Baicalein inhibits mycobacterium tuberculosis-induced macrophage M1 polarization depending on the regulation of YY1/RAB10/TLR4 pathway

Abstract

Background

Baicalein (Bai) has been found to alleviate the progression of tuberculosis (TB) by inhibiting mycobacterium tuberculosis (M.tb)-induced macrophage pyroptosis, so it may be used as an adjuvant treatment for TB. However, the underlying molecular mechanism of Bai remains unclear.

Methods

THP-1 macrophages were infected with M.tb and treated with Bai. The viability and apoptosis of macrophages were examined with CCK8 assay, flow cytometry and TUNEL staining. The levels of inflammatory cytokines were tested by ELISA. Macrophage M1 polarization was assessed by detecting CD86⁺ cell rate using flow cytometry. The protein levels of RAB10, YY1, TLR4, MYD88, p-P65/P65 and p-IκBα/IκBα were determined using western blot. The interaction between RAB10 and YY1 or TLR4 was confirmed by ChIP assay, dual-luciferase reporter assay and Co-IP assay.

Results

M.tb promoted macrophage M1 polarization, apoptosis and inflammation, while this effect was abolished by Bai treatment. Bai decreased RAB10 expression, and RAB10 overexpression reversed the anti-TB effect of Bai. YY1 enhanced the transcription of RAB10, and YY1 knockdown inhibited M.tb-induced macrophage M1 polarization by reducing RAB10 expression. Also, Bai could decrease YY1 expression, and YY1 overexpression eliminated the regulation of Bai on M.tb-induced macrophage M1 polarization. Moreover, RAB10 could interact with TLR4 to activate TLR4/MYD88/NF-κB pathway, thus promoting M.tb-induced macrophage M1 polarization.

Conclusion

Bai might play anti-TB effect by regulating YY1/RAB10/TLR4/MYD88/NF-κB pathway, providing a novel idea for the treatment of TB.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12950-025-00466-6.

Introduction

Tuberculosis (TB) is a chronic infectious disease caused by mycobacterium tuberculosis (M.tb), which poses a serious threat to human health [1, 2]. As a kind of immune cells, macrophages play a role in fighting infectious diseases by maintaining the homeostasis of body [3]. Studies have shown that macrophages are the main host cells of M.tb, which regulate cellular inflammation and apoptosis by secreting cytokines after polarization, thus playing a role in anti-TB infection [4, 5]. Therefore, clarifying the molecular mechanism of M.tb-induced macrophage polarization may provide new ideas for TB treatment.

Baicalein (Bai), a flavonoid extracted from Scutellaria baicalensis, has various biological activities, such as anti-inflammatory and anti-cancer [6, 7]. Bai mediates the progression of many human diseases by regulating macrophage functions. For example, Bai might relieve the development of atherosclerosis, which could inhibit lipid accumulation and pro-inflammatory cytokine release [8]. In TB-related study, Ning et al. reported that Bai inhibited M.tb-infected macrophage pyroptosis by promoting autophagy and suppressing inflammasome assembly [9], confirming the anti-TB role of Bai. However, the underlying molecular mechanisms of Bai resistance to TB still need to be further revealed.

RAB10, a member of the RabGTPase family, is involved in many biological processes, including lysosome exocytosis and plasma membrane repair [10]. Aberrant RAB10 expression has been verified to be associated with the development of human diseases, such as breast cancer [11] and Alzheimer’s disease [12]. Previous studies had shown that RAB10 was highly expressed in M.tb-infected macrophages, which could promote NF-κB pathway activation and pro-inflammatory cytokine secretion to affect M.tb survival [13]. This study confirms the positive role of RAB10 in TB progression. This study found that Bai significantly reduced RAB10 expression, but whether Bai mediated TB progression through the regulation of RAB10 is still unknown. In addition, our study discovered that transcription factor YY1 might mediate RAB10 transcription by analyzing jaspar software, and RAB10 could interact with TLR4 using STRING protein interaction website. However, whether YY1/RAB10/TLR4 pathway is involved in Bai-mediated anti-TB effect has not been explored.

This study aimed to reveal the underlying molecular mechanism of Bai against TB. Based on the above, our study hypothesized that Bai might inhibit M.tb-induced macrophage polarization by regulating the YY1/RAB10/TLR4 axis, thereby alleviating the progression of TB.

Materials and methods

Cell culture and treatment

THP-1 cells (Procell, Wuhan, China) were grown in RPMI-1640 medium plus 10% FBS, 1% penicillin/streptomycin and 0.05 mM β-mercaptoethanol. To induce macrophage-like state, THP-1 cells were treated with PMA (100 ng/mL; Sigma-Aldrich, St Louis, MO, USA) for 20 h. To mimic TB cell models, macrophages were treated with M.tb (H37Rv, virulent strain) at 0, 1, 5 and 10 multiplicity of infection (MOI) for 24 h. For Bai treatment, macrophages were infected with 5 MOI M.tb for 4 h, and then treated with various concentrations of Bai (20, 40 and 60 µM; purity > 98%; Sigma-Aldrich) for 24 h.

Human monocyte-derived macrophages (hMDMs) were isolated from the blood of healthy donors (n = 8) recruited from Zhuzhou Hospital Affiliated to Xiangya School of Medicine, Central South University as previously described [14]. Each donor signed written informed consent, and our study was approved by the Ethics Committee of Zhuzhou Hospital Affiliated to Xiangya School of Medicine, Central South University.

Cell transfection

The pcDNA RAB10/YY1 overexpression vector, siRNAs against RAB10/YY1 (si-RAB10/si-YY1), and their negative controls (Vector and si-NC) were obtained from RiboBio (Guangzhou, China). They were transfected into PMA-induced THP-1 macrophages using Lipofectamine 3000 (Invitrogen, Waltham, MA, USA). After transfection for 24 h, macrophages were infected with M.tb or treated with Bai.

CCK8 assay

Macrophages were re-seeded into 96-well plates. After 48 h, cells were incubated with CCK8 reagent (Beyotime, Shanghai, China) for 2 h, and cell viability was determined at 450 nm using a microplate reader.

Flow cytometry

For measuring cell apoptosis rate, transfected or treated macrophages were collected and re-suspended with Annexin V-Binding Buffer. Afterwards, cells were incubated with Annexin V-FITC and PI Solution (Elabscience, Wuhan, China) for 15 min, followed by measuring cell apoptosis rate using flow cytometer.

For detecting cell surface antibody, transfected or treated macrophages were labelled with anti-CD86⁺ (PE/Cy7; ab233571, Abcam, Cambridge, MA, USA) for 30 min, and then CD86⁺ cell ratio was evaluated by flow cytometer.

TUNEL staining

Macrophages in 6-well plates were fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton X-100. Then, macrophages were incubated with TUNEL reaction mixture and counterstained with DAPI using TUNEL In situ Apoptosis Kit (Elabscience). Finally, TUNEL-positive cells were observed under a fluorescence microscope.

ELISA

The supernatant of macrophages was collected to measure IL-1β and TNF-α levels according to the instructions of Human IL-1β ELISA Kit (ab214025, Abcam) and Human TNF-α ELISA Kit (ab181421, Abcam), respectively.

Colony forming unit (CFU)

M.tb-infected macrophages were lysed with 1% SDS followed by 20% BSA. CFUs were determined by plating serially diluted lysates onto Middlebrook 7H10 plates.

qRT-PCR

Total RNAs were extracted by TRIzol reagent and synthesized into cDNA using PrimeScript RT Reagent Kit (Takara, Tokyo, Japan). PCR amplification was performed by SYBR Green (Takara) with specific primers: RAB10, F 5’-CTCGGTTTCCTGGGGCTATG-3’, R 5’-TGAGGACAGTCTCTCTCCCG-3’; GAPDH, F 5’-AAGGCTGTGGGCAAGGTCATC-3’, R 5’-GCGTCAAAGGTGGAGGAGTGG-3’. Relative RAB10 expression was calculated by 2^−ΔΔCt method.

Western blot (WB)

Extracted proteins were separated and transferred onto PVDF membranes. Membranes were incubated with antibodies (Abcam), including: anti-RAB10 (1:1000, ab237703), anti-YY1 (1:25000, ab245365), anti-TLR4 (1:1000, ab13556), anti-MYD88 (1:500, ab2064), anti-P65 (1:1000, ab32536), anti-p-P65 (1:1000, ab76302), anti-IκBα (1:10000, ab32518), anti-p-IκBα (1:1000, ab133462), anti-GADPH (1:2500, ab9485) and Goat anti-Rabbit IgG (1:50000, ab205718). Finally, protein signals were visualized by ECL reagent (Beyotime).

ChIP assay

Jaspar software predicted the binding sites of YY1 in RAB10 promoter region. Using Magna ChIP A/G Kit (Millipore, Billerica, MA, USA), THP-1 cells were fixed with formaldehyde and sonicated into fragments. Magnetic beads and anti-YY1 (ab245365, Abcam) or anti-IgG (ab172730, Abcam) were used to precipitate the specific DNA fragment. The enrichment of RAB10 promoter was detected by qRT-PCR.

Dual-luciferase reporter assay

The wild-type or mutant-type fragments of RAB10 promoter site 1 were inserted into the pGL3-basic vectors to generate WT-RAB10 or MUT-RAB10 vectors. 293 T cells were transfected with the vectors and si-NC/si-YY1. After 48 h, cell luciferase activity was measured to confirm the interaction between YY1 and RAB10 promoter.

Co-IP assay

The lysates of macrophages were incubated with protein A/G agarose beads pre-coated with anti-IgG, anti-TLR4, or anti-RAB10. Then, the signals of TLR4 and RAB10 in immunoprecipitated proteins were measured by WB.

Statistical analysis

Data are presented as mean ± SD and analyzed by GraphPad Prism 8.0 software. Student’s t-test or ANOVA was performed to analyze data. P < 0.05 was considered statistical significance.

Results

M.tb induced macrophage M1 polarization, apoptosis and inflammation

To screen for the optimal dose of M.tb to treat macrophages, our study evaluated the function of THP-1 macrophages under M.tb infection at different MOI. As shown in Fig. 1A, macrophage viability was significantly reduced with the increasing of M.tb dose. Besides, the apoptosis rate, TUNEL-positive cells, IL-1β and TNF-α levels were markedly enhanced in M.tb-induced macrophages in a dose-dependent manner (Fig. 1B-D). Furthermore, the cell ratio of M1 polarization marker CD86⁺ was gradually enhanced in macrophages after infected with M.tb (Fig. 1E). Based on the above results, M.tb at 5 MOI was selected for subsequent studies.

Fig. 1.

Effect of M.tb on macrophage M1 polarization. PMA-induced THP-1 cells were infected with 0, 1, 5 and 10 MOI of M.tb. CCK8 assay (A), flow cytometry (B) and TUNEL staining (C) were used to assess cell viability and apoptosis. (D) ELISA was used to detect the levels of IL-1β and TNF-α. (E) CD86⁺ cell ratio was analyzed using flow cytometry. *P < 0.05

Bai suppressed M.tb-induced macrophage M1 polarization

To explore the role of Bai in TB progression, M.tb-induced macrophages were treated with Bai. Different concentrations of Bai could significantly improve the viability of M.tb-induced macrophages (Fig. 2A), and 40 µM Bai was selected for following experiments. Through flow cytometry, TUNEL staining and ELISA, our study found that Bai treatment significantly inhibited apoptosis rate, TUNEL-positive cells, IL-1β, TNF-α, and CD86⁺ cell ratio, while enhanced the cell ratio of M2 polarization marker (CD206⁺) in M.tb-induced macrophages (Fig. 2B-E and Supplementary Fig. 1). Furthermore, CFU assay determined that Bai could inhibit CFU in M.tb-induced macrophages, suggesting that Bai effect might also involve antimicrobial activity (Supplementary Fig. 2). Moreover, Bai enhanced viability, while reduced IL-1β, TNF-α and CD86⁺ cell ratio in M.tb-induced hMDMs (Supplementary Fig. 3A-C). These results confirmed the inhibition effect of Bai on M.tb-induced macrophage M1 polarization, suggesting that Bai might restrain TB progression.

Fig. 2.

Effect of Bai on M.tb-induced macrophage M1 polarization. PMA-induced THP-1 cells were infected with 5 MOI of M.tb and treated with Bai. Cell viability and apoptosis were determined using CCK8 assay (A), flow cytometry (B) and TUNEL staining (C). (D) The levels of IL-1β and TNF-α were examined by ELISA. (E) Flow cytometry was used to examine CD86⁺ cell ratio. *P < 0.05

Fig. 3.

Effects of Bai and RAB10 on M.tb-induced macrophage M1 polarization. (A-B) RAB10 mRNA and protein expression was tested by qRT-PCR and WB in PMA-induced THP-1 cells infected with 0, 1, 5 and 10 MOI of M.tb. (C-J) PMA-induced THP-1 cells were transfected with Vector/RAB10, infected with M.tb and treated with Bai. (C) RAB10 protein expression was detected using WB. CCK8 assay (D), flow cytometry (E) and TUNEL staining (F-G) were performed to measure cell viability and apoptosis. (H) IL-1β and TNF-α levels were tested by ELISA. (I-J) CD86⁺ cell ratio was assessed by flow cytometry. *P < 0.05

Bai reduced RAB10 expression to affect M.tb-induced macrophage M1 polarization

RAB10 expression was gradually promoted with the increasing dose of M.tb in macrophages (Fig. 3A-B). Also, Bai decreased RAB10 protein level in M.tb-induced hMDMs (Supplementary Fig. 3D). To investigate whether Bai regulated TB progression by mediating RAB10 expression, M.tb-induced macrophages were transfected with RAB10 overexpression vector and treated with Bai. The detection of RAB10 protein expression showed that Bai treatment markedly reduced RAB10 level in M.tb-induced macrophages, and this effect could be abolished by RAB10 overexpression vector (Fig. 3C). Through functional experiments, our study found that RAB10 overexpression reversed the promoting effect of Bai on viability, as well as the inhibitory effect on apoptosis rate, TUNEL-positive cells, IL-1β, TNF-α, and CD86⁺ cell ratio in M.tb-induced macrophages (Fig. 3D-J). Thus, our study believed that Bai alleviated TB progression by decreasing RAB10 expression.

YY1 activated the transcription of RAB10

Jaspar software predicted that there were 2 binding sites between transcription factor YY1 and RAB10 promoter region (Fig. 4A). ChIP analysis showed that RAB10 promoter site 1 was significantly enriched by anti-YY1 (Fig. 4B), confirming the interaction between YY1 and RAB10 promoter site 1. In addition, si-YY1 was designed to reduce YY1 expression (Fig. 4C). Also, YY1 knockdown significantly reduced the luciferase activity of WT-RAB10 vector rather than the MUT-RAB10 vector (Fig. 4D), further verifying the interaction between YY1 and RAB10 promoter. Besides, silencing of YY1 obviously inhibited RAB10 protein expression (Fig. 4E), and YY1 protein expression was gradually enhanced in macrophages after treated with the increasing dose of M.tb (Fig. 4F). Therefore, our study hypothesized that YY1 might mediate TB progression by regulating RAB10 transcription.

Fig. 4.

YY1 activated RAB10 transcription. (A) Jaspar software predicted the binding sites between YY1 and RAB10 promoter region. (B) The interaction between them was confirmed by ChIP assay. (C) The transfection efficiency of si-YY1 was assessed by WB. (D) Dual-luciferase reporter assay was used for assessing interaction. (E) RAB10 protein expression was tested by WB in PMA-induced THP-1 cells transfected with si-NC/si-YY1. (F) WB was used to detect YY1 protein expression in PMA-induced THP-1 cells infected with 0, 1, 5 and 10 MOI of M.tb. *P < 0.05

YY1 knockdown alleviated M.tb-induced macrophage M1 polarization by regulating RAB10

Following, M.tb-induced macrophages were transfected with si-YY1 and RAB10 overexpression vector to explore whether YY1 regulated RAB10 expression to mediate TB progression. As presented in Fig. 5A-C, downregulation of YY1 promoted viability, reduced apoptosis rate and decreased TUNEL-positive cells in M.tb-induced macrophages, while these effects were eliminated by RAB10 overexpression. Furthermore, overexpressed RAB10 also overturned the inhibition effect of YY1 knockdown on IL-1β, TNF-α, and CD86⁺ cell ratio in M.tb-induced macrophages (Fig. 5D-E). All data revealed that YY1 might promote RAB10 expression to accelerate TB progression.

Fig. 5.

Effects of si-YY1 and RAB10 on M.tb-induced macrophage M1 polarization. PMA-induced THP-1 cells were transfected with si-NC/si-YY1/RAB10 and infected with 5 MOI of M.tb. Cell viability and apoptosis were examined by CCK8 assay (A), flow cytometry (B) and TUNEL staining (C). (D) IL-1β and TNF-α levels were determined using ELISA. (E) Flow cytometry was performed to analyze CD86⁺ cell ratio. *P < 0.05

Bai inhibited M.tb-induced macrophage M1 polarization through downregulating YY1

Bai decreased YY1 protein level in M.tb-induced hMDMs (Supplementary Fig. 3E). Then, M.tb-induced macrophages were transfected with YY1 overexpression vector and treated with Bai for investigating whether Bai regulated YY1 expression to mediate TB process. YY1 protein expression could be suppressed by Bai, and YY1 overexpression vector reversed this effect (Fig. 6A). Bai-mediated the promotion on viability, as well as the suppression on apoptosis rate, TUNEL-positive cells, IL-1β, TNF-α, and CD86⁺ cell ratio, could be abolished by YY1 overexpression in M.tb-induced macrophages (Fig. 6B-F). These results illuminated that Bai treatment inhibited YY1 expression to relieve TB progression.

Fig. 6.

Effects of Bai and YY1 on M.tb-induced macrophage M1 polarization. PMA-induced THP-1 cells were transfected with Vector/YY1, infected with M.tb and treated with Bai. (A) WB was used for analyzing YY1 protein expression. CCK8 assay (B), flow cytometry (C) and TUNEL staining (D) were utilized to test cell viability and apoptosis. (E) The levels of IL-1β and TNF-α were determined using ELISA. (F) CD86⁺ cell ratio was evaluated using flow cytometry. *P < 0.05

RAB10 activated TLR4/MYD88/NF-κB pathway

The STRING protein interaction website predicted that RAB10 could interact with TLR4 (Fig. 7A), and further Co-IP assay confirmed this interaction (Fig. 7B). Following, RAB10 expression was decreased by the transfection of si-RAB10 (Fig. 7C). The detection of TLR4 protein expression showed that TLR4 level was enhanced in M.tb-induced macrophages, and RAB10 knockdown eliminated this effect (Fig. 7D). Given the important role of TLR4/MYD88/NF-κB pathway in TB progression, our study explored whether Bai regulated RAB10 to mediate the activity of TLR4/MYD88/NF-κB pathway. The results suggested that Bai reduced the protein levels of TLR4, MYD88, p-P65/P65 and p-IκBα/IκBα, which could be reversed by RAB10 overexpression (Fig. 7E-G). Above all, Bai inhibited RAB10 expression to inactivate TLR4/MYD88/NF-κB pathway.

Fig. 7.

Bai and RAB10 regulated TLR4/MYD88/NF-κB pathway. (A) STRING protein interaction website predicted the relationship between RAB10 and TLR4. (B) The interaction of RAB10 and TLR4 was confirmed by Co-IP assay. (C) The transfection efficiency of si-RAB10 was assessed by WB. (D) TLR4 protein expression was tested by WB in PMA-induced THP-1 cells transfected with si-NC/si-RAB10 and infected with M.tb. (E-G) The protein levels of TLR4, MYD88, p-P65/P65 and p-IκBα/IκBα were detected by WB in PMA-induced THP-1 cells transfected with si-NC/si-RAB10, infected with M.tb and treated with Bai. *P < 0.05

NF-κB pathway inhibitor reversed the effect of RAB10 on M.tb-induced macrophage M1 polarization

To further determine the importance of the NF-κB pathway, our study used NF-κB pathway inhibitor BAY11-7085 (5 µM) to treat M.tb-induced macrophages transfected with RAB10 overexpression vector. Our results revealed that RAB10 overexpression suppressed viability, promoted apoptosis rate, TUNEL-positive cells, IL-1β, TNF-α, and CD86⁺ cell ratio in M.tb-induced macrophages, while these effects were reversed by BAY11-7085 (Fig. 8A-E). Therefore, RAB10 contributed to TB progression by activating NF-κB pathway.

Fig. 8.

Effects of RAB10 and BAY11-7085 on M.tb-induced macrophage M1 polarization. PMA-induced THP-1 cells were transfected with Vector/RAB10, treated with BAY11-7085 and infected with M.tb. Cell viability and apoptosis were measured using CCK8 assay (A), flow cytometry (B) and TUNEL staining (C). (D) ELISA was used for measuring IL-1β and TNF-α levels. (E) Flow cytometry was used for detecting CD86⁺ cell ratio. *P < 0.05

Discussion

Macrophages plays an important role in TB development, and M1 polarization mainly occurs in the early and active stage of M.tb infection [15, 16]. Therefore, a better understanding of the mechanism by which M.tb induces macrophage polarization will help to develop new methods for TB treatment. Bai, the major active ingredient extracted from Scutellaria baicalensis, has been shown to regulate various human diseases, including neurodegenerative diseases [17], cardiovascular diseases [18] and malignant tumors [19]. Bai has anti-apoptosis and anti-inflammation roles in many types of cells [20, 21]. Importantly, the inhibitory effect of Bai on macrophage M1 polarization has been widely demonstrated [22, 23]. In M.tb-induced macrophages, our study found that Bai restrained apoptosis, inflammation and M1 polarization, verifying that Bai could suppress TB progression. Consistent with the results of Ning et al. [9], our data also confirmed the anti-TB role of Bai, providing a new evidence for Bai as an adjuvant treatment strategy of TB.

RAB10 mediates disease progression by regulating multiple cellular functions. RAB10 alleviated cardiomyocyte oxidative stress and apoptosis to protect against doxorubicin-induced cardiotoxicity [24]. RAB10 suppressed THP-1 cell apoptosis and enhanced proliferation to promote the progression of acute myeloid leukemia [25]. Also, RAB10 inhibited renal tubular epithelial cell apoptosis to relieve ischemia/reperfusion-induced acute kidney injury [26]. Although Zhu et al. identified that RAB10 mediated TB progression by regulating M.tb-infected macrophage function [13], the specific mechanism still needs to be further elucidated. Consistent with this reports, our data detected the high RAB10 expression in M.tb-infected macrophages. Through analyzing, our study confirmed that Bai decreased RAB10 expression, and ectopic RAB10 expression reversed Bai-mediated inhibition on M.tb-infected macrophage M1 polarization. These results further revealed that Bai indeed suppressed TB progression via downregulating RAB10.

YY1, a member of zinc finger transcription factor Gli-kruppel family, is widely expressed in various tissues as a regulator [27, 28]. YY1 can regulate macrophage function to mediate disease development. For example, YY1 inhibited macrophage migration and phagocytosis to regulate inflammatory diseases via suppressing CXCR4 expression [29]. Besides, knockdown of YY1 alleviated ox-LDL-induced macrophage inflammation and lipid accumulation by binding to PCSK9 promoter, thus relieving atherosclerosis process [30]. In TB, a recent study showed that YY1 activated TLR4 transcription to promote M.tb-induced macrophage injury and inflammation [31], confirming that YY1 might be a vital regulator for TB. Through prediction and analyzing, our study determined that YY1 bound to RAB10 promoter to increase its transcription. Furthermore, RAB10 overexpression eliminated the suppressive effect of YY1 silencing on M.tb-induced macrophage apoptosis, inflammation and M1 polarization, suggesting that YY1 promoted M.tb infection in macrophages to aggravate TB progression by activating RAB10. In addition, our study found that Bai reduced YY1 expression, and YY1 overexpression also overturned the anti-TB effect of Bai. These data refine our hypothesis regarding the Bai/YY1/RAB10 axis.

TLR4 belongs to the TLRs family and is a pattern recognition receptor in the body’s innate immune system. TLR4/MYD88/NF-κB, an important pathway that regulates inflammatory responses, is confirmed to be activated during TB progression [32, 33]. Previous study indicated that RAB10 promoted membrane TLR4 expression to increase the production of inflammatory cytokines and interferons in LPS-induced macrophages [34]. FAM49B facilitated proliferation, metastasis, and chemoresistance in breast cancer by activating RAB10/TLR4/NF-κB pathway [35]. Besides, Bai has been confirmed to inhibit NF-κB activation to mediate human disease process [36, 37]. In this study, our study confirmed the interaction between RAB10 and TLR4, and determined that Bai inhibited the TLR4/MYD88/NF-κB pathway via reducing RAB10 expression. NF-κB pathway inhibitor BAY11-7085 reversed RAB10-mediated the promotion on macrophage M1 polarization under M.tb infection, further suggesting that RAB10 enhanced TB progression by activating TLR4/MYD88/NF-κB pathway.

Autophagy is involved in the regulation of the body’s inflammatory response, and is related to the pathogenesis and treatment of inflammatory diseases, including TB [38–40]. Beyond autophagy, emerging evidence highlights the complex crosstalk between multiple cell death modalities during M.tb infection. Recent studies demonstrate that M.tb can simultaneously modulate pyroptosis (via NLRP3 inflammasome activation), apoptosis (through caspase-8/9 pathways), and necroptosis (mediated by RIPK1/MLKL) to evade host immunity [41]. Notably, our data suggest that Bai’s suppression of YY1/RAB10 axis may also intersect with these pathways, as NF-κB is a known regulator of PANoptosis [41]. Future studies should explore whether Bai’s anti-TB effects involve coordinated regulation of these death programs.

Due to the limitations of funds, conditions and time, our study are temporarily unable to carry out animal studies (such as mice infected with M.tb). In the future, this study will construct animal models to further determine whether Bai can indeed regulate macrophage polarization and control TB progression through the YY1/RAB10/TLR4 axis in living organisms to provide more solid evidence for the results of this study. Although Bai can regulate cell function at a dose range of 20–60 µM, in vitro experiments may require higher initial concentrations due to the lack of pharmacokinetic processes such as tissue accumulation and prolonged exposure time. In addition, nanoformulations (such as liposomal baicalein) may increase bioavailability, potentially bridging the gap between in vitro and in vivo. Therefore, in future animal experiments, concentration gradients need to be developed to screen for effective concentrations of Bai, and the use of nanoformulations can be considered to optimize Bai dosing regimens.

In conclusion, our study reveals a potential molecular mechanism by which Bai regulates TB progression. This research showed that Bai inhibited M.tb-induced macrophage M1 polarization, apoptosis and inflammation by inactivating the TLR4/MYD88/NF-κB pathway through inhibiting YY1/RAB10 axis. The molecular mechanism of Bai against TB provides new ideas for TB treatment, which has important clinical significance.

Authors’ contributions

Q.Z. and S.C. conducted the experiments and drafted the manuscript. L.S. and T.Y. prepared figures, collected and analyzed the data. Q.W. contributed the methodology. Y.O. and L.Q. operated the software and edited the manuscript. X.H. designed and supervised the study. All authors reviewed the manuscript.

Funding

None.

Data availability

The data are available from the corresponding author on reasonable request.

Declarations

Ethical approval and consent to participate

Our study was approved by the Ethics Committee of Zhuzhou Hospital Affiliated to Xiangya School of Medicine, Central South University, and each participate signed written informed consent.

Competing interests

The authors declare no competing interests.

Consent for publication

Not applicable.