The inhibition of deep peritoneal endometriosis by Andrographolide through macrophage M1 activity in an endometriosis mice model

By day 14, ectopic lesions presented as hyperaemic, thin-walled, vascularised nodules on the peritoneum. The mean lesion area (mm 2 ) differed across the groups (overall P  < 0.001), with the smallest mean area in P3 and larger areas in KN/KP (exact group means ± SD shown in  Figure 1 ). Post-hoc testing confirmed P3 < KN ( P  = 0.002). Endometriosis is macroscopically evident on the peritoneum, appearing pink with reddish patches, thin-walled cysts infiltrated by blood vessels, and attached to the peritoneum. Andrographolide is a natural compound with anti-inflammatory properties that can modulate M1 and M2 macrophages, components of the innate immune system that can alter their phenotype in response to stimuli. There are two types of macrophages: M1 macrophages are pro-inflammatory and release cytokines to combat pathogens and inhibit tumour growth, while M2 macrophages, being anti-inflammatory, produce cytokines that assist in tissue repair and promote tumour growth. According to calculations using Motic Image software, the endometriosis area in the P3 group is smaller than that in the KP group, measuring 7.91 mm 2 . The statistical test revealed a significant difference in the area of peritoneal endometriosis between the study groups overall ( P  < 0.001). Table 1 demonstrates the M1 CD86 and M2 CD163 macrophage percentage proportions in the BALB/c mice peritoneal fluid in the research group ( n  = 42). In the flow cytometry procedure analysing peritoneal macrophages, the percentages of the M1 (CD86 + ) and M2 (CD163 + ) populations do not necessarily add up to 100%, because macrophages can exist in different activation states, including an M0 (resting) state, and some markers may be expressed on multiple populations. 100% refers to all the gated macrophages, not all the cells in the peritoneal cavity fluid. The area of peritoneal endometriosis in the P3 group is smaller than in the negative control group ( P  = 0.002). The comparison of the peritoneal endometriosis area in each BALB/c mice group is shown in Figure 2 . Lesion area quantified with Motic Image; circles delineate ROIs of each group (N, KN, KP, P1, P2, and P3) KN = endometriosis without therapy group; KP = endometriosis + dienogest group; N = normal group; P1 = endometriosis with 0.05 mg/kg Andrographolide group; P2 = endometriosis + 0.1 mg/kg Andrographolide group; P3 = endometriosis + 0.2 mg/kg Andrographolide group Values are mean ± SD and median (min–max); Percentages need not sum to 100% due to resting/other phenotypes and marker overlap ( n  = 42) KN = endometriosis without therapy group; KP = endometriosis + dienogest group; N = normal group; P1 = endometriosis with 0.05 mg/kg Andrographolide group; P2 = endometriosis + 0.1 mg/kg Andrographolide group; P3 = endometriosis + 0.2 mg/kg Andrographolide group The results in  Table 1 from flow cytometry examinations of the peritoneal fluid samples show that the highest percentage of M1 CD86 macrophages was in group P2 (80.02 ± 4.423%), while the lowest was in group P3 (20.42 ± 1.864%). The number of M2 CD163 macrophages in the peritoneal fluid of mice was greatest in the negative control group at 67.67 ± 3.269%, and lowest was in the P3 group at 15.48 ± 4.168%. The results of the statistical tests indicated that, overall, there were significant differences in the number of M2 CD163 macrophages in the peritoneal fluid between the study groups ( P  < 0.001). The number of M2 CD163 ( Figure 3 ) macrophages in the peritoneal fluid of the P1 group was significantly lower than that of the negative control group ( P  = 0.002). The statistical test results indicated a significant difference in the number of M1 CD86 macrophages in the peritoneal fluid between the study groups ( P  < 0.001; Kruskal–Wallis test). The intergroup comparison of M1 CD86 macrophage counts in the mice’s peritoneal fluid also revealed significance. Endometriosis lesions in the P3 group had a significantly higher M1/M2 ratio than in the KN group and were nearly identical to those in the KP group. The M1 CD86 macrophages were higher than the M2 CD163 macrophages in the andrographolide therapy group compared with the positive group ( Figure 4 ). Additionally, the number of M2 CD163 macrophages in the P2 group was markedly lower than in the negative control group ( P  = 0.002). Likewise, the number of M2 CD163 macrophages in the P3 group was significantly lower than in the negative control group ( P  = 0.002) ( Figure 5 ). KN = endometriosis without therapy group; N = normal group; KP = endometriosis + dienogest group; ns = not significant; P1 = endometriosis with 0.05 mg/kg Andrographolide group; P2 = endometriosis + 0.1 mg/kg Andrographolide group; P3 = endometriosis + 0.2 mg/kg Andrographolide group; s = significantly different; *extreme outlier; o mild outlier KN = endometriosis without therapy group; KP = endometriosis + dienogest group; N = normal group; P1 = endometriosis with 0.05 mg/kg Andrographolide group; P2 = endometriosis + 0.1 mg/kg Andrographolide group; P3 = endometriosis + 0.2 mg/kg Andrographolide group KN = endometriosis without therapy group; KP = endometriosis + dienogest group; N = normal group; ns = not significant; P1 = endometriosis with 0.05 mg/kg group; P2 = endometriosis + 0.1 mg/kg Andrographolide group; P3 = endometriosis + 0.2 mg/kg Andrographolide group; s = significant; *extreme outlier; o mild outlier

This experimental study employed a post-test-only design with a control group. A total of 42 female BALB/c mice, weighing between 20 and 30 g and aged 2 to 3 months, were obtained from the Animal Engineering Laboratory at the Faculty of Veterinary Medicine, Airlangga University in Surabaya, Indonesia that treated with dienogest as the standard therapy that served as the positive control group (KP) and andrographolide for the treatment groups (P1, P2, P3) were prepared before making a suspension from powder to liquid. The study’s methodology was approved by the Medical Faculty’s medical ethics committee at Diponegoro University (License number: 108/EC/KEPK/FK-UNDIP/XI/2024). After the mice acclimated for one week, cyclosporine A was injected at a dose of 0.2 cc per mouse to suppress the immune system, facilitating the growth of endometriosis implants in the peritoneal cavity. The origin of the endometriosis cells collected from human adenomyosis nodules, then the adenomyosis tissue was excised, minced into small fragments, and suspended in phosphate-buffered saline (PBS) prior to intraperitoneal injection. This syngeneic approach is widely used to establish an immunologically compatible endometriosis model in BALB/c mice by injecting endometrial cells into the peritoneal layer of the BALB/c mice’s abdomen. The mice were divided into six groups, each consisting of seven mice (Federer’s formula): group N (normal) with no treatment, the negative control group (KN) injected with endometrial cells, the positive control group (KP) injected with endometrial cells into the peritoneal layer of the mice abdomen and treated with dienogest (a synthetic progestogen) at a dose of 0.005 mg/kg body weight, treatment group 1 (P1) injected with endometrial cells into the peritoneal layer of the mice abdomen and treated with andrographolide at a dose of 0.05 mg/kg body weight, treatment group 2 (P2) injected with endometrial cells into the peritoneal layer of the mice abdomen and treated with andrographolide at a dose of 0.1 mg/kg body weight, and treatment group 3 (P3) injected with endometrial cells and treated with andrographolide at a dose of 0.2 mg/kg body weight. Additionally, the mice were injected intramuscularly in the thigh with ethinylestradiol at a dose of 0.2 μg per mouse using a 1 ml disposable syringe. On the 15 th  day, the mice were euthanised prior to dissection to determine the number of endometriosis implants within the peritoneum ( Figure 1 ). KN = endometriosis without therapy group; KP = endometriosis + dienogest group; N = normal group; P1 = endometriosis with 0.05 mg/kg Andrographolide group; P2 = endometriosis + 0.1 mg/kg Andrographolide group; P3 = endometriosis + 0.2 mg/kg Andrographolide group Before euthanising the mice, peritoneal lavage macrophages were isolated from the peritoneal cavity and processed to remove debris and non-cellular material. A peritoneal lavage was performed to collect macrophages using specific chemicals and fluorochromes for cell isolation and analysis with phosphate-buffered saline (PBS), followed by two centrifugations at 769  g . The isolation technique using centrifugation separates components of the lavage fluid based on density, without nylon mesh filtration, yielding a very clean peritoneal macrophage cell suspension. Different speeds and durations can be used to pellet cells, debris, or other particulate matter, enabling the collection of supernatant or pellet fractions. The abdominal wall and peritoneum were then detached, followed by cutting and stretching the peritoneum on millimetre paper, which was thoroughly documented to observe the extent of the endometriosis. An anatomical pathology examination was conducted on the reddest peritoneal tissue obtained during preparation, which was preserved in 10% formalin. The peritoneum was examined using a Nikon H600L microscope equipped with a 300-megapixel DS Fi2 digital camera. The area of endometriosis implants was macroscopically evaluated for hyperaemic regions. This study compared the formation of ectopic endometrial lesions in BALB/c mice treated with andrographolide at various therapeutic doses with those in the normal, negative control, and positive control groups that received standard therapy. Peritoneal fluid collection occurred 14 days after the intraperitoneal injection of the syngeneic endometrial tissue. M1 and M2 represent forms of mononuclear phagocytes that differentiate in vivo , characterised by distinct phenotypic patterns and functional properties influenced by the microenvironment and natural signals received by macrophages from the antibodies kit. Antibodies against CD86 and CD163 were used in their commercially available forms, each directly conjugated to fluorochromes for flow cytometry detection. After centrifuging the peritoneal fluid, we developed and optimised a colour polychromatic flow cytometric assay to identify the macrophage subtypes. CD86 (Biolegend) was used as a marker for the M1 macrophages, which were labelled with a red marker, while CD163 (Biolegend) acted as a marker for the M2 macrophages. After centrifugation of the peritoneal fluid, cell pellets were resuspended in phosphate-buffered saline (PBS) with a 2% foetal bovine serum (FBS). To distinguish the macrophage subtypes, we employed fluorochrome-conjugated primary antibodies from BioLegend. CD86 (clone GL1, PE-conjugated) was used as a marker of classically activated macrophages (M1), while CD163 (clone GHI/61, APC-conjugated) was used as a marker of alternatively activated macrophages (M2). Antibodies were used according to the manufacturer’s recommended dilutions (typically 1–2 μg per 10 6  cells in 100 μl). Cells were incubated with antibodies for 30 min at 4 °C in the dark, washed twice with PBS, and resuspended in the acquisition buffer. Compensation controls were established using single-stained samples to correct for spectral overlap. Isotype-matched controls were included to exclude non-specific binding. Flow cytometric acquisition was performed using BD FACSCalibur or BD FACSCanto II, and data were analysed with FlowJo/BD CellQuest Pro (v11, Germany) software. Gating was performed sequentially: (1) forward and side scatter to select live mononuclear cells, (2) CD45 + and F4/80 + gating to identify macrophages, and (3) within the macrophage population, analysis of the CD86 + (M1) and CD163 + (M2) subsets. Double-negative and double-positive populations were recorded, but not included in the M1/M2 percentages. Percentages were calculated relative to the total macrophage gate, not the total peritoneal cells. The implantation of endometriosis lesions was measured by calculating the area of red lesions on the peritoneal wall in mm 2 using Motic Image software, which specialises in area calculation. The data were tested for normality, which indicated a normal distribution, so a one-way analysis of variance (ANOVA) followed by a post hoc test was conducted to analyse the differences. All the calculations were performed using SPSS software (v19; IBM, USA).

In this BALB/c mouse peritoneal endometriosis model, andrographolide decreased the lesion size and shifted the macrophage balance away from the CD163 + M2 phenotype. The intermediate dose (P2) was associated with the highest M1 percentage, while the highest dose (P3) showed simultaneous reductions in both M1 and M2, with the smallest lesions. Overall, these findings support a dose-dependent immunomodulatory effect that inhibits pro-angiogenic M2 activity. Ono et al. (2021) stated that fractalkine (FKN), secreted by eutopic endometrial stromal cells, increases the IL-10 production and inhibits the IL-12 production, thereby inducing the M2 polarisation of macrophages that benefits endometriosis proliferation and invasion. Additionally, Lu et al. (2021) found that Smad2/Smad3 was upregulated in macrophages exposed to eutopic and ectopic endometrial homogenates from women with endometriosis, supporting the hypothesis of M1 to M2 macrophage polarisation via the Smad2/Smad3 pathway. Li et al. (2021) argued that M1 macrophages constitute the most abundant macrophage population in the endometrium of endometriosis patients. Macrophages are phagocytic cells of the immune system distributed across various tissues and play a crucial role in several diseases, including inflammatory disorders and tumour growth. Depending on their function, macrophages are broadly classified into M1 (classically activated) and M2 (alternatively activated). M1 macrophages are significantly involved in the pathogenesis of peritoneal endometriosis by increasing the pro-inflammatory cytokines to suppress chronic peritoneal inflammation associated with endometriosis ( Moni and Uddin 2018 ; Jeljeli et al. 2020 ; Liang et al. 2023 ). In contrast, M2 macrophages contribute to tumour growth and to diseases characterised by tissue remodelling in endometriosis through their regenerative capabilities. The macrophages present in endometriosis lesions form a heterogeneous population within the peritoneal fluid, triggering immune responses. Notably, some immune and inflammatory responses closely tied to angiogenesis in endometriosis include TNF-α and COX-2-Induced hypoxia-inducible factor 1α (HIF-1), which lead to extracellular matrix degradation and are critical components in the adhesion and angiogenesis associated with endometriosis ( Calmon et al. 2024 ; Peng et al. 2024 ; Quan et al. 2024 ; Wong et al. 2024 ). Andrographolide is the primary active component found in the herb sambiloto ( Andrographis paniculata ). This active ingredient exhibits several pharmacological activities, including immunosuppressant, antithrombotic, antiviral, antioxidant, and anti-inflammatory effects ( Lagana et al. 2020 ; Calmon et al. 2024 ; Peng et al. 2024 ; Quan et al. 2024 ; Wong et al. 2024 ). Andrographolide, an anti-inflammatory and antioxidant, works by increasing the expression of phosphoinositide 3-kinase (p-PI3K), protein kinase B (p-AKT), and endothelial nitric oxide synthase polymorphism (p-eNOS) enzymes that are inhibited by endothelial dysfunction. This mechanism suppresses the gene expression of interleukin 1β (IL-1β), interleukin 6 (IL-6), tumour necrosis factor alpha (TNF-α), vascular endothelial growth factor (VEGF), and tumour growth factor-α (TGF-α) ( Lagana et al. 2020 ; Calmon et al. 2024 ; Peng et al. 2024 ; Quan et al. 2024 ; Wong et al. 2024 ). Sambiloto ( Andrographis paniculata Nees.) is widely recognised for its pharmacological effects, including its anti-inflammatory properties. One class of compounds found in sambiloto is flavonoids, which help inhibit the inflammatory process, a normal response that protects tissue from injury caused by physical trauma, toxic chemicals, or microbial substances ( Lotfizadeh et al. 2020 ; Li et al. 2021 ; Mishra et al. 2021 ; Prasetyo et al. 2021 ). Our findings suggest that andrographolide functions as an immunomodulator rather than a unidirectional M1 agonist. The intermediate dose (P2) increased the proportion of M1, while the higher dose (P3) was associated with reductions in both M1 and M2 markers, with a disproportionately greater decrease in M2, which aligned with the smallest lesion area. Therefore, the therapeutic effect in this model seems to involve shifting away from pro-angiogenic M2 activity, rather than simply amplifying M1 at all doses. These dose-dependent effects imply that andrographolide adjusts macrophage activation states and might expand the non-polarised/resting (M0) pool at higher doses. Future research should include viability tests and additional surface or functional markers to better understand this mechanism. The limitation of this study is that it focuses on specific markers, such as CD86 and CD163 in peritoneal fluid, thereby neglecting other important factors contributing to macrophage polarisation and the pathogenesis of endometriosis in peritoneal tissue. This includes variations in cytokine levels and tumour marker expression that have not been thoroughly examined. Additionally, the study utilised BALB/c endometriosis mice that received only andrographolide therapy for 7 days. Andrographolide, known for its anti-inflammatory effects, indeed suppresses the M1 macrophage activation. However, claiming it “thus prefers M2 activation” is not accurate. Although M1 activation is reduced, its effect on the M2 activation is more complex and not clearly shown to promote M2. Additionally, while M2 macrophages are associated with certain angiogenic growth factors, this does not directly cause endometriosis development. Andrographolide may affect the M1/M2 macrophage ratio in endometriosis. This research indicates that andrographolide can shift the balance toward more M1 macrophages and fewer M2 macrophages, similar to the effects of dienogest, a common treatment for endometriosis. M1 macrophages in peritoneal fluid regulate environmental differentiation, acting as a precursor to the development of endometriosis in the peritoneum. The immunomodulatory effects of andrographolide are associated with a favourable M1/M2 ratio and smaller lesions, especially in the P2 and P3 groups. These findings are significant for several reasons. First, the activation of M1 macrophages highlights heterogeneity in endometriosis growth in the peritoneum. Deep endometriosis, triggered by environmental stimuli, exhibits established variations in immune responses that may result from complex individual reactions to endogenous alarm signals and infectious agents. Second, andrographolide can act as a local inhibitor of the reparative actions of M1 macrophage therapy in endometriosis and can serve as an alternative herbal treatment. This preliminary study, conducted with experimental animals, lays the foundation for future research on andrographolide as an alternative treatment for endometriosis. Andrographolide therapy in endometriosis can improve the regulation of M1 macrophage differentiation in peritoneal fluid, potentially inhibiting endometriosis growth by suppressing M2 phenotypes that promote vascularisation and disease progression.