles in modulating the GI motility. As we mentioned before, the interstitial cells form two axes in muscular layers, the ENS-ICC-SMC axis and the ENS-PDGFRα+ cell-SMC axis, by which the force and frequency of smooth muscle contractions are regulated. Therefore, the factors that may affect the ICCs and PDGFRα+ cells are likely to influence the contractility of smooth muscles. According to the two axes, the experiments were designed to clarify the role of interstitial cells from two angles. First, we focused on ANO1, the functional channels highly expressed in ICCs. ANO1 channels are responsible for the generation of slow waves and postjunctional response in the GI tract. , Thus, the antagonist of ANO1, Ani9, was used in contractile experiments to abolish the effect of ANO1. Our results show that the administration of ANO1 antagonist did not affect Kaempferol’s inhibitory effect of colonic contractions, which suggests that ANO1 in ICCs were not the key by which Kaempferol inhibited the colonic contractions (Figure ). Second, SK3 channels in PDGFRα+ cells are the key factors we should pay attention to, for its role of electrical basis in regulating the potentials of PDGFRα+ cells and excitability of smooth muscle cells. Additionally, purinergic responses to the ENS are transduced by PDGFRα+ cells via P2Y1 receptors on them, activate SK3 channels, lead to the hyperpolarization of PDGFRα+ cells, and eventually result in decreased excitability of smooth muscle cells. ,, The use of Apamin, the blocker of SK3 channels, in the contractile experiments is meant to eliminate the effect of SK3 channels. However, when we blocked the SK3 channels by Apamin, the amplitude of Kaempferol’s suppression did not change significantly (Figure ). These data mean that the SK3 channels did not participate in Kaempferol’s effects on colonic contractions. In summary, as the functional channels, ANO1 in ICCs and SK3 channels in PDGFRα+ cells, did not play roles in Kaempferol’s effects, we think that the interstitial cells are not the pathways by which Kaempferol works.

The smooth muscle cells are the working cells to generate the contractions in the GI tract. So, we hypothesize that Kaempferol acts directly on smooth muscle cells to affect its contractions. The contractility of smooth muscle cells is closely related to channels in smooth muscle cells. It has been reported that most inhibitory agents regulate the contractions by activating potassium channels. , Meanwhile, in previous study, it has been investigated that the relaxation of blood vessels by Kaempferol resulted from the opening of KATP and calcium-activated channels in vascular smooth muscles. So, we used the KATP inhibitor, Glibenclamide, to explore whether KATP participates in Kaempferol’s effect. The results show that blocking KATP did not change the amplitude of Kaempferol’s suppression (Figure ). Potassium channels play important roles in regulating the membrane potentials and contractility of smooth muscle cells. There are different potassium channels in smooth muscle cells, including calcium-activated channels, delayed rectifying potassium channels, and so on. Therefore, we used a nonselective potassium channel blocker (TEA) to abolish the influence of potassium channels. In the present study, the data suggests that the administration of TEA could not affect Kaempferol’s suppression (Figure ). To sum up, potassium channels did not mediate Kaempferol’s suppression of colonic contractions.

The action potentials and excitation–contraction (E–C) coupling of GI smooth muscle cells owe to Ca2+ entry, especially via L-type VDCCs in them. , When slow waves propagate passively to smooth muscle cells, the depolarization activates VDCCs and causes Ca2+ entry, which leads to the action potentials. Meanwhile, the influx of Ca2+ launches the E–C coupling in the colonic smooth muscle, which generates the contractions. Given the importance of L-type VDCCs, we should pay attention to it and explore the role of VDCCs in Kaempferol’s suppression of colonic contractions. As Bay K8644 could activate VDCCs, it was used in contractile experiments. When we activated the VDCCs in smooth muscles with Bay K8644, Kaempferol’s effect was significantly decreased, indicating the mediation of VDCCs (Figure ). For further explanation, barium currents (I Ba) via L-type VDCCs were recorded with a whole-cell patch clamp. Step depolarizations from −20 to +40 mV evoke large amplitude of inward I Ba. However, when Kaempferol was added to external solutions, I Ba was significantly decreased, which means that Kaempferol could inhibit the VDCC currents (Figure ). These findings demonstrate that Kaempferol inhibits colonic contractions through decreasing Ca2+ entry via L-type VDCCs.

In conclusion, the present study focused on the effects of Kaempferol and its mechanism. Kaempferol suppressed colonic contractions significantly. However, this was not mediated by the ENS and interstitial cells in the GI tract, even though they were important parts of the SIP syncytium. Kaempferol directly inhibited the Ca2+ entry via L-type VDCCs and then affected the contractility of colonic smooth muscles. Thus, Kaempferol may have therapeutic potential for hypermotility disorders (such as diarrhea-predominant irritable bowel syndrome). However, this study has not found out how Kaempferol inhibits L-type VDCCs and the underlying mechanisms. Meanwhile, this study cannot exclude the effects of other channels on smooth muscles. So, we may need to pay more attention to explore the underlying mechanisms of Kaempferol’s effects on colonic motility.

Acknowledgments

This work was supported by the Key project of Jingchu University of Technology (ZD202211), Jiangsu Training Program of Innovation and Entrepreneurship for Undergraduates (202210313016Z), Hubei Training Program of Innovation and Entrepreneurship for Undergraduates (S202311336026), and Funding of National Demonstration Center for Experimental Basic Medical Science Education (Xuzhou Medical University).

Meng Yang: conception and design of experiments; collection, analysis, and interpretation of data; drafting of the article; and critical revision of the paper for important intellectual content. Rui Xiao, Wen Yu, Ying Wang, Sijia Chen, Song Zhao, Ziyu Liu, Jiayu Gu, and Zibo Zhao: collection, analysis, and interpretation of data. Nina Song: conception and design of experiments; analysis and interpretation of data; and critical revision of the paper for important intellectual content. All authors approved the final version of the manuscript.

The authors declare no competing financial interest.

References

  1. Rha C. S., Jeong H. W., Park S., Lee S., Jung Y. S., Kim D. O.. Antioxidative, Anti-Inflammatory, and Anticancer Effects of Purified Flavonol Glycosides and Aglycones in Green Tea. Antioxidants. 2019;8(8):278. doi: 10.3390/antiox8080278. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Gouda N. A., Alshammari S. O., Abourehab M. A. S., Alshammari Q. A., Elkamhawy A.. Therapeutic potential of natural products in inflammation: underlying molecular mechanisms, clinical outcomes, technological advances, and future perspectives. Inflammopharmacology. 2023;31(6):2857–2883. doi: 10.1007/s10787-023-01366-y. [DOI] [PubMed] [Google Scholar]
  3. Periferakis A., Periferakis K., Badarau I. A., Petran E. M., Popa D. C., Caruntu A., Costache R. S., Scheau C., Caruntu C., Costache D. O.. Kaempferol: Antimicrobial Properties, Sources, Clinical, and Traditional Applications. Int. J. Mol. Sci. 2022;23(23):15054. doi: 10.3390/ijms232315054. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Wang J., Mao J., Wang R., Li S., Wu B., Yuan Y.. Kaempferol Protects Against Cerebral Ischemia Reperfusion Injury Through Intervening Oxidative and Inflammatory Stress Induced Apoptosis. Front. Pharmacol. 2020;11:424. doi: 10.3389/fphar.2020.00424. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Silva Dos Santos J., Gonçalves Cirino J. P., de Oliveira Carvalho P., Ortega M. M.. The Pharmacological Action of Kaempferol in Central Nervous System Diseases: A Review. Front. Pharmacol. 2021;11:565700. doi: 10.3389/fphar.2020.565700. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Nigusse T., Zhang L., Wang R., Wang X. N., Li J., Liu C.. Flavonoids in a crude extract of Catha edulis inhibit rat intestinal contraction via blocking Ca­(2+) channels. Neurogastroenterol Motil. 2019;31(7):e13602. doi: 10.1111/nmo.13602. [DOI] [PubMed] [Google Scholar]
  7. Al-Hebshi N. N., Skaug N.. Khat (Catha edulis)-an updated review. Addict Biol. 2005;10(4):299–307. doi: 10.1080/13556210500353020. [DOI] [PubMed] [Google Scholar]
  8. Gharzouli K., Holzer P.. Inhibition of guinea pig intestinal peristalsis by the flavonoids quercetin, naringenin, apigenin and genistein. Pharmacology. 2004;70(1):5–14. doi: 10.1159/000074237. [DOI] [PubMed] [Google Scholar]
  9. Xu Y. C., Leung S. W., Leung G. P., Man R. Y.. Kaempferol enhances endothelium-dependent relaxation in the porcine coronary artery through activation of large-conductance Ca­(2+) -activated K­(+) channels. Br. J. Pharmacol. 2015;172(12):3003–3014. doi: 10.1111/bph.13108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Duarte J., Pérez Vizcaíno F., Utrilla P., Jiménez J., Tamargo J., Zarzuelo A.. Vasodilatory effects of flavonoids in rat aortic smooth muscle. Structure-activity relationships. Gen. Pharmacol. 1993;24(4):857–862. doi: 10.1016/0306-3623(93)90159-u. [DOI] [PubMed] [Google Scholar]
  11. Wahid M., Saqib F., Akhtar S., Ali A., Wilairatana P., Mubarak M. S.. Possible Mechanisms Underlying the Antispasmodic, Bronchodilator, and Antidiarrheal Activities of Polarity-Based Extracts of Cucumis sativus L. Seeds in In Silico, In Vitro, and In Vivo Studies. Pharmaceuticals. 2022;15(5):641. doi: 10.3390/ph15050641. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Revuelta M. P., Cantabrana B., Hidalgo A.. Mechanisms involved in kaempferol-induced relaxation in rat uterine smooth muscle. Life Sci. 2000;67(3):251–259. doi: 10.1016/S0024-3205(00)00627-5. [DOI] [PubMed] [Google Scholar]
  13. Sanders K. M., Ward S. M., Koh S. D.. Interstitial cells: regulators of smooth muscle function. Physiol Rev. 2014;94(3):859–907. doi: 10.1152/physrev.00037.2013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Sanders K. M., Koh S. D., Ro S., Ward S. M.. Regulation of gastrointestinal motility--insights from smooth muscle biology. Nat. Rev. Gastroenterol Hepatol. 2012;9(11):633–645. doi: 10.1038/nrgastro.2012.168. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Harrington A. M., Hutson J. M., Southwell B. R.. Immunohistochemical localisation of cholinergic muscarinic receptor subtype 1 (M1r) in the guinea pig and human enteric nervous system. J. Chem. Neuroanat. 2007;33(4):193–201. doi: 10.1016/j.jchemneu.2007.03.001. [DOI] [PubMed] [Google Scholar]
  16. Lies B., Beck K., Keppler J., Saur D., Groneberg D., Friebe A.. Nitrergic signalling via interstitial cells of Cajal regulates motor activity in murine colon. J. Physiol. 2015;593(20):4589–4601. doi: 10.1113/JP270511. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Sanders K. M., Santana L. F., Baker S. A.. Interstitial cells of Cajal - pacemakers of the gastrointestinal tract. J. Physiol. 2023:10.1113/JP28474. doi: 10.1113/JP284745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hwang S. J., Blair P. J., Durnin L., Mutafova-Yambolieva V., Sanders K. M., Ward S. M.. P2Y1 purinoreceptors are fundamental to inhibitory motor control of murine colonic excitability and transit. J. Physiol. 2012;590(8):1957–1972. doi: 10.1113/jphysiol.2011.224634. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hwang S. J., Durnin L., Dwyer L., Rhee P. L., Ward S. M., Koh S. D., Sanders K. M., Mutafova-Yambolieva V. N.. β-nicotinamide adenine dinucleotide is an enteric inhibitory neurotransmitter in human and nonhuman primate colons. Gastroenterology. 2011;140(2):608–617e606. doi: 10.1053/j.gastro.2010.09.039. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Horváth V. J., Vittal H., Lörincz A., Chen H., Almeida-Porada G., Redelman D., Ordög T.. Reduced stem cell factor links smooth myopathy and loss of interstitial cells of cajal in murine diabetic gastroparesis. Gastroenterology. 2006;130(3):759–770. doi: 10.1053/j.gastro.2005.12.027. [DOI] [PubMed] [Google Scholar]
  21. Wu Y. S., Lu H. L., Huang X., Liu D. H., Meng X. M., Guo X., Kim Y. C., Xu W. X.. Diabetes-induced loss of gastric ICC accompanied by up-regulation of natriuretic peptide signaling pathways in STZ-induced diabetic mice. Peptides. 2013;40:104–111. doi: 10.1016/j.peptides.2012.12.024. [DOI] [PubMed] [Google Scholar]
  22. Song N.-N., Lu H.-L., Lu C., Tong L., Huang S.-Q., Huang X., Chen J., Kim Y.-C., Xu W.-X.. Diabetes-induced colonic slow transit mediated by the up-regulation of PDGFRα­(+) cells/SK3 in streptozotocin-induced diabetic mice. Neurogastroenterol Motil. 2018;30:e13326. doi: 10.1111/nmo.13326. [DOI] [PubMed] [Google Scholar]
  23. Lu H., Zhang C., Song N., Lu C., Tong L., Huang X., Kim Y. C., Chen J., Xu W.. Colonic PDGFRα Overexpression Accompanied Forkhead Transcription Factor FOXO3 Up-Regulation in STZ-Induced Diabetic Mice. Cell. Physiol. Biochem. 2017;43(1):158–171. doi: 10.1159/000480335. [DOI] [PubMed] [Google Scholar]
  24. He X., Wu T., He H., Chen L., Han K., Zheng J., Zhang Z., Yuan S., Wang Y., Zhang Y.. et al. Study of kaempferol in the treatment of rheumatoid arthritis through modulation of the NLRP3/CASP1/GSDMD axis and T-cell activation: Based on network pharmacology, single-cell analysis, and experimental validation. Int. Immunopharmacol. 2024;143(Pt 1):113357. doi: 10.1016/j.intimp.2024.113357. [DOI] [PubMed] [Google Scholar]
  25. Dabeek W. M., Marra M. V.. Dietary Quercetin and Kaempferol: Bioavailability and Potential Cardiovascular-Related Bioactivity in Humans. Nutrients. 2019;11(10):2288. doi: 10.3390/nu11102288. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Chen M., Xiao J., El-Seedi H. R., Woźniak K. S., Daglia M., Little P. J., Weng J., Xu S.. Kaempferol and atherosclerosis: From mechanism to medicine. Crit Rev. Food Sci. Nutr. 2024;64(8):2157–2175. doi: 10.1080/10408398.2022.2121261. [DOI] [PubMed] [Google Scholar]
  27. Su H., Liu Z., Zhang Z., Jing X., Meng L.. Development of a Deep Eutectic Solvent-Assisted Kaempferol Hydrogel: A Promising Therapeutic Approach for Psoriasis-like Skin Inflammation. Mol. Pharmaceutics. 2023;20(12):6319–6329. doi: 10.1021/acs.molpharmaceut.3c00729. [DOI] [PubMed] [Google Scholar]
  28. Su H., Chen Y., Jing X., Zhao X., Sun H., Liu Z., Qiu Y., Zhang Z., Guan H., Meng L.. Antimicrobial, Antioxidant, and Anti-Inflammatory Nanoplatform for Effective Management of Infected Wounds. Adv. Healthcare Mater. 2024;13(5):e2302868. doi: 10.1002/adhm.202302868. [DOI] [PubMed] [Google Scholar]
  29. Leeya Y., Mulvany M. J., Queiroz E. F., Marston A., Hostettmann K., Jansakul C.. Hypotensive activity of an n-butanol extract and their purified compounds from leaves of Phyllanthus acidus (L.) Skeels in rats. Eur. J. Pharmacol. 2010;649(1–3):301–313. doi: 10.1016/j.ejphar.2010.09.038. [DOI] [PubMed] [Google Scholar]
  30. Mañé N., Gil V., Martínez-Cutillas M., Clavé P., Gallego D., Jiménez M.. Differential functional role of purinergic and nitrergic inhibitory cotransmitters in human colonic relaxation. Acta Physiol. 2014;212(4):293–305. doi: 10.1111/apha.12408. [DOI] [PubMed] [Google Scholar]
  31. Koh S. D., Drumm B. T., Lu H., Kim H. J., Ryoo S. B., Kim H. U., Lee J. Y., Rhee P. L., Wang Q., Gould T. W.. et al. Propulsive colonic contractions are mediated by inhibition-driven poststimulus responses that originate in interstitial cells of Cajal. Proc. Natl. Acad. Sci. U.S.A. 2022;119(18):e2123020119. doi: 10.1073/pnas.2123020119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Gomez-Pinilla P. J., Gibbons S. J., Bardsley M. R., Lorincz A., Pozo M. J., Pasricha P. J., de Rijn M. V., West R. B., Sarr M. G., Kendrick M. L.. et al. Ano1 is a selective marker of interstitial cells of Cajal in the human and mouse gastrointestinal tract. Am. J. Physiol Gastrointest Liver Physiol. 2009;296(6):G1370–1381. doi: 10.1152/ajpgi.00074.2009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Kurahashi M., Zheng H., Dwyer L., Ward S. M., Don Koh S., Sanders K. M.. A functional role for the ’fibroblast-like cells’ in gastrointestinal smooth muscles. J. Physiol. 2011;589(3):697–710. doi: 10.1113/jphysiol.2010.201129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Koh S. D., Ward S. M., Sanders K. M.. Ionic conductances regulating the excitability of colonic smooth muscles. Neurogastroenterol Motil. 2012;24(8):705–718. doi: 10.1111/j.1365-2982.2012.01956.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Kuriyama H., Kitamura K., Itoh T., Inoue R.. Physiological features of visceral smooth muscle cells, with special reference to receptors and ion channels. Physiol Rev. 1998;78(3):811–920. doi: 10.1152/physrev.1998.78.3.811. [DOI] [PubMed] [Google Scholar]
  36. Sun J. B., Huang X., Xu H. Y., Li X. L., Gao L., Kim Y. C., Xu W. X.. Inhibitory effect of C-type natriuretic peptide on L-type calcium channel currents in gastric antral myocytes of guinea pigs. Gen. Physiol. Biophys. 2006;25(4):365–377. [PubMed] [Google Scholar]
  37. Sanders K. M.. Regulation of smooth muscle excitation and contraction. Neurogastroenterol Motil. 2008;20(s1):39–53. doi: 10.1111/j.1365-2982.2008.01108.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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