To investigate the mechanism of anti-inflammatory and anti-adhesive effects of pelvic inflammatory granules on chronic pelvic inflammatory disease in rats caused by mixed bacteria plus mechanical stimulation. METHODS: The rat chronic pelvic inflammatory disease model was replicated by mixed bacterial plus mechanical stimulation method, and the rats were randomly divided into normal group, model group, sham-operated group, pelvic inflammatory net group and pelvic inflammatory granules high, medium and low dose groups, and the concentrations of serum IL-1β, IL-10, IL-8 and MCP-1 in each group were measured by enzyme-linked immunosorbent assay.
RESULTS: Pelvic inflammatory pellets significantly reduced the serum IL-1β, IL-8 and MCP-1 levels and significantly increased the serum IL-10 levels in the model rats, and the levels in the high and medium dose groups were close to those in the normal group. Conclusion: The mechanism of anti-inflammatory and anti-adhesive effects of pelvic inflammatory granules on model rats may be to reduce the levels of serum pro-inflammatory factors and chemokines, while increasing the levels of anti-inflammatory factors, thus restoring the balance of inflammatory factors and reducing local inflammatory reactions and fibroplasia.
Pelvic inflammatory granules; chronic pelvic inflammatory disease; inflammatory cytokines; chemokines
Chronic pelvic inflammatory disease is a common and frequent disease in gynecology, characterized by abdominal pain, hypochondrium, pelvic tissue thickening, adhesions and mass formation, which often recurs and is difficult to be cured. In this study, we observed the effects of pelvic inflammatory granules on serum inflammatory cytokines and chemokines in rats, based on the previous experimental study, in order to investigate some of the mechanisms of the anti-inflammatory and anti-adhesive effects of blood circulation and kidney tonic method.
1. Materials and methods
1. 1 Experimental animals
Seventy healthy female uncrossed SD rats. Body weight 180~220g, provided by the Animal Experiment Center of Shandong University, license number: SCXK LU20030004.
1. 2 Experimental drugs
Pelvic inflammatory granules (Salvia miltiorrhiza, Radix Paeoniae, Semen Cuscutae, Phellodendron sinensis, fried Wu Ling Li, forsythia, etc.) were provided by the Preparation Department of the Affiliated Hospital of Shandong University of Traditional Chinese Medicine; Pelvic inflammatory granules (Lonicera japonica, Gynostemma lucidum, Yimouzuo, Chuanxiong, Dog’s spine, etc.) were produced by Kunming Jieda Pharmaceutical Co. No. 003301.
1. 3 Experimental reagents
Strain: Escherichia coli, Staphylococcus aureus and Streptococcus hemolyticus B were dissolved in sterile saline in the ratio of 2:1:1 to form a mixed bacterial suspension with a concentration of 15×109/mL, which was provided by the strain room of Shandong Provincial Health and Epidemic Prevention Station. Interleukin-1β (IL-1β), interleukin-8 (IL-8), interleukin-10 (IL-10) and monocyte chemotactic protein-1 (MCP-1) enzyme immunization kits were purchased from ADL, USA.
1. 4 Experimental methods
1. 4. 1 Model replication Rats were acclimatized for 3 days and then molded by mixed bacterial injection on day 4. After the rats were anesthetized by intraperitoneal injection of sodium pentobarbital (concentration 0.4%) (50 mg/kg), an incision of about 0.8-1 cm was made in the lower abdomen under aseptic conditions, and the uterus was exposed and fixed after opening. 0.2 mL of mixed bacterial suspension was extracted with a 1 mL syringe, and the endometrial tissue was mechanically damaged with the syringe before injecting the bacterial solution, and then the bacterial solution was injected into the uterus bilaterally, and the incision was sutured. In the sham-operated group, the abdomen was opened after anesthesia, and 0.1 mL of sterile injection water was injected into each side of the uterus, and then the abdomen was closed.
1. 4. 2 Grouping and drug administration Eight rats were randomly selected from 65 rats as the normal group, and the remaining 52 rats participated in the modeling, among which eight rats were randomly selected as the sham-operated group. The remaining rats were kept for 14 days after modeling, and were randomly divided into model group, pelvic inflammatory disease high dose group, pelvic inflammatory disease medium dose group, pelvic inflammatory disease low dose group and pelvic inflammatory disease net group.
After 2 weeks of rearing after modeling, the drug administration was started. The treatment group was given pelvic inflammatory pellet preparation (100mg/mL), 2g/kg for rats in the high-dose group, 1g/kg for rats in the medium-dose group and 0.5g/kg for rats in the low-dose group; the pelvic inflammatory net group was given pelvic inflammatory net pellet preparation (54mg/mL) by gavage, 540mg/kg for each rat; the model group was given 2mL of saline by gavage, and the normal group was kept normally without any treatment. The normal group was kept normally without any treatment. The rats were administered once a day for 14 days.
After 24h of the last dose, the animals were fixed and 3~4mL of blood was taken from the heart, centrifuged (3000r/min) for 10min and the supernatant was separated and stored at -30℃. The concentrations of cytokines IL-1, IL-8, IL-10 and MCP-1 in serum were determined by enzyme-linked immunosorbent assay in strict accordance with the operating instructions of the kit. The uterus was removed from both sides and fixed in 10% formalin fixative, and the histopathological changes of the uterus were observed under conventional sectioning, HE staining and light microscopy.
1. 4. 4 Statistical methods The results were statistically analyzed by SPSS 16. 0 software with one-way ANOVA, and q test was used for comparison between groups.
2. Results
2. 1 Effect of pelvic inflammatory pellets on the serum IL-1βIL-10 level of model rats
The experimental results showed that the level of IL-1β increased significantly (P<0. 01) and the level of IL-10 decreased significantly (P<0. 01) in the model rats compared with the normal group. All dose groups of pelvic inflammatory pellets could reduce IL-1β level and increase IL-10 level to different degrees, and the medium and high dose groups were close to the normal level (P>0. 05). The IL-1β level was significantly lower in the pelvic inflammatory granules high-dose group compared with the pelvic inflammatory net granules group (P<0. 05). See Table 1.
Table 1 Effect of pelvic inflammatory pellets on serum IL-1β IL-10 levels in model rats (pg/mL,
±s)
Group
n
IL-1β
IL-10
Normal control group
8
35.73±4.43??
42.43±4.48??
Model group
8
45.88±4.93※※
27.35±2.99※※※
Sham surgery group
8
36.43±3.74?
41.23±4.68?
Pelvic inflammatory net group
8
41.01±3.69? The pelvic inflammatory drug group
36.75±3.87?
Pelvic inflammatory disease high dose group
9
36.49±3.09? ? △
39.32±4.00?
Pelvic inflammatory disease medium dose group
9
38.82±2.44? ?
39.97±3.81?
Pelvic inflammatory disease low dose group
9
40.58±3.61?※
35.71±3.72※※?
Note: Compared with the normal control group,*P<0. 05,**P<0. 01; compared with the model group,*P<0. 05,*? P<0. 01; compared with pelvic inflammatory net group, △P<0. 05, △△P<0. 01; the same below.
2.2 Effect of pelvic inflammatory pellets on serum IL-8MCP-1 level of model rats
The experimental results showed that the levels of IL-8 and MCP-1 in the model rats were significantly higher than those in the normal group (P<0. 01), and all dose groups of pelvic inflammatory granules could reduce the levels of IL-8 and MCP-1 to different degrees, while the medium and large dose groups were close to the normal level (P>0. 05), and the degree of effect was dose-related. The levels of IL-8 and MCP-1 in the medium and high dose groups of pelvic inflammatory granules were significantly lower than those in the pelvic inflammatory granules group (P<0. 05 or P<0. 01). See Table 2.
Table 2 Effect of pelvic inflammatory granules on serum IL-8 MCP-1 levels in model rats (pg/mL.
±s)
Group
n
IL-8
MCP-1
Normal control group
8
22.47±1.96? ?
19.04±1.89??
Model group
8
31.50±2.67※※※
26.05±2.27※※※
Sham surgery group
8
22.57±2.98?
18.08±1.82?
Pelvic inflammatory net group
8
25.87±2.45? The group of pelvic inflammatory purification 8 25.87±2.45?
22.90±2.02? ※※
Pelvic inflammatory disease high dose group
9
21.14±2.96? ? △△
20.66±1.66? ? △△
Pelvic inflammatory medium dose group
9
23.22±2.11? ? △
21.63±1.68?
Pelvic inflammatory disease low dose group
9
27.00±3.32※※? ?
24.37±2.07※※?
Note: Compared with the normal control group,*P<0. 05,**P<0. 01; compared with the model group,?P<0. 05,? P<0. 01; compared with pelvic inflammatory net group, △P<0. 05, △△P<0. 01; the same below.
3. Discussion
It has been suggested that some chronic pelvic inflammatory disease has no pathogens in the chronic development process, and the pathological changes in the chronic stage are caused by the immune response following bacterial infection, which is related to abnormal changes in immune factors such as relevant cytokines, inflammatory mediators, free radicals and apoptotic cells. Pro-inflammatory cytokines such as interleukin-1, IL-8, tumor necrosis factor-α, etc., and anti-inflammatory cytokines such as interleukin-4, IL-10, transforming growth factor-β, etc., can be classified into two major categories according to their functions. Pro-inflammatory cytokines can promote the development of inflammation, while anti-inflammatory cytokines can inhibit the inflammatory response and promote the repair and regeneration of tissues. The balance of these two factors will influence the development and outcome of tissue trauma and inflammation.
IL-1 is a monoclonal factor, one of the main cytokines secreted by macrophages, and one of the inflammation initiators. IL-1β can further promote the release of inflammatory factors by activating neutrophils and endothelial cells, and produce metabolites such as oxygen radicals, proteases, and arachidonic acid, resulting in a cascade waterfall effect that causes damage to endothelial cells.
Monocyte chemotactic protein-1 (MCP-1) and IL-8 are both chemokines and pro-inflammatory factors. It promotes the release of lysosomes and superoxide negative particles, leukotrienes and 5hydroxytryptamine from neutrophils, and enhances the travel ability of monocytes by increasing the expression of adhesion molecules; at the same time, IL-8 is a pro-inflammatory factor, and neutrophils change their morphology after contact with IL-8, and then travel to the reaction site and release a series of active products, leading to local inflammation. IL-10 is a very important class of neutrophils.
IL-10 is a very important class of endogenous anti-inflammatory factors, mainly produced by T cells, and is a key member of the negative feedback regulatory loop of inflammation, which has the effect of reducing the over-activated state of inflammatory cells, and is activated in parallel with the activation of pro-inflammatory factors, and widely inhibits the synthesis of various pro-inflammatory cytokines such as TNF-a, IL-1β and IL-6 [4]. inhibit monocyte-dependent Th cell proliferation and suppress the synthesis and activity of Th1-like cytokines, especially the production of interferon (IFN-γ). It has been found that IL-10 down-regulates the expression of inflammatory factors by inhibiting the activity of IκB kinase and impairing the ability of nuclear factor-κB (NF-κB) to bind to DNA-specific sites.
IL-1 has a wide range of effects on angiogenic activity of endothelial cells, proliferation of fibroblasts and collagen synthesis; IL-1 is released after trauma and binds to receptors to promote proliferation of epidermal cells, fibroblasts and collagen synthesis by autocrine and paracrine means. il-8 regulates endometrial stromal cells Mulayim et al. showed that IL-8 increased the activity of MMPs in endometrial stromal cells (ESCs) cultured in vitro and increased their invasive capacity.
IL-8 also induced the adhesion of endometrial cells to fibronectin, which in turn induced the expression of IL-8, and these two promoted each other to further promote the development of abdominal inflammation. The mechanism of IL-l0 inhibiting the progression of fibrosis may be related to the fact that IL-10 deficiency can upregulate type I collagen synthesis and promote collagenase gene expression to remodel the extracellular matrix, and also prevent the production of pro-fibrotic factor TGF-β from being effectively inhibited.
Pelvic inflammatory granule is an effective drug for the treatment of chronic pelvic inflammatory disease. According to the theory of Chinese medicine, “long-standing disease has many stasis” and “long-standing disease has kidney”, it is used to activate blood circulation and remove blood stasis, tonify kidney and cultivate kidney. Previous studies have shown that this drug can improve humoral immunity and cellular immunity of patients.
The results of this experiment showed that the levels of IL-1β, IL-8 and MCP-1 in rats with pelvic inflammatory disease were significantly higher than those in the normal group, and the levels of IL-10 were significantly lower than those in the normal group, indicating that the balance between pro-inflammatory factors and anti-inflammatory factors was disrupted in the pathological process of chronic pelvic inflammatory disease, and the excessive production of pro-inflammatory factors including chemokines and the lack of counteracting anti-inflammatory factors led to the development of inflammation causing tissue damage and fibroplasia. In contrast, the dose of pelvic inflammatory granules reduced the levels of pro-inflammatory factors and chemokines to different degrees, and increased the level of anti-inflammatory factor IL-10, and brought the inflammatory factors close to normal levels, i.e. restoring the balance between pro-inflammatory factors and anti-inflammatory factors, thus reducing the local inflammatory response and fibroplasia.
It is thus inferred that pelvic inflammatory granules may exert their anti-inflammatory and anti-adhesive effects by restoring the balance of the imbalanced inflammatory factor network, which may also be part of the mechanism of action of the blood activation and kidney tonic method in the treatment of this disease.