Study On The Analgesia, Healing And Anti-inflammation Mechanisms Of Simiao Junyi Ointment Based On The Neuroendocrine-immunoregulatory Network

Objective: Previous study of this project has proved that Simiao Junyi Ointment has good effects of analgesia on postoperative dressings in anorectal diseases together with some healing and anti-inflammation effects. After dozens of tests, it becomes a kind of patent medicine. This study further explores the drug’s mechanism of action according to the multi-linked and multi-targeted efficacy of traditional Chinese medicine(TCM) compounds in order to invent some new TCM compounds different from chemicals. Method: Based on the theory of neuroendocrine-immunoregulatory(NEI) network, experiments are designed from the three aspects of pain, wound healing and inflammation, and related common chemical “languages”— microscopic molecules such as cytokine, receptor, hormone and neurotransmitter—are picked out according to previous studies. This study preliminarily reveals possible paths in which TCM compounds regulate the whole body by affecting different microscopic molecules.1. Experiment 1: To build the acetic acid-induced pain model of mice. The mice are divided into the control group, the naloxone hydrochloride group, the Simiao Junyi Ointment group, and the naloxone hydrochloride + Simiao Junyi Ointment group. The writhing frequency of mice in 15 minutes is observed in each group.2. Experiment 2: To build the acetic acid-induced pain model of mice. The enzyme-linked immunosorbent assay(ELISA) is adopted to testify the expression of analgesic β-endorphin(β-EP) and pain-producing prostaglandin E2(PGE2) in the peripheral blood serum of mice in each group; ELISA is also adopted to determine the contents of two neurotransmitters, 5-hydroxy trptamine(5-HT) and gamma-aminobutyric acid(GABA), in the mice brain tissue homogenate of each group; and the expression levels of 5-hydroxy trptamine receptor(5-HT2AR) in hypothalamus and gamma-aminobutyric acid A receptor(GABAAR) in pallium are tested by immunohistochemistry assay on paraffin sections.3. Experiment 3: To build the full-thickness excision cutaneous wound model of rats. The Sprague-Dawley(SD) rats are divided into the model group(M Group), the control group(K Group), the recombinant human epidermal growth factor gel group(CZ Group), the Longzhu Ointment group(LZ Group), and the Simiao Junyi Ointment group(SM Group). Rats of each group are randomly divided into 3 equal teams. The general healing conditions, healing areas and healing rates of wounds at the back of rats in the first team as well as the total healing time are macroscopically observed in the 7th and 14 th day after modeling; the other two teams of rats are killed in the 7th and 14 th day respectively for sampling, and the pathological wound healing conditions, the protein concentration on the surface of wounds and the expression levels of epidermal growth factor receptor(EGFR), transforming growth factor alpha(TGF-α) and interleukin-1 beta(IL-1β) are microscopically studied.4. Experiment 4: To build the postoperative pain model of rats in anorectal diseases. The general conditions, partial conditions of anuses and pathological wound healing conditions of rats in each group are observed; ELISA is employed to detect the concentration of tumor necrosis factor- alpha(TNF-α), interleukin-6(IL-6) and interleukin-10(IL-10) in rat serum of each group. Result:1. Experiment 1: The writhing frequency of mice in the Simiao Junyi Ointment group is the lowest, followed by that in the naloxone hydrochloride + Simiao Junyi Ointment group. The difference between these two groups is statistically significant(P<0.05).2. Experiment 2:(1)Concentration of β-EP: the expression levels of β-EP in three treatment groups are lower than that in the model group. Specifically, the expression level in the Simiao Junyi Ointment group is the lowest, which is of high statistical significance(P<0.01).(2)Concentration of PGE2: the concentration of PGE2 decreases in three treatment groups and the model group. That in the Simiao Junyi Ointment group falls the most, which has high statistic significance(P<0.01).(3)Concentration of GABA in mice brains: the expression levels of GABA in three treatment groups are higher than that in the model group, and their differences are significant(P<0.01), but the comparisons between the three treatment groups have no statistical significance(P>0.05).(4)Concentration of 5-HT in mice brains: the expression level of 5-HT grows in three treatment groups when compared with that in the model group, and their differences are significant(P<0.01), but the comparisons between the three treatment groups are not statistically significant(P>0.05).(5)Grayscale values of 5-HT2 AR in hypothalamus: the grayscale values of 5-HT2 AR in three treatment groups are apparently lower than that in the model group, and their differences are of high statistical significance(P<0.01) while those between the three treatment groups have little statistical significance(P>0.05).(6)Grayscale values of GABAAR in pallium: the grayscale values of GABAAR in three treatment groups are apparently lower than that in the model group, and their differences are of high statistical significance(P<0.01) while those between the three treatment groups have little statistical significance(P>0.05).3. Experiment 3:1) Macroscopic Observation(1)The healing rate of wounds in SM Group is higher than those in M Group both in the 7th and 14 th day after modeling, which is of high statistical significance(P<0.01).(2)The total healing time of wounds at the right side of rats in SM Group is shorter than that in M Group, which is of high statistical significance(P<0.01).2) Microscopic Observation(1)Comparison of scores for pathological wound healing conditions in each group of rats at different time intervals: scores in the 7th day—CZ Group>SM Group>LZ Group>K Group>M Group; the comparison between SM Group and M Group is of high statistical significance(P<0.01), that between SM Group and CZ Group of statistical significance(P<0.05), and that between SM Group and LZ Group of statistical significance(P<0.05). Scores in the 14 th day—CZ Group>SM Group>LZ Group>K Group>M Group; the comparison between SM Group and M Group is of high statistical significance(P<0.01), that between SM Group and CZ Group of statistical significance(P<0.05), and that between SM Group and LZ Group of little statistical significance(P>0.05).(2)Comparison of protein concentrations on the surface of wounds in each group of rats at different time intervals: the result trend of protein concentration is similar in the 7th and 14 th day—SM Group>LZ Group>CZ Group>K Group>M Group. In the 7th day, the difference between SM Group and CZ Group has high statistical significance, and that between SM Group and LZ Group little statistical significance(P>0.05). In the 14 th day, the difference between SM Group and CZ Group is of high statistical significance(P<0.01), the same as that between SM Group and LZ Group(P<0.01).(3)Grayscale values of EGFR in each group of rats at different time intervals: grayscale values in the 7th day—CZ Group<SM Group<LZ Group<K Group<M Group; the difference between SM Group and M Group has high statistical significance(P<0.01), so does that between SM Group and CZ Group(P<0.01), while that between SM Group and LZ Group has not statistical significance(P>0.05). Grayscale values in the 14 th day—CZ Group<SM Group<LZ Group<M Group<K Group; the comparison between SM Group and M Group as well as that between SM Group and CZ Group is highly significant(P<0.01), but that between SM Group and LZ Group is not statistically significant(P>0.05).(4)Expression levels of TGF-α in rat serum of each group at different time intervals: expression levels in the 7th day—SM Group>LZ Group>CZ Group>K Group>M Group; the difference between SM Group and M Group has high statistical significance(P<0.01), so does that between SM Group and M Group(P<0.01); the comparison of SM Group and LZ Group is statistical significant(P<0.05). Expression levels in the 14 th day—SM Group>LZ Group>CZ Group>K Group>M Group; there is highly significant difference between SM Group and M Group(P<0.01), significant difference between SM Group and CZ Group(P<0.05), and no significant difference between SM Group and LZ Group(P>0.05).(5)Comparison of IL-1β contents in rat serum of each group at different time intervals: IL-1β contents in the 7th day—SM Group<LZ Group<CZ Group<K Group<M Group; the difference between SM Group and M Group has high statistical significance(P<0.01) while there is no significant difference between SM Group, LZ Group and CZ Group. In the 14 th day, the contents of IL-1β in rat serum of each group are too small to be obviously detected by ELISA.4. Experiment 4:(1)Concentration of IL-10: Tetracaine hydrochloride gel group >Simiao Junyi Ointment group>normal group>carbomer group>model group; the comparison between the Simiao Junyi Ointment group and the tetracaine hydrochloride gel group has statistical significance(P<0.05) and that between the Simiao Junyi Ointment group and the model group is of high statistical significance(P<0.01).(2)Concentration of IL-6: normal group<Simiao Junyi Ointment group<tetracaine hydrochloride gel group<carbomer group<model group; the difference between the Simiao Junyi Ointment group and the tetracaine hydrochloride gel group has statistical significance(P<0.05) and that between the Simiao Junyi Ointment group and the model group is of high statistical significance(P<0.01).(3)Concentration of TNF-α: normal group<Simiao Junyi Ointment group<tetracaine hydrochloride gel group<carbomer group<model group; there is no statistical significance of differences between the Simiao Junyi Ointment group and the tetracaine hydrochloride gel group(P>0.05) but high statistical significance between the Simiao Junyi Ointment group and the model group(P<0.01). Conclusion:1. Simiao Junyi Ointment gives effect to peripheral analgesia by regulating analgesic β-EP and pain-producing PGE2 in peripheral blood serum, and to analgesia in central nervous system by regulating the expression of 5-HT, 5-HT2 A, GABA and GABAAR. Thus, Simiao Junyi Ointment is a new type of analgesics with combined mechanism of action, which is different from the mechanism of opioid analgesics and nonsteriodal anti-inflammatory drugs.2. Simiao Junyi Ointment facilitates the expression of TGF-α and EGFR, the combination of which will induce the formation and repair of blood vessels to help with the formation of granulation tissue and wound healing. Besides, the ointment can lower the concentration of IL-1β in serum to control local inflammation so as to construct a “good” microenvironment for immune cells and trigger the release of relevant growth factors and cytokines beneficial for wound healing. Therefore, Simiao Junyi Ointment is able to improve local microenvironment by regulating inflammatory cytokines in order to adjust the expression of growth factors together with their receptors, which will be of great help to the formation of granulation tissue and the functioning of healing mechanism.3. Simiao Junyi Ointment might improve microenvironment by regulating the balance between IL-6 and TNF-α(inflammatory cytokines) and IL-10(anti-inflammatory cytokine) so as to take effect in anti-inflammation.In summary, according to the theory of NEI network, Simiao Junyi Ointment has effect to analgesia, healing and anti-inflammation by regulating the multi-targeted expression of β-EP, PGE2, 5-HT, 5-HT2 AR, GABA, GABAAR, TGF-α, EGFR, IL-1β, IL-6, TNF-α and IL-10.