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ORIGINAL ARTICLE
Year : 2023 | Volume
: 9
| Issue : 2 | Page : 123-130
Effect of electroacupuncture on NLRP3 inflammasome and morphology of uterine in rats with primary dysmenorrhea based on meridian acupoint viscera correlation theory
Shao-Hua Wang, Han-Yu Yuan, Juan Li, Si-An Pan, Xiao Xue, Zeng-Hui Yue, Yu Liu
College of Acupuncture-Moxibustion, Tui-na and Rehabilitation, Hunan University of Chinese Medicine, Changsha, China
Correspondence Address:
Dr. Yu Liu
College of Acupuncture-Moxibustion, Tui-na and Rehabilitation, Hunan University of Chinese Medicine, No. 300 Xueshi Road, Yuelu 410208, Changsha
China
Dr. Zeng-Hui Yue
College of Acupuncture-Moxibustion, Tui-na and Rehabilitation, Hunan University of Chinese Medicine, No. 300 Xueshi Road, Yuelu 410208, Changsha
China
Source of Support: None, Conflict of Interest: None
DOI: 10.4103/2311-8571.378174
Objective: To explore the relationship between acupoints and the uterus in primary dysmenorrhea (PDM) and observe the effects of electroacupuncture (EA) on NLRP3 inflammasome and morphology of uterine tissues in rats. Materials and Methods: Rats were divided into four groups: Control, PDM, EA, and nonacupoint EA (sham) groups. Writhing reactions in the rats were carefully monitored. Prostaglandin E2 (PGE2) and prostaglandin F2α (PGF2α) levels in the rat serum were measured using the enzyme-linked immunosorbent assay. To assess the damage, the histomorphology of the rat uterus was examined using the optical and electron microscopy. Western blotting was performed to determine the nucleotide-binding oligomerization domain carboxy-terminal leucine-rich repeat domain and amino-terminal pyrin domain-containing protein 3 (NLRP3), apoptosis-associated speck-like protein containing a caspase recruitment domain(ASC), and cysteinyl aspartate specific proteinase-1(caspase-1), interleukin-1β (IL-1β), and IL-18. Results: Compared with the control group, the PDM group exhibited significant increases in both writhing times and scores, with a high pathological score of the uterine tissue. Serum PGF2α levels were significantly elevated, whereas PGE2 levels were significantly reduced. In addition, the expression levels of NLRP3, ASC, caspase-1, IL-1β, and IL-18 significantly increased (P < 0.001). The EA group showed opposite effects from the PDM group (all P < 0.05). The sham group exhibited significantly higher writhing time, serum PGF2α levels, and expression levels of NLRP3, ASC, caspase-1, IL-1β, and IL-18 than the EA group. In contrast, serum PGE2 levels in the sham group were significantly lower than those in the EA group (all P < 0.05). Conclusions: NLRP3 inflammasome activation in PDM triggers pathological inflammation. Sanyinjiao(SP6) and Guanyuan(CV4) had specific effects on PDM. EA of SP6 and CV4 may treat PDM by inhibiting inflammation and protecting the morphological structure of the uterus.
Keywords: Electroacupuncture, primary dysmenorrhea, meridian acupoint-viscera correlation, morphology, NLRP3 inflammasome
Primary dysmenorrhea (PDM), defined as painful menstruation without pelvic pathology, is usually characterized by spastic suprapubic pain.[1] PDM affects approximately 50%–90% of women worldwide and is common in women who do not seek medical attention.[2] Acupuncture can effectively treat PDM and is safer than nonsteroidal anti-inflammatory drugs.[3] Acupuncture is recommended as a treatment for PDM in the Clinical Practice Guidelines reviewed by the Canadian Society of Obstetricians and Gynecologists.[4] Clinically, the development of PDM is associated with increased prostaglandin (PG) levels.[5]
The nucleotide-binding oligomerization domain carboxy-terminal leucine-rich repeat domain and amino-terminal pyrin domain-containing protein 3 (NLRP3) inflammasome is a group of proteins comprising NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and cysteinyl aspartate-specific proteinase-1 (caspase-1), which work together to initiate inflammatory responses. NLRP3 binds to ASC, which recruits caspase-1, leading to complex assembly.[6] The expression of NLRP3 inflammasome was detected in endometrial epithelial cells and basal layer fibroblasts of birthing cattle.[7] In the uterus of parturient cows, endometrial epithelial cells are destroyed, and stromal fibroblasts are exposed, producing a pathological inflammatory immune response. NLRP3 inflammasome activation in matrix-exposed fibroblasts leads to the production and release of interleukin-1β (IL-1β) into the cavity and surrounding endometrial tissue, leading to inflammation.
Acupoints are special sites that contain structures, such as muscles, nerves, and blood vessels. The relationship between acupoints and viscera in PDM has rarely been studied. Previous studies by our research group showed that NLRP3 inflammasome and IL-1β were highly expressed in the uterine tissue of PDM rats, and the endometrium was exfoliated.[8] Catgut embedding at the acupoint suppresses NLRP3 inflammasome expression. To study the relationship between acupoints and viscera, we used estradiol benzoate and oxytocin to construct a rat model of PDM and observed the effects of acupoints and nonacupoints on the uterus.
Materials and MethodsAnimals and groups
Twenty female Sprague–Dawley rats, aged 6–8 weeks and weighing between 180 and 220 g, were obtained from Beijing HFK Bioscience Co. Ltd. The rats were healthy and of specific pathogen-free grade (SCXK[Jing] 2019–0008) and were not expecting. All rats were housed at a suitable temperature of 20°C–25°C with free access to water and food. One week later, the rats were randomly divided into four groups: Control, PDM, electroacupuncture (EA), and nonacupoint EA (sham) groups. The use of animals in this study was approved by the Laboratory Animal Ethics Committee of the Hunan University of Chinese Medicine (approval number LL2021040703). All procedures were performed in strict adherence to the scientific animal welfare guidelines.
Instruments and chemicals
Estradiol benzoate was purchased from Ningbo No. 2 Hormone Factory (China) (veterinary drug approval number (2016) 110252511); oxytocin (catalog No. P1029; batch number: 07; molecular formula: C45H70N12O14S2; molecular weight: 1067.2; purity: 98.52%) was purchased from Selleck (USA). Other reagents and materials used included a rat prostaglandin E2 (PGE2) enzyme-linked immunosorbent assay (ELISA) kit (production lot number: 20211111-J2944, Hunan Aifang Biotechnology Co. Ltd., China), rat prostaglandin F2α (PGF2α) ELISA kit (production lot number: 20211111-J3106, Hunan Aifang Biotechnology Co. Ltd., China), 812 epoxy resin monomers (CAS number: #90529-77-4, SPI Supplies, USA), NLRP3 antibodies (production lot number: Ab263899, Abcam, UK), caspase-1 antibodies (catalog number: #2225, Cell Signaling Technology, USA), ASC antibodies (production lot number: Bs-6741R, Bioss Antibodies, USA), IL-1β antibodies (catalog number: #12242, Cell Signaling Technology, USA), IL-18 antibodies (catalog number: 10663-1-AP, Proteintech, USA), and β-actin antibodies (catalog number: AF5001, Beyotime Biotechnology, China). The main instruments used were a Hwato acupuncture needle (0.25 mm × 13 mm, Suzhou Medical Supplies Factory Co. Ltd., China), an EA instrument (SDZ-V, Suzhou Medical Supplies Factory Co. Ltd., China), and an electron microscope (HT7700, Hitachi, Japan).
Construction of the primary dysmenorrhea rat model and treatments
We used a combination of estradiol benzoate and oxytocin to establish a PDM rat model. Estrogen increases uterine sensitivity by regulating cell proliferation and angiogenesis[9] and increasing oxytocin receptor mRNA expression.[10] Oxytocin can effectively induce uterine contractions,[11] causing relative ischemia and hypoxia in the uterus, which leads to pain. Rats in the control group were subcutaneously injected with 0.9% sodium chloride (NaCl) for 10 consecutive days and intraperitoneally injected with 0.9% NaCl on the 11th day. Rats in the other three groups were subcutaneously injected with estradiol benzoate at 9 o'clock every morning (dose: 2 mg/kg body weight on the 1st day, 0.8 mg/kg from the 2nd to 9th day, and 2 mg/kg on the 10th day). The dose and timing of estradiol benzoate were based on in vivo pharmacokinetic studies.[12] At 9:00 am on the 11th day, rats in the PDM, EA, and sham groups were intraperitoneally injected with 2 U/rat oxytocin (concentration: 0.5 mg/mL).
After fixing the rats in the EA group, Guanyuan (CV4) and Sanyinjiao (SP6) were selected for treatment. The locations of CV4 and SP6 are based on the latest standard.[13] CV4 is situated 25 mm below the level of the umbilicus, at the point where the lower 1/4 and upper 3/4 of the sternoclavicular symphysis and symphysis pubis intersect. SP6 was located 10 mm above the medial malleolus of the hind limb. The acupuncture needle penetrated CV4 by approximately 2 mm and SP6 by approximately 5 mm. The handles of the acupuncture needle were connected to an SDZ-V EA Instrument with the following settings: Dense wave, frequency of 50 Hz, and current of 1 mA. Thirty min after the injection of estradiol benzoate, rats were continuously stimulated with EA for 20 min each time from the 1st to the 10th day.
In the sham group, approximately 5 mm above the front of SP6 and next to CV4 was used as a nonacupoint after the rats were fixed. The selection principles for nonacupoints refer to high-quality randomized controlled trials.[14] The SDZ-V EA Instrument was set up with the same parameters as those for the EA group. Thirty min after the injection of estradiol benzoate, rats were continuously stimulated by EA for 20 min each time from the 1st to the 10th day.
Measurement of writhing times and scores of rats
The rat writhing response was tested as reported by Wei et al.[15] We recorded the number of writhing events and scores for 30 min after injecting the rats with oxytocin. After the writhing reaction occurred, all rats were anesthetized. Blood was obtained from the abdominal aorta, and uterine tissue was extracted before euthanizing the rats. Using sterile surgical scissors, uterine tissues (approximately 5 mm × 5 mm × 5 mm in size) were placed in 4% paraformaldehyde solution, and tissues (approximately 1 mm × 1 mm × 1 mm in size) were placed in an electron microscope fixative for microscopic examination. The remaining tissue was placed in the liquid nitrogen for subsequent examination. The writhing degree score was determined as follows: 0 points for normal movement, exploratory behavior, and the resulting extension of the trunk and hindlimbs; 1 point for abdominal depression and body tilt; 2 points for hind leg extension, hind paw dorsiflexion, and body stretching with rotation of the pelvis; and 3 points for abdominal muscle retraction, rear extension of the hind limbs, and twisting of the rigid body.
Microscopy
Uterine tissue from the same segment on the same side of each rat was collected for microscopy. Rat uterine tissue was soaked in 4% paraformaldehyde at 25°C for 24 h, dehydrated, washed with xylene, embedded in paraffin, and sectioned (5 μm). The tissues were deparaffinized using xylene and ethanol, dried, stained with hematoxylin and eosin, mounted using rhamsan gum, and viewed under an optical microscope to observe the histomorphology of uterine tissue. Pathological damage to the rat uterus was scored based on a previous report by Wei et al.[16] Based on the morphological features of the uterus observed under the microscope, a uterus without pathology was scored as 0, endometrial degeneration and necrosis as 1, lamina propria edema as 2, glandular increase in the lamina propria as 3, inflammatory cell infiltration into the lamina propria as 4, and myometrial inflammation as 5 points.
The rat uterine tissue was placed in an electron microscope fixative for 2–4 h and fixed with osmic acid and phosphate buffer for 2 h. The samples were dehydrated with an ethanol and acetone solution and then infiltrated with acetone and 812 epoxy resin monomers. Polymerization was performed in a 60°C oven for 48 h and sliced with a microtome to a thickness of 60–80 nm. The uterine tissue of rats was observed under an electron microscope after double-staining with uranium (U) and lead (Pb).
Enzyme-linked immunosorbent assay
Approximately 5 mL of blood was collected from the abdominal aortas of five randomly selected rats. The blood was left to settle for approximately 20 min at 25°C before being centrifuged at 3 000 r/min for 20 min. The serum obtained from centrifugation was carefully collected, and the levels of PGE2 and PGF2α were measured using a rat ELISA kit according to the manufacturer's instructions.
Western blot assay
Approximately 30 mg of uterine tissue from the same side of the rats in each group was collected and ground with a homogenizer at a temperature of 4°C. Precooled RIPA lysis buffer, protease inhibitor, and phosphorylase inhibitor were then added and placed on ice for 30 min. The samples were centrifuged at 4°C at 12,000 r/min for 10 min, the supernatant containing the protein was removed, sample buffer was added, and the samples were heated in a boiling water bath for 10 min to completely denature the protein. A bicinchoninic acid protein assay kit was used to determine the protein standard curve and calculate the protein concentration. After electrophoresis, the separated proteins were transferred onto polyvinylidene fluoride (PVDF) membranes and blocked with PVDF membranes, and the specific primary antibody was diluted with Tris-buffered saline Tween-20 (TBST). PVDF membranes were immersed in a diluent containing primary antibodies and incubated overnight at 4°C. The primary antibodies used were anti-NLRP3 (1:1000), anti-ASC (1:3000), anti-caspase-1 (1:3000), anti-IL-1β (1:3000), anti-IL-18 (1:3000), and anti-β-actin (1:2000). The membranes were then washed with TBST and incubated with a secondary antibody for 60 min. Enhanced electroluminescence color exposure was performed. AlphaEaseFC software was used to analyze and calculate the gray values of the strips.
Statistical analysis
The data collected during the experiment were analyzed using IBM SPSS Statistics (version 26.0, USA), and the results are presented as mean ± standard deviation. One-way analysis of variance was used for the statistical analysis if the data met the criteria of normality and homogeneity of variance. If these criteria were not met, the Kruskal–Wallis H-test was used. The level of statistical significance was set at α =0.05, where P < 0.05 was considered statistically significant.
ResultsElectroacupuncture reduced the writhing times and scores in primary dysmenorrhea rats
The control group did not show any signs of discomfort after the administration of oxytocin for 30 min. In contrast, the PDM group demonstrated a significant increase in the frequency and intensity of writhing movements, indicating the successful establishment of the PDM rat model. However, the EA group showed a significant reduction in the number of writhing movements and scores compared with the PDM group, suggesting the efficacy of EA in alleviating PDM. Nonetheless, the sham group exhibited a significantly higher number of writhing movements and scores than the control group, indicating a difference between the sham EA and EA groups. In addition, the writhing scores were significantly higher in the sham group than in the EA group shown in [Figure 1].
Figure 1: Comparison of writhing times and writhing scores 30 min after oxytocin injection in each group. (a) Each writhing behaviour was documented as one time. (b) The highest score described in Section 2.4 within 30 min was recorded as the rat writhing score. All experiments were randomized and double-blinded. The data in Figure 1 were analysed by the Kruskal-Wallis H test and are represented as the mean ± SD, n=5. ▲▲▲P<0.001 compared to the control group; *P<0.05 compared to the PDM group; ♦P<0.05 compared to the EA group)Electroacupuncture alleviated endometrial cell injury and reduced uterine pathological damage score in primary dysmenorrhea rats
The rat uterus was stained with hematoxylin-eosin staining (HE) to evaluate the protective effect of EA against rat endometrial cell injury. Under an optical microscope, no obvious histological changes were observed in the uteri of the control group. The endometrial epithelial cells were neatly arranged and columnar with little physiological degeneration, and no obvious inflammatory cell infiltration was observed. Endometrial epithelial cells in the PDM group were massively vacuolated and necrotic, many spiral arterioles were congested, and neutrophil infiltration was observed. In the EA group, there was a small amount of vacuolar degeneration and necrosis in the uterine epithelium, with no congestion and only a small amount of neutrophil infiltration. In the sham EA group, endometrial epithelial cells showed vacuolar degeneration and necrosis, some spiral arterioles were congested, and neutrophil infiltration was observed. The pathological score was based on microscopic evaluation, and scores that conformed to the standard were cumulatively assessed. The results indicated that the PDM group had a significantly higher uterine histopathological damage score than the control group (P < 0.001). However, the pathological score of the EA group was significantly lower than that of the PDM group (P < 0.05). Notably, the sham group had a significantly higher pathological score than the control group, and there was no significant difference between the sham and PDM groups. These findings suggest that EA treatment has a protective effect against uterine injury in PDM rats, whereas EA treatment at nonacupoint did not appear to have a similar effect shown in [Figure 2].
Figure 2: Histopathological changes and comparison of pathological damage scores in the uterine tissue of rats in each group. (a- d) were designated as the control, PDM, EA, and Sham groups, respectively. Additionally, E was utilized as a means of comparing the extent of pathological damage between each group using HE staining. a, b, c and d were at 100× magnification. In the first four panels of Figure 2, blue, black, red, and orange arrows referred to vacuolar degeneration, dead cells, hyperaemia, and neutrophil infiltration, respectively. The scale of 5 grids in this figure represents 100μm. In e, the HE score was analysed by the Kruskal–Wallis H test, and the data is presented as the mean plus or minus the standard deviation with a sample size of 5. Statistical analysis shows that the experimental group has a significant difference compared to the control group with a P value of less than 0.01 (▲▲) and less than 0.001 (▲▲▲). Additionally, the experimental group showed a significant difference compared to the PDM group with a P value of less than 0.05 (*)Electroacupuncture protected the endometrial epithelial subcellular structure of primary dysmenorrhea rats
To further examine the effects of EA on the uterus of rats, we used an electron microscope to observe its ultrastructure. In the control group, endometrial epithelial cells had uniform cytoplasm and oval nuclei. The structures of the rough endoplasmic reticulum (RER) and mitochondria were normal. In the PDM group, the cytoplasm of epithelial cells showed obvious edema, cytosolic electron density was reduced, the cell membrane was complete and continuous, and a large number of collagen fibers were observed around the cell membrane. The nucleus was slightly irregular and locally pitted, the nuclear membrane was clear, and the perinuclear space did not widen significantly. Mitochondria were ovoid and severely swollen, the matrix dissolved, ridge fractures disappeared, and cavitation occurred. The RER was slightly dilated, and no significant degranulation was observed. In the EA group, the cell nucleus was intact, cytoplasmic electron density was reduced, mitochondria were oval, crista was continuous and complete, and no obvious enlargement was observed. RER dilated, with slight degranulation. In the sham group, the electron density of endometrial epithelial cells increased, the cell membrane blurred, and a large number of collagen fibers were observed. The nuclei showed severe irregularities, increased heterochromatin, and slight condensation. Mitochondria were ovoid, and most of them had no obvious swelling; the matrix was uniform, the crest was slightly broken and shortened, and a few mitochondria were swollen. The RER was moderately dilated, with slight degranulation shown in [Figure 3].
Figure 3: Ultrastructural changes in the endometrial epithelial cells of four groups: (a) the control group, (b) the PDM group, (c) the EA group, and (d) the Sham group. Images were taken at 8000× magnification and the diagrams included abbreviations for collagen fibre, nucleus, mitochondria, and rough endoplasmic reticulum. Abbreviations in the diagrams: CF, collagen fibre; N, nucleus; M, mitochondria; RER, rough endoplasmic reticulumElectroacupuncture down-regulated the content of prostaglandin F2α and up-regulated that of prostaglandin E2 in the serum of rats with primary dysmenorrhea
PG is involved in the mechanism of PDM.[5] A human trial demonstrated the efficacy of PGF2α inhibitor in treating PDM.[17] During dysmenorrhea, PGF2α promotes uterine contractions, while PGE2 inhibits them.[18] Therefore, PG can be used to evaluate the success of PDM in animal models. Compared with the control group, serum PGF2α levels [[Figure 4]a, P < 0.001] were significantly increased, and PGE2 levels were [[Figure 4]b, P < 0.001] significantly decreased in the PDM group. Compared to the PDM group, serum PGF2α levels [[Figure 4]a, P < 0.01] decreased significantly in the EA group, and PGE2 levels [[Figure 4]b, P < 0.01] increased significantly. Serum PGF2α levels [[Figure 4]a, P < 0.05] was significantly higher in the sham group compared to the EA group, whereas the level of serum PGE2 was significantly lower in the sham group than in the EA group as shown in [Figure 4]b (P < 0.05). These results indicated the differences in the regulation of PG between EA and nonacupoint EA.
Figure 4: Levels of PGF2α (a) and PGE2 (b) were compared in the serum of rats in each group using ELISA. The data were analyzed using one-way ANOVA and presented as mean ± SD, with n=5. Results indicated that levels of PGF2α and PGE2 were significantly different between groups. Specifically, the ▲▲▲P<0.001 compared to the control group, **P<0.01 compared to the PDM group, and ♦P<0.05 compared to the EA groupElectroacupuncture suppressed the expression of NLRP3 inflammasome component proteins
To investigate the impact of EA on NLRP3 inflammasome, we conducted a Western blot (WB) analysis of the key inflammasome proteins. Our results indicated that the expression levels of NLRP3, ASC, and caspase-1 were significantly elevated in the uterine tissue of the PDM group compared with those in the control group [[Figure 5], P < 0.001]. Conversely, in the EA group, the expression levels of these proteins were significantly reduced compared to those in the PDM group [[Figure 5], P < 0.01 NLRP3 and caspase-1, and P < 0.001 for ASC]. Notably, the content of these proteins in the uterine tissue of the EA group was significantly higher than that in the sham group [[Figure 5], P < 0.05 NLRP3 and P < 0.01 for ASC and caspase-1].
Figure 5: The protein expression levels of the NLRP3 inflammasome components, including NLRP3, ASC, and cleaved caspase-1, were compared among the different groups of rats. The results were analyzed using ANOVA, and all values are presented as mean ± standard deviation (SD), with n=5. The control group was used as a reference, and statistical significance was indicated by different symbols. For instance, ▲▲P<0.01 or ▲▲▲P<0.001 indicated a significant difference between the control group and the other groups, whereas **P<0.01, or ***P<0.001 indicated a significant difference between the PDM group and the other groups. Similarly, ♦P<0.05 or ♦♦P<0.01 indicated a significant difference between the EA group and the other groupsElectroacupuncture down-regulated the expression of interleukin-1β and interleukin-18
To investigate the underlying factors contributing to uterine tissue damage in PDM rats, we conducted a WB analysis to evaluate the levels of IL-1β and IL-18 protein expression. Results showed that IL-1β (P < 0.001) and IL-18 (P < 0.01) protein levels were markedly increased in the uterine tissue of PDM rats compared to those in the control group. However, in the EA group, IL-1β (P < 0.01) and IL-18 (P < 0.001) protein expression levels were significantly lower than those in the PDM group. Furthermore, the levels of IL-1β (P < 0.05) and IL-18 (P < 0.05) in the uterine tissues of rats in the EA group were significantly higher than those in the sham group shown in [Figure 6]. These results indicated differences between EA and nonacupoint EA in the regulation of inflammatory factors in PDM rats.
Figure 6: The levels of inflammatory factors in the uterine tissue of rats were compared among different groups. PDM rats showed a significant increase in the levels of IL-1β and IL-18, as determined by WB analysis. However, the expression of IL-1β and IL-18 was suppressed in the uterus of rats treated with EA. The results were analysed using ANOVA, and all data are presented as mean ± SD, with n=5. Statistical analysis revealed that the levels of IL-1β and IL-18 were significantly different between the groups. Specifically, the control group had the lowest levels, while the PDM group had the highest levels. EA treatment significantly reduced the levels of IL-1β and IL-18 in comparison to the PDM group Discussion The results of this study showed that the expression of NLRP3, ASC, and caspase-1 significantly increased in PDM rats. Activation of NLRP3 inflammasome involves two processes. The first involves the upregulation of the expression levels of NLRP3, ASC, and caspase-1 components, along with modifications to NLRP3 to enhance its activation. The second pathway involves the identification of an NLRP3 activator, which leads to the complete activation and assembly of the inflammasome.[19] Activation of NLRP3 inflammasome in PDM can be triggered by pattern-recognition receptors, including toll-like receptors. This in turn activates nuclear factor-κB (NF-κB), leading to the transcription of specific genes[20] or mitochondrial dysfunction.[21] NLRP3 inflammasome activation causes caspase-1 to undergo processing and cleavage, which was also demonstrated in gene knockout experiments.[22],[23] Our results showed that the expression levels of the inflammatory factors IL-1β and IL-18 in the uterus of the PDM group were significantly higher than those in the control group. The release of IL-1β and IL-18 exhibited two patterns. Cleaved caspase-1 cleaves gasderminD (GSDMD) and mediates pyroptosis, whereas IL-1β and IL-18 are released from pyrotic cells by cleaved caspase-1 processing.[24],[25] Another pattern is that IL-1β and IL-18 processed by cleaved caspase-1 are directly released without undergoing pyroptosis.[26] IL-1β and IL-18 are the source of pain.[27],[28] Therefore, tissue damage and pain in PDM may be mediated by IL-1β and IL-18 expression. Our results showed that serum PGF2α content was increased and PGE2 content was decreased in PDM rats. When dysmenorrhea occurs, PGF2α promotes uterine contractions and PGE2 inhibits them.[18] The study of Lim and Lappas[29] showed that during labor, human uterus contracted, NLRP3 protein expression and PGF2α expression were increased. PGF2α expression was significantly decreased after NLRP3 knockout. Our results and the above studies suggest that the increase of PGs induced by NLRP3 inflammasome activation is involved in the pathogenesis of PDM.
We observed organelle damage in the ultrastructure of endometrial epithelial cells in PDM rats. Endoplasmic reticulum changes were observed in PDM rat uterine epithelial cells. Protein kinase R-like endoplasmic reticulum kinase is activated when endoplasmic reticulum stress (RRS) occurs. PERK, inositol-requiring enzyme 1, and activating transcription factor 6 (ATF6) pathways induce transcription of pro-inflammatory cytokine genes.[30] Overexpression of PERK can increase the expression of NLRP3, and degradation of ATF6 can lead to endoplasmic reticulum stress (ERS); all of these mechanisms are involved in NLRP3 inflammasome activation.[31],[32] The results of this study showed that mitochondria were severely damaged in the PDM group. Mitochondrial dysfunction, ROS production, and DNA release in the cytoplasm are key factors in NLRP3 activation.[19] After treatment with mitochondrial autophagy inhibitors, the number of damaged and dysfunctional mitochondria increases, thereby increasing NLRP3 inflammasome activation.[33] The development of microcephaly and NLRP3 inflammatory response in endometrial epithelial cells may be linked to the presence of mitochondrial disorders and endoplasmic RRS. The study showed that the expression of NLRP3 inflammasome in endometrial cells and fibroblasts of postpartum cattle caused the death of endometrial cells.[7] Since the endometrial microenvironment of postpartum cattle is similar to that during menstruation, IL-1β released by NLRP3 inflammasome activation may mediate tissue damage. Our findings revealed that the EA group demonstrated a notable decrease in the endoplasmic reticulum and mitochondrial damage compared to the sham and PDM groups. This result indicates that EA has the potential to preserve the structural integrity of endometrial tissue, while nonacupoint EA may not provide the same level of protection.
The combination of CV4 and SP6 has the highest support of acupoints selection.[34] Therefore, CV4 and SP6 were selected as acupoint combinations for the EA treatment of PDM. At the time of treatment, the effect of the treatment before menstruation was better than that during menstruation.[35] Hence, we chose to perform EA therapy before PDM modeling was completed. Our results showed that the levels of NLRP3 inflammasome component proteins significantly decreased in the EA group. In our previous study, EA inhibited the expression of NF-κB, NLRP3, and caspase-1 in PDM rats.[36] EA may suppress the activation of NLRP3 inflammasome by interrupting the NF-κB signaling pathway, leading to a reduction in the expression of IL-1β and IL-18 in the uterine tissue of rats in the EA group. This decrease was associated with a reduction in the activity of caspase-1, which is responsible for the processing of these inflammatory cytokines. As a result, the release of IL-1β and IL-18 decreased. Besides, the content of PGF2α was decreased and the content of PGE2 was increased in PDM rats under EA treatment. Based on above results, EA can treat or relieve PDM by inhibiting NLRP3 inflammasome activation and thereby reducing PGs production. Given that the uterus is the affected organ in PDM, it is noteworthy that the SP6 acupoint, located on the spleen channel of foot-taiyin, connects the foot-shaoyin kidney and foot-jueyin liver channels. SP6 has a regulatory effect on the uterus, reflecting the specificity of acupoints. The connection between SP6 and the uterus was achieved through meridians.[37] SP6 is innervated by L4-S1[38] and may therefore be associated with the uterine connection. The afferent projection of the uterus and CV4 overlapped at L3-L6, which may be the neural mechanism of the connection between the uterus and CV4.[39] Therefore, the treatment of PDM with EA SP6 and CV4 was achieved by regulating the uterus.
In this study, we investigated whether EA was more effective than sham EA. Therefore, a sham EA group was used in this study. No significant reduction was observed in the writhing response or pathological damage scores of uterine tissue in the sham group, indicating that nonacupoints did not improve the symptoms of PDM rats. Similarly, the levels of NLRP3 inflammasome, IL-1β, and IL-18 were significantly higher in the sham group than in the control group. However, there were no significant differences in the levels of these markers between the sham and PDM groups. These findings suggest that PDM may not have a significant effect on the levels of these inflammatory markers. At the molecular level, nonacupoints do not control uterine inflammation in rats. There are potential limitations to our experiments because we did not study the effects of individual acupoints. In addition, there is no unified standard for the selection of nonacupoints. Nonmeridian, nonacupoint controls can be used to study the specificity of acupoints.[40] In addition, the mechanism underlying the difference between acupoints and nonacupoints has not been studied.
ConclusionsNLRP3 inflammasome activation in PDM mediates inflammation. SP6 and CV4 have specific effects on PDM. EA with SP6 and CV4 may treat PDM by inhibiting uterine inflammation and protecting the morphological structure of the uterus.
Financial support and sponsorship
The funds for this study were provided by several sources, including the National Natural Science Foundation of China's Youth Programme (No. 82004490), the Natural Science Foundation of Hunan Province's Youth Programme (No. 2021JJ40402), the Education Department of Hunan Province's Innovation Platform Open Fund Project (No. 20K091), and the Hunan University of Chinese Medicine's Graduate Innovation Project (2022CX100).
Conflicts of interest
There are no conflicts of interest.
References
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