The anti-inflammatory effect of Andrographis paniculata (Burm. f.) Nees on pelvic inflammatory disease in rats through down-regulation of the NF-κB pathway

Pelvic inflammatory disease (PID) is a common gynecological disease that usually causes ectopic pregnancy, tubal factor infertility and chronic pelvic pain, which has been deemed as a great threat for life quality of woman. It includes endometritis, salpingitis, peritonitis, etc., whose etiopathogenisis is the infection of pathogenic microorganisms in upper genital tract [1]. Infiltrations of neutrophils and lymphocytes in the upper genital tract could be employed as a criteria to diagnose PID in clinic, which could be observed on hematoxylin and eosin (H & E)-stained biopsy sections [2]. With the inflammatory cell recruitment, amount of proinflammatory cytokines, such as interleukin (IL)-1β, IL-6, etc., are released excessively in local tissue and play an important role in the pathogenesis of PID [3]. The important reason for the excessive production and release of proinflammatory cytokines is the activation of nuclear factor-kappa B (NF-κB) signaling pathway when pathogens are recognized by their receptors [4].

According to the guidelines from the Center for Disease Control and Prevention (CDC) in US, antibiotics is the first choice for the treatment of PID [5], but the bacterial drug resistance and drug side effects are shortages for clinical use of antibiotics. Therefore, new complementary medicines used for PID are needed for further improvement in clinical outcomes. Andrographis paniculata (Burm. f.) Nees (APN) is a famous traditional medicine, widely used to treat sore throat, flu, and upper respiratory tract infections in many Asian countries [6]. Phytochemical studies on APN have found the principal bioactive compound andrographolide [7] and many other constituents, including diterpenoids, flavonoids, quinic acids, xanthones [8, 9], and noriridoids [10, 11]. Based on the abundant bioactive constituents, APN showed many salutary effects, such as anticancer [12], hepatoprotective [13], antiviral [14, 15], antipyretic and analgesic [16] effects, etc. Interestingly, the immunomodulatory [17], antioxidant [18], anti-inflammatory [19] and antimicrobial [20, 21] activities of APN were also reported, which may suggest the clinical use of APN in treatment for PID. Besides, APN is the principal constituent in a famous traditional Chinese medicine Fukeqianjin tablet which is used to treat PID. However, whether APN has pharmacological effect on PID has not been eliminated yet. In this study, we test the anti-inflammatory effect of APN on PID rats, and illustrate a potential mechanism of this activity.

In clinic, the common pathogens, whose infections in upper genital tract can lead to PID, include Chlamydia trachomatis, Neisseria gonorrhoeae, genital mycoplasmas, some gram-negative and gram-positive bacteria, etc. [26‐29]. Each pathogen could be recognized by one or more members of Toll-like receptor (TLR) family and then initiate inflammation. TLR2 and TLR4 are two important members of TLR family in upper genital tract [30], and U. urealyticum and E. coli can be recognized by TLR2 and TLR4, respectively [31, 32]. Therefore, we attempted to use U. urealyticum and E. coli mixed solution to provoke an augmented inflammation in upper genital tract.

Neutrophils in blood circulation could firmly adhere to the endothelium cell barrier, cross it, and are recruited into sites of inflammation in different pathogen-infected tissues, which is the first line of innate immune defense against pathogens [33]. Recently, study also showed the infiltration of neutrophils into the endometrium of mice with lipopolysaccharides (LPS)-induced endometritis [34]. When mass neutrophils reach the tissue at the site of infection, they will release abundant inflammatory factors, oxygen free radical and proteolytic enzyme to kill pathogens. However, the excessive products will also cause tissue damage and lead to structural disease in upper genital tract. Infiltration of lymphocyte in genital tract of mice were observed after chronic pathogen genital infection, and this infiltration play a significant role in controlling the infection [35]. Additionally, researchers found more T lymphocyte and fewer plasma cells throughout the stroma and within the epithelium [36]. Unfortunately, Patton et al. reported that the tissue damage including epithelial cell degeneration occurred close approximation to lymphocytes [37]. In the present study, after chronic pathogen infections, large number of neutrophils and lymphocytes infiltrated into the epithelium of upper genital tract, and APN showed a good activity in attenuating the infiltration of these inflammatory cells, avoiding tissue damages which is due to the inflammation.

The proinflammatory cytokines, such as IL-1β and IL-6, play essential roles on the initiation and propagation of inflammatory response, whose level in upper genital tract were increased with the pathogen infection and the recognition of immunogens by local TLRs [38‐40]. At the site of inflammation, these proinflammatory cytokines stimulate the proliferation and activation of leukocyte, and enhance the production of chemokines (i.e., CXCL-1, MCP-1, and RANTES) leading to the recruitment of hematopoietic immune cells. Then, activated neutrophils will release inflammatory cytokines and chemokines, and further intensify the inflammatory response [41]. At the same time, various proinflammatory cytokines will enhance the survival or function of neutrophil [42]. In this case, the tissue damage and structural disease in upper genital tract may occur due to the intensified inflammatory response. In the present study, elevated productions of cytokines and chemokines including IL-1β, IL-6, CXCL-1, MCP-1 and RANTES were observed in upper genital tract of PID rats, indicating an obvious local inflammatory response. Besides, APN could significantly lower the levels of these cytokines and chemokines in a dose-dependent manner, suggesting a potent anti-inflammatory effect of APN for PID. As andrographolide is the major bioactive substance and has a critical effect on inhibiting the release of an important proinflammatory factor TNF-α in APN [43], it may also contribute importantly to suppressing the inflammatory response in this study.

NF-κB is a pivotal factor in promoting the transcription of genes involved in inflammatory and immune responses [44]. In most resting cells, NF-κB family members are covalently bound to IκB family members and located in the cytoplasm with no activity [45]. When the TLRs on surface of these cells recognize pathogens, resulting in the phosphorylation and degradation of IκB members, the NF-κB members will translocate to the nucleus and bind to the cis-acting NF-κB enhancer element of genes, promoting the expression of inflammatory mediators, such as IL-1β, IL-6, etc. [31]. These produced proinflammatory cytokines act as a positive autocrine feedback so as to the further activation of NF-κB, and subsequently more proinflammatory mediators are produced [46]. NF-κB p65 and IκB-α are representative members of NF-κB and IκB family in uterus and fallopian tube, respectively [47], and therefore were chosen as indexes to test whether the NF-κB pathway was activated in the present study. Our results demonstrated the activation of NF-κB signal pathway after multi-infection of pathogens in uterus. In the rat PID model, APN exerted its anti-inflammatory with a potential mechanism of blocking the activation of NF-κB pathway. Previous studies reporting that both of APN and its main substance andrographolide showed the effect on inhibiting NF-κB pathway in vitro support this result [48, 49]. On the other hand, andrographolide also showed the other anti-inflammatory effects, such as inhibiting JAK/STAT signaling [49], inhibiting p38 MAPK pathway [50], suppressing TLRs family expressions [49], etc., and whether APN have the same or more effects in upper genital tract should be verified in the further studies to facilitate its clinical use.

In this study, oral administration of APN showed significant anti-inflammatory activity in pathogen-induced PID rats, including suppressing the infiltration of neutrophils and lymphocytes and reducing excessive production of cytokines or chemokines. A potential mechanism of this effect was involved in inhibiting the activation of NF-κB pathway.