Bushen Wenyang Huayu Decoction Targets TLR4/NF-κB Mediated Autophagy to Treat Endometriosis Effectively

Endometriosis has been found to be closely related to autophagy. This study aimed to elucidate the possible mechanism of Bushen Wenyang Huayu Decoction (BWHD) in treating endometriosis (EMs) by targeting TLR4/NF-κB-mediated autophagy. Autologous grafting was used to generate the EMs model in rats. Once the model was developed, BWHD high-dose and low-dose groups received intragastric administration of BWHD, and the gestrinone group served as a positive control. Immunofluorescence labeling and Western blotting were used for the protein expression of toll-like receptor 4 (TLR4), nuclear transcription factor-κB (NF-κB), Beclin-1, and selective autophagy connector protein P62 (P62). Quantitative real-time polymerase chain reaction (qRT-PCR) was used to analyze mRNA levels of TLR4, NF-κB, Beclin-1, and P62. We found that BWHD significantly reduced the size of ectopic lesions in rats with EMs, regulated reproductive hormone levels, and alleviated the cell autophagy level. It suggested that BWHD could be an effective treatment of EMs by targeting TLR4/NF-κB signaling pathway.


Introduction
Endometriosis (EMs) is a chronic condition that causes discomfort and disability and afects about 15% of women of childbearing age; it is also believed to afict 30%-50% of infertile women [1]. Aberrant proliferation, infltration, and recurrent bleeding of stromal cells and endometrial epithelial cells outside of the uterine cavity lead to the formation of nodules and masses, which can result in a wide range of clinical symptoms, including chronic pelvic discomfort, dysmenorrhea, menorrhagia, and infertility [2]. EMs is a frequent gynecological condition with a high recurrence rate [3], which has a detrimental efect on the quality of life for those afected by it. Accordingly, more research into EMs is required to address this pressing problem better [4]. Despite extensive research, the etiology and pathogenesis of EMs remain unclear; however, several studies have shown that the occurrence of EMs may be related to autophagy [5][6][7].
Autophagy is a highly conserved cellular process involved in decomposition, metabolism, and recycling, which plays a pivotal role in maintaining cellular homeostasis as well as the normal function of organelles [8,9], and autophagy has been linked to several human diseases, such as Parkinson's disease, metabolic diseases, EMs, and cancers [10][11][12][13][14].
Autophagy is believed to act as a housekeeping regulator to keep the intracellular environment stable by degrading and recycling damaged or unneeded parts [15]. Hypoxia, endoplasmic reticulum stress, starvation, cytotoxicity, and infection are all regulators that infuence the process of autophagy [16]. However, despite abundant literature about autophagy, its role in EMs is still controversial. Several reports have suggested that EMs has increased autophagy [17][18][19]; however other researchers have come to the opposite result [7,20]. It is important to fnd novel strategies to cure EMs by modulating autophagy.
TLR4/NF-κB constitutes the classical infammatory signaling pathway, can play crucial roles in mediating immune and infammatory responses, and participate in the pathological process of EMs [21,22]; furthermore, autophagy was thought to be regulated via TLR4/NF-κB signaling pathway [23,24]. Recently, TLR4/NF-κB has been implicated in EMs due to the critical regulatory roles of infammation, indicating that targeting TLR4/NF-κB might be a strategy for EMs therapy. However, it has not been reported to detect the induction of autophagy by TLR4/NF-κB in EMs. Terefore, this study aimed to investigate the role of TLR4/NF-κB in EMs and its potential mechanism.
Te importance of TCM's efcacy in the management of EMs has emerged in recent years [25,26]. Previous research by our team demonstrated that BWHD has great clinical efectiveness in treating EMs and can play a crucial role in supporting the atrophy of ectopic lesions by infuencing many channels and targets [27]. In this study, the potential mechanisms of BWHD on TLR4/NF-κB signaling pathway and the levels of autophagy-related factors Beclin-1 and P62 were observed to clarify the mode of action of BWHD for EMs treatment, thus providing a useful theoretical basis for deep application in this disease.

Materials and Methods
To perform this study, a total of 100 SPF female SD rats aged between 8 and 10 weeks and weighing 220 ± 20 g were purchased from the Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. (SCXK (Beijing) 2016-0011). Te experiment was carried out in the Scientifc Research Center of Hebei University of Chinese Medicine (SYXK (Hebei) 2017-005). Good ventilation and adaptive feeding were used during the one-week experiment, including the regular pellet meal and drinking water at room temperature (23 ± 2°C). All the diferent procedures were performed according to the ethical standards of the Ethics Committee of Hebei University of Chinese Medicine (no. DWLL2020031).

Grouping and Treatment
. Following a week of adapted feeding, 100 SPF female SD rats were randomly assigned to 5 groups with 20 rats each: Group 1 = sham group, Group 2 = model group, Group 3 = BWHD high-dose group, Group 4 = BWHD low-dose group, and Group 5 = gestrinone group. Except for the sham group, the EMs model was established by autologous transplantation as described in the literature [28]. All rats had their estrus cycles synchronized three days before the modeling by injecting them with 1 mg/kg of estradiol valerate.
After administering isofurane inhalation anesthesia and using iodine for standard disinfection, a 2.5 cm ventral midline incision was done to expose the uterus. After ligating the left horn of the uterus, the organ was preserved in a solution of normal saline. Te uterine horn was slit longitudinally and then sutured diagonally to the internal right abdominal wall using 4-0 absorbable thread. In the sham group, the left uterine horn was removed, and the abdomen was stitched shut. To prevent an infection from developing, gentamicin (0.1 ml) was administered intraperitoneally three days following the operation. Intragastric administration (1 ml/100 g) was started on the frst day after the surgery. Te therapeutically equivalent dose was determined to serve as a standard dose (a factor of 6.3 of the adult unit body weight). Te high-dose group received 46.25 g/kg of BWHD (2.5 times the clinical equivalent dosage), while the low-dose group received 18.5 g/kg of BWHD (clinical equivalent dosage), once daily. Te gestrinone group received 0.25 mg/kg of gestrinone twice a week, and an equal volume of pure water intragastric administration at other times. Te model and sham groups were given an equal volume of pure water for three weeks.

Specimen
Collection. Te size and shape of the ectopic foci in the rats were observed after the administration of anesthesia. Te uterus and ectopic foci were surgically removed promptly. To preserve the tissue, some of it was placed in a 4% paraformaldehyde solution, some in a 2.5% glutaraldehyde fxative solution, and the remainder were instantly frozen in liquid nitrogen and stored at a temperature of -80°C.

Measuring of Serum CA125, E 2 , P, FSH, and LH.
Te serum CA125, E 2 , P, FSH, and LH concentrations were detected by IRMA and ELISA, according to the instructions included in the purchased kits.

Immunofuorescence Staining.
Tissue chips were dewaxed with xylene, dehydrated with gradient alcohol, and then subjected to antigen repair and PBS washing. Te agents A and B naturally quench fuorescence were added. After washing with PBS, 0.5% Triton-PBS and 10% goat serum were added in jars and sealed correctly. After incubation, the wash was given with PBS and sealed in the pot along with an antifuorescence quenching agent. After 0.5% Triton PBS and PBS washing, 4',6-diamidino-2-phenylindole (DAPI) was incubated for 15 min. Leica SP8 laser scanning confocal microscope was used to observe the various images (Leica, Wetzlar, Germany).

Transmission Electron Microscopy.
Tissues were fxed right away in 4% glutaraldehyde at 4°C for 2 hours, then fxed again in 1% osmium acid for another 2 hours. After washing the fxed tissue with 0.1 M PBS, the tissue was embedded in a 1 : 1 acetone/812 mixture for 2-4 hours, a 1 : 2 acetone/812 combination overnight, and a pure 812 embedding agent for 5-8 hours before being dehydrated in ethanol and acetone gradients after dehydration resin block sections were cut with a Leica UC7 ultramicrotome of 60-80 nm (Leica, Wetzlar, Germany). After treating the samples with 2% uranyl acetate-saturated aqueous solution and then treating them with citrate solution for 15 minutes, the ultrastructural alterations were analyzed by looking at the samples with a Hitachi HT7700 transmission electron microscope (Hitachi, Changlunake, Japan).

Expression
2.11. Statistical Analysis. SPSS 26.0 statistical software was used for the statistical analysis; as a result of the measurement, the data are expressed as x ± s. For comparisons within the multiple groups, one-way ANOVA was used, while SNK tests were used for comparisons between the groups. A statistically signifcant diference was determined by p < 0.05.

Comparison of Volume and Weight of Ectopic
Foci. It was found that compared with the model group, the volume and weight of the ectopic foci in the high-dose and low-dose groups and the gestrinone group decreased, and the differences were statistically signifcant (p < 0.05 and p < 0.01). Tere were no signifcant diferences noted between the various treatment groups (p > 0.05) ( Table 1).

Comparison of Serum CA125, E 2 , P, FSH, and LH.
Serum concentrations of CA125, E 2 , P, FSH, and LH were all shown to be higher in the model group compared to the sham group, with the increases being statistically signifcant (P < 0.01). BWHD high-dose, low-dose, and gestrinone groups all showed signifcant reductions in serum CA125, E 2 , P, FSH, and LH as compared to the model group (P < 0.01) ( Table 2).

Ultrastructural Changes in the Eutopic and Ectopic
Endometrium. Transmission electron microscopy (TEM) revealed a low number of autophagosomes in the sham group and a signifcant rise in the model group. Te number Evidence-Based Complementary and Alternative Medicine of autophagosomes decreased signifcantly after BWHD treatment, and a normal bilayer membrane structure and undegraded cytoplasm were discovered (Figure 1).

Expression of TLR4, NF-κB, Beclin-1, and P62 Proteins by
Immunofuorescence. Te results of the immunofuorescence assay demonstrated that TLR4, Beclin-1, and P62 were primarily found in the cytoplasm, whereas NF-κB was mainly localized in the cytoplasm and nucleus. Te results showed that the red fuorescence of TLR4, Beclin-1, and NF-κB was brighter in the model group as compared to the sham group and darker after treatment. However, the expression of P62 displayed the opposite change ( Figure 2).

Expression of TLR4, NF-κB, Beclin-1, and P62 Proteins by
Western Blot. It was observed that compared with the sham group, the expressions of TLR4, NF-κB, and Beclin-1 in the eutopic endometrium of the model group were increased, while the expression of P62 was decreased (P < 0.05 and P < 0.01) (Figure 2). In addition, compared with the model group, the expressions of TLR4, NF-κB, and Beclin-1 in the endometrium of the high-dose group, low-dose group, and gestrinone group were decreased, while the expression of P62 was increased (P < 0.05, P < 0.01). Moreover, compared with the model group, the expressions of TLR4, NF-κB, and Beclin-1 in the ectopic endometrium of high-dose group, low-dose group, and gestrinone groups were decreased, while P62 was increased (P < 0.05 and P < 0.01) (Figures 3-4).

TLR4, NF-κB, Beclin-1, and P62 mRNA by qRT-PCR.
It was noted that compared with the sham group, mRNA levels of TLR4, NF-κB, and Beclin-1 were increased in the model group, while mRNA levels of P62 were decreased (P < 0.05 and P < 0.01). In addition, compared with the model group, mRNA levels of TLR4, NF-κB, and Beclin-1 in the endometrium of high-dose group, low-dose group, and gestrinone groups were decreased, and the mRNA level of P62 was increased (P < 0.05 and P < 0.01). However, compared with the model group, mRNA levels of TLR4, NF-κB, and Beclin-1 in the ectopic endometrium in the high-dose group, low-dose group, and gestrinone group were decreased, while the mRNA level of P62 was increased (P < 0.05 and P < 0.01) (Tables 3 and 4).

Discussion
In this study, we found that TLR4/NF-κB signaling pathway can participate in promoting EMs pathology by increasing autophagy. Terapeutic efects against EMs can be achieved using BWHD due to its ability to efectively suppress TLR4 and NF-κB expression, alleviate autophagy, decrease energy supply, and subsequently increase the cell death of ectopic lesions ( Figure 6).
EMs is considered a benign gynecological illness. However, it has been shown to have growth, invasion, metastasis, and recurrence patterns comparable to malignant tumors [29]. Because of the unclear pathophysiology, there are numerous unknown aspects of EMs treatment. Western medicine mostly uses medications and surgery to treat EMs, but major side efects and recurrence rates are problems that cannot be overlooked in the treatment process. In TCM theory, EMs are classifed as "zhengjia," "dysmenorrhea," and "infertility." Its underlying pathophysiology is blood stasis, which is caused mostly by kidney  Note. Compared with the model group, * P < 0.05 and * * P < 0.01. yang insufciency. BWHD can notify the kidney, warm Yang, remove blood stasis, and relieve pain. In our previous research, combining clinical observation and animal experiments, we discovered that BWHD could alleviate clinical symptoms, lower VAS scores, attenuate the release of pain substances, relieve pain, reduce the size of lesions, and block the growth of EMs [27,30]. A metaanalysis found that tonifying kidneys and activating blood stasis therapy are efective and safe for EMs, suggesting that it may be a potential method for treating EMs [31]. Tis study found that during a 3-week treatment period, BWHD can dramatically reduce the volume and weight of ectopic lesions while also promoting their dissipation and absorption, establishing BWHD as an efective medicine in the clinical treatment of EMs. It has a clear therapeutic efect and can efectively lower serum CA125 levels. It can also lower E 2 , P, FSH, and LH levels while also regulating hormone levels.
Ectopic lesions eliminate their foreign bodies by producing infammatory responses, thus EMs can be thought of as a chronic immunological infammatory illness. TLR4, belonging to the TLR family, is widely expressed on the cell membranes of immune cells as a receptor and an infammatory mediator that can play a critical role in the signal transduction throughout the innate immune system. It is found in endometrial cells and acts as a crucial factor in the development of EMs [32]. TLR4, as we know, is essential for initiating the innate immune response through activating the diferent intracellular signaling pathways NF-κB [33].
TLR4 signaling causes infammation and can greatly enhance autophagosome production [34,35]. NF-κB is a crucial transcription factor that acts downstream of the TLR4 signaling pathway [36], which regulates immunological responses and is thought to be the beginning factor of several infammatory reactions. Its activation can cause Evidence-Based Complementary and Alternative Medicine 5 infammation as well as the aggregation of the microtubuleassociated protein light chain 3 (LC3) and the ubiquitination of Beclin-1, which can then activate autophagy [37]. Te TLR4/NF-κB pathway is a signal transduction system that has been linked to infammatory immunological mechanisms and the regulation of infammation as well as the regulation of autophagy [38]. Some researchers have discovered that the TLR4/NF-κB signaling pathway can promote autophagy in the pathological phase of idiopathic pulmonary fbrosis, demonstrating that TLR4/NF-κB is required for autophagy [23]. Beclin-1 is a critical regulator of autophagy, which mediates the initiation of autophagy and is considered as a hallmark of autophagy initiation. P62 is an autophagic substrate conjunctin, which contains multiple domains and can bind to the protein to be degraded to transport it to autophagosomes, and eventually to be degraded in lysosomes [39]. P62 is an autophagy adaptor protein that can be destroyed but accumulates when autophagy is blocked. As a result, P62 is inversely associated with autophagy [40].
In this study, we assessed the expression of TLR4, NF-κB, and autophagy-associated markers like Beclin-1 and P62 to see if they play a role in EMs. TLR4 and NF-κB protein and mRNA levels were higher in the eutopic and ectopic endometrium of EMs animals. Furthermore, these two parameters were shown to be positively connected, suggesting that the overactivation of the TLR4/NF-κB signaling pathway was associated with the incidence of EMs, and the two parameters can have a synergistic efect, which was consistent with the previous research [21]. TLR4 activation can eventually result in NF-κB activation at the mRNA and protein levels, and activation of the NF-κB signaling pathway      Evidence-Based Complementary and Alternative Medicine can stimulate the production of numerous proinfammatory cytokines and contribute to the formation of EMs. In order to evaluate the amount of cellular autophagy, we used Western blot, qRT-PCR, and immunofuorescence to determine the expression of autophagy markers such as Beclin-1 and P62. Results showed that Beclin-1 was upregulated and P62 was downregulated in the model group and that TEM imaging revealed ultrastructural changes in both the eutopic and ectopic endometrium, including the disappearance or vacuolization of some mitochondrial cristae and an increase in the number of autophagosomes in both types of the endometrium.

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A correlation study revealed that TLR4 was highly correlated with NF-κB. At the same time, NF-κB was also positively correlated with autophagy, suggesting that the TLR4/NF-κB signaling pathway is critical in the regulation of EMs autophagy. TLR4 and NF-κB protein and mRNA expression levels reduced dramatically following BWHD treatment, demonstrating that this prescription can reduce infammation by suppressing the activation of the TLR4/NF-κB signaling pathway and thus can signifcantly enhance the local microenvironment. Furthermore, it was discovered that after TCM treatment, the expression of Beclin-1 was decreased considerably while that of P62 was signifcantly increased, implying that this prescription can reduce the level of autophagy and efectively decrease the energy supply of ectopic cells, resulting in death. It has been reported that autophagy suppression can increase apoptosis in recent years [41]. P62 activation has also been shown to suppress NF-κB activation, reducing infammation development [42]. Ravanan et al. [43] revealed that inhibiting autophagy can signifcantly diminish the infammatory response generated by the NF-κB pathway. Our fndings suggested that BWHD could reduce autophagy and increase the shrinkage of ectopic lesions by blocking the TLR4/NF-κB signaling pathway, which could be one of the mechanisms of BWHD in treating EMs. Furthermore, according to prior research [42], activation of P62 could block NF-κB activity, hence controlling the development of EMs infammation and significantly improving the internal environment of EMs, which requires additional validation in the future.
As a result of its ability to modulate various targets and pathways with relatively fewer adverse efects, TCM is currently widely employed for the treatment and prevention of endometriosis [44]. Overall, our results show that BWHD can dramatically lower the autophagy level of abnormally increased in EMs and that the mechanism may be linked to the suppression of the TLR4/NF-κB signaling pathway. By its good clinical efcacy and fewer side efects in treating EMs, Chinese herbal therapy has gained a lot of attention as a supplemental medicine. However, more research is required to determine whether TCM delays or reduces the frequency of EMs recurrence.

Data Availability
Te datasets used and analyzed during the current study are available from the corresponding author upon reasonable request.

Conflicts of Interest
Te authors declare that there are no conficts of interest regarding the publication of this paper.  . Note: the protein and mRNA expressions of autophagy-related genes Beclin-1 were activated and p62 was downregulated in EMs models; BWHD may play the function of alleviating autophagy by inhibiting the TRL4/NF-κB signaling pathway, thereby reducing the autophagy level, to achieve the purpose of ameliorating the progress of EMs. Tese results proved that there was an inhibition efect of BWHD on autophagy in EMs by regulating TLR4/NF-κB signaling pathway.
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