To investigate the mechanism of anti-inflammatory effect of pelvic inflammatory granules on chronic pelvic inflammatory disease in rats caused by mixed bacteria plus mechanical injury. METHODS: The rat chronic pelvic inflammatory disease model was replicated by mixed bacteria plus mechanical injury method, and randomly divided into normal group, sham-operated group, model group, pelvic inflammatory net group and pelvic inflammatory granules high, medium and low dose groups. 2 weeks after modeling, each treatment group was administered by gavage for 2 weeks, and the serum IL-4 and IL-6 concentrations of each group were measured by enzyme-linked immunosorbent assay. RESULTS: Pelvic inflammatory granules could significantly reduce the serum IL-6 level and significantly increase the serum IL-4 level in the model rats.
CONCLUSION: The mechanism of anti-inflammatory effect of pelvic inflammatory granules on model rats may be to reduce serum pro-inflammatory factor levels and simultaneously increase anti-inflammatory factor levels, thus restoring inflammatory factor balance and reducing local inflammatory response and fibroplasia.
Chronic pelvic inflammatory disease; pelvic inflammatory granules; inflammatory cytokines
Chronic pelvic inflammatory disease is a common and frequent disease in gynecology, mainly characterized by abdominal pain, hypochondrium, pelvic tissue thickening, adhesions and mass formation. The prevention and treatment of chronic pelvic inflammatory disease has become an important issue to be solved in the field of gynecology because it is often recurrent and difficult to be cured. In this study, we observed the effect of pelvic inflammatory granules on serum inflammatory cytokines in rats based on the previous experimental study, so as to investigate the mechanism of anti-inflammatory effect of blood activation and kidney tonic method.
1. Materials and methods
1.1 Experimental animals
Sixty-five female Wistar rats, weighing 200±20g, were purchased from the Experimental Animal Center of Shandong University of Traditional Chinese Medicine, animal certificate number: SCXK(LU)20050015.
1.2 Experimental drugs
Pelvic inflammatory granules: provided by the preparation room of the Affiliated Hospital of Shandong University of Traditional Chinese Medicine, the drug composition is Salvia miltiorrhiza, Paeonia lactiflora, raw Puhuang, fried Lingliao, Cuscuta sinensis, Cornu Cervi Pantotrichum, forsythiae, and Aromatic herb. Pelvic inflammatory pellets: produced by Kunming Jieda Pharmaceutical Co., Ltd, with the approval number: State Drug Registration No. Z20013124. The main ingredients are Lonicera japonica, Gynostemma lucidum, Yimouzuo, Chuanxiong, Dog’s spine, etc. The adult dosage of pelvic inflammatory granules and pelvic inflammatory net granules are both 30g/day, prepared as a suspension with distilled water, containing 325mg of drug per ml.
1.3 Experimental reagents
Interleukin-4 (IL-4) and interleukin-6 (IL-6) ELISA test kits, produced by ADL, USA; strains: Escherichia coli, Staphylococcus aureus, Streptococcus hemolyticus type B in 2:1:1 dissolved in sterile saline to make a mixed bacterial suspension of 3 billion/ml, provided by the strain room of Shandong Province Health Prevention Station.
1.4 Test method
1.4.1 Model replication method [After abdominal anesthesia with 3% sodium pentobarbital (40mg/kg), under aseptic conditions, take a lower abdominal median incision, 1cm long, open the abdomen to expose and fix the uterus, extract 0.2ml of mixed bacterial suspension with a 1ml syringe, mechanically damage the endometrial tissue with a syringe, then inject 0.1mL of bacterial solution into the bilateral uterus respectively, and suture the incision. In the sham-operated group, the abdomen was opened after anesthesia, and 0.1mL 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 method Eight rats were randomly selected from 65 rats as the normal control group, eight rats as the sham-operated group, and the remaining 49 rats participated in the modeling. Six rats died during the modeling process due to anesthesia overdose, etc. The remaining rats were reared for 14 days after modeling and randomly divided into 8 rats each in the pelvic inflammatory pellets high-dose group, pelvic inflammatory pellets medium-dose group, pelvic inflammatory pellets small-dose group, pelvic inflammatory pellets group and model group. After modeling, they were kept for 2 weeks and the drug administration was started.
In the pelvic inflammatory granules large, medium and small dose groups, 6.48g/kg/day, 3.24g/kg/day and 1.62g/kg/day pelvic inflammatory granules prepared solution was given by gavage respectively; in the pelvic inflammatory net group, 3.24g/kg/day pelvic inflammatory net granules prepared solution was given by gavage; in the control group, model group and sham operation group, 1ml/100g/day distilled water was given by gavage without special treatment. The drug was administered once a day for 14 days.
1.4.3 Indicator detection The rats in each group were fasted in the early morning of the 15th day after drug administration, and 4-5 ml of blood was taken from the heart by fixing the animals in a test tube with anticoagulant. The supernatant was separated by centrifugation (3000 r/m in) for 10 min and stored at -30℃ for the detection of immunological parameters by enzyme-linked immunosorbent assay (ELISA).
1.4.4 Statistical methods Statistical software SPSS17.0 was used, and the measurement data were expressed in the form of mean ± standard deviation ( ± s), and t-test was used, and ANOVA was used for comparison between multiple groups, with P<0.05 as significant difference and P<0.01 as very significant difference.
2. Results
2.1 The results of serum IL-6 concentration detection in rats in each group
The experimental results showed that the serum concentration of IL-6 in rats with pelvic inflammatory disease model was significantly higher than that in normal rats (P<0.01), while there was no significant difference between the sham-operated rats and the normal control group (P>0.05). The concentrations of pelvic inflammatory pellets and pelvic inflammatory pellets in all dose groups could be significantly reduced (P<0.01), among which the effect of pelvic inflammatory pellets in medium and large dose groups and pelvic inflammatory pellets group was the most significant, and the concentration was close to the normal level (P>0.05), and the small dose group of pelvic inflammatory pellets was significantly higher than that of pelvic inflammatory pellets group (P<0.01).
Table 1 Expression of serum interleukin-6 (IL-6) in rats in each group ( ±s)
Group
n
Interleukin-6(ng/L)
Normal group
8
118.23±10.82||
Sham surgery group
8
122.23±23.22||
Model group
8
205.08±24.67※※▲▲
Pelvic inflammatory granules high dose group
8
126.91±11.94||
Pelvic inflammatory granules medium dose group
8
131.99±14.82||
Pelvic inflammatory granules small dose group
8
177.34±9.98※※||▲▲
Pelvic inflammatory net group
8
130.88±13.29||
Note: Compared with the normal group, ※P<0.05, ※※P<0.01; compared with the model group, |P<0.05, ||P<0.01; compared with the pelvic inflammatory net group, ▲P<0.05, ▲▲P<0.01; the same below.
2.1 The results of serum IL-4 concentration detection in each group of rats (see Table 2)
The experimental results showed that the serum IL-4 level of pelvic inflammatory model rats was significantly lower than that of normal rats (P<0.01), while the rats in the sham-operated group were not significantly different from the normal control group (P>0.05). The levels of pelvic inflammatory granules were significantly increased in all dose groups and pelvic inflammatory net group (P<0.01), and the levels of pelvic inflammatory granules in high dose group were close to normal (P>0.05), and the levels of pelvic inflammatory granules in medium and low dose groups were close to those of pelvic inflammatory net group (P>0.05).
Table 2 Serum interleukin-4 (IL-4) concentration of rats in each group ( ±s)
Group
n
Interleukin-4 (ng/L)
Normal group
8
130.62±21.14||▲▲▲
Sham surgery group
8
124.70±18.48||▲▲
Model group
8
55.42±10.99※※▲▲▲
Pelvic inflammatory granules high dose group
8
130.19±11.43||▲▲
Pelvic inflammatory granules medium dose group
8
89.07±16.64※※||
Pelvic inflammatory granules small dose group
8
78.61±12.15※※||
Pelvic inflammatory net group
8
83.71±10.63※※||
3.Discussion
It has been suggested that some chronic pelvic inflammatory diseases have become pathogen-free during the chronic development and that the pathological changes in the chronic phase are the result of immune responses secondary to bacterial infection [2] and are associated with abnormal changes in immune factors such as relevant cytokines, inflammatory mediators, free radicals and apoptosis. Inflammatory cytokines can be classified into two major groups: pro-inflammatory cytokines such as IL-6, IL-1, IL-8, IL-12, and tumor necrosis factor (TNF) and anti-inflammatory cytokines such as IL-4, IL-10, IL-13, etc. Pro-inflammatory cytokines promote the development of inflammation, while anti-inflammatory cytokines inhibit the inflammatory response and promote tissue repair and regeneration. The balance of the two will influence the outcome of tissue trauma and inflammation.
IL-6 is a cytokine with inflammation-mediating activity and is an important inflammatory factor in the initiation phase of inflammation. It promotes neutrophil production and activation, B-cell proliferation, differentiation and immunoglobulin production, and T-cell proliferation and differentiation during infection by inducing the synthesis and secretion of various acute phase proteins by various cells. In the chronic inflammatory phase, IL-6 acts as a growth and maturation factor for lymphocytes to cause local tissue immune dysregulation and further mediates tissue damage and fibrosis, while IL-4 activates cytotoxic T cells, a cytokine typically produced by Th2 cells, and is important for the development of T and B lymphocytes and for driving humoral immune responses and antibody production.
Chronic pelvic inflammatory disease often recurs and is difficult to be cured. According to the theory of Chinese medicine that “chronic diseases have more stasis” and “chronic diseases and kidney”, we have created pelvic inflammatory granules by activating blood stasis and tonifying the kidney to cultivate the yuan, which are clinically used to treat chronic pelvic inflammatory disease of blood stasis and kidney deficiency with remarkable efficacy. It has been shown that this drug can improve the humoral immunity and cellular immunity of patients. It has been proved that pelvic inflammatory granules can significantly reduce serum IL-1B, IL-8 and MCP-1 levels and significantly increase serum IL-10 levels in model rats, suggesting that the mechanism of anti-inflammatory and anti-adhesive effects of pelvic inflammatory granules on model rats may be to reduce serum pro-inflammatory factors and chemokines levels, while increasing anti-inflammatory factors levels, thus restoring the balance of inflammatory factors and reducing local inflammatory response and fibroplasia. Fibroplasia.
The results of this experiment showed that the level of IL-6 in rats with pelvic inflammatory disease was significantly higher than that in the normal group, and the level of IL-4 was significantly lower than that 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 and the lack of counteracting anti-inflammatory factors led to the development of inflammation causing tissue damage and fibroplasia. In contrast, pelvic inflammatory pellets reduced the level of pro-inflammatory factor IL-6 and increased the level of anti-inflammatory factor IL-4 to different degrees, which led to the dynamic balance of inflammation/anti-inflammation in the direction of disappearance of inflammation and reduction of pathological changes, thus promoting the healing of inflammation. This may be another evidence that pelvic inflammatory granules exert their anti-inflammatory effect by restoring the balance of the imbalanced inflammatory factor network, which is part of the mechanism of action of the blood activation and kidney tonic method in the treatment of this disease.