An experimental study of acute toxicity and pharmacology of fermented Platycodonis Radix / 中国中药杂志

This study investigated the acute toxicity of fermented Platycodonis Radix on mice and its effect on coughing in mice infected with Mycoplasma pneumoniae. The maximum dosage(MAD) was used in the acute toxicity experiment on mice to observe the signs of mice. After 14 days, dissection, blood biochemical examination, and pathological tissue section observation were conducted. In the pharmacological experiment of fermented Platycodonis Radix, 60 healthy BALB/c mice, 30 males and 30 females, were randomly divided into a blank group, a model group, a carbetapentane group(0.013 g·kg~(-1)·d~(-1)), and high-, medium-, and low-dose fermented Platycodonis Radix groups(5.2, 2.6, and 1.3 g·kg~(-1)·d~(-1)), with 10 mice in each group. Except for the blank group, the mice in the other five groups underwent model induction by intranasally instilling 20 μL of 1×10~6 CCU M. pneumoniae for 3 days, and the mice in each group were orally administered the corresponding drugs for 7 days. Cough induction experiment was conducted to observe and record the cough latency and total cough count within 3 min for each group. Hematoxylin-eosin(HE) staining and Masson staining were used to observe the pathological changes in lung tissues. Immunohistochemistry was performed to observe the protein expression of transient receptor potential A1(TRPA1), calcitonin gene-related peptide(CGRP), and substance P(SP) in the lung tissues of mice in each group. Real-time fluorescence-based quantitative polymerase chain reaction(qRT-PCR) was used to elucidate the changes in the mRNA levels of cough-related factors TRPA1, CGRP, and SP in mice treated with fermented Platycodonis Radix. No mice died in the acute toxicity experiment, and there were no changes in general behavior and major organ histopathological examinations. Compared with the blank group, there were no statistically significant differences in blood biochemical indexes. In the pharmacological experiment of fermented Platycodonis Radix, compared with the model group, the high-and medium-dose fermented Platycodonis Radix groups showed improved lung tissue structure of mice, with clear structure and regular tissue morphology. The qRT-PCR and immunohistochemical detection showed a decrease in the expression of TRPA1, CGRP, and SP in the fermented Platycodonis Radix groups. Fermented Platycodonis Radix can exert an inhibitory effect on cough by suppressing the expression of TRPA1, CGRP, and SP in lung tissues, thereby identifying the target of the drug.

Expression of calcitonin gene-related peptide and pulp sensitivity tests in irreversible pulpitis

Abstract The aim of the present study was to identify the relationship between the expression of calcitonin gene-related peptide (CGRP) and the responses of pulp sensitivity tests in healthy pulps and irreversible pulps by performing a cross-sectional study on patients. Two hundred subjects were evaluated. A total of 75 subjects complied with the criteria. The participants were divided into two groups: a) Healthy pulp (subjects [n = 35] having posterior teeth with clinically normal pulp tissue), and b) Irreversible pulpitis (subjects [n = 40] having posterior teeth with irreversible pulpitis). All participants were evaluated using the following variables: a) medical and dental history, b) pulp sensitivity tests, c) expression of CGRP by the enzyme-linked immunosorbent assay (ELISA), and d) expression levels of mRNA CGRP and mRNA CGRP receptor genes. We determined that the responses of the cold test between 4 and ≥12 s presented a higher average of the expression of CGRP in the group having irreversible pulpitis (p = 0.0001). When we compared the groups with the value of the electrical impulse, we found statistically significant differences (p = 0.0001), observing positive responses to the test with electrical impulses of 7 to 10, with an average of 72.15 ng/mL of CGRP in the irreversible pulpitis group. High values of CGRP expression were observed in that group in the responses of pulp sensitivity.

The effect of capsaicin on expression patterns of CGRP in trigeminal ganglion and trigeminal nucleus caudalis following experimental tooth movement in rats

ABSTRACT Objectives The aim of this study was to explore the effect of capsaicin on expression patterns of calcitonin gene-related peptide (CGRP) in the trigeminal ganglion (TG) and trigeminal subnucleus caudalis (Vc) following experimental tooth movement. Material and Methods Male Sprague-Dawley rats were used in this study and divided into small-dose capsaicin+force group, large-dose capsaicin+force group, saline+force group, and no force group. Closed coil springs were used to mimic orthodontic forces in all groups except for the no force group, in which springs were inactivated. Capsaicin and saline were injected into periodontal tissues. Rats were euthanized at 0 h, 12 h, 1 d, 3 d, 5 d, and 7 d following experimental tooth movement. Then, TG and Vc were obtained for immunohistochemical staining and western blotting against CGRP. Results Immunohistochemical results indicated that CGRP positive neurons were located in the TG, and CGRP immunoreactive fibers were distributed in the Vc. Immunohistochemical semiquantitative analysis and western blotting analysis demonstrated that CGRP expression levels both in TG and Vc were elevated at 12 h, 1 d, 3 d, 5 d, and 7 d in the saline + force group. However, both small-dose and large-dose capsaicin could decrease CGRP expression in TG and Vc at 1 d and 3 d following experimental tooth movement, as compared with the saline + force group. Conclusions These results suggest that capsaicin could regulate CGRP expression in TG and Vc following experimental tooth movement in rats.

Sensory Neuronal Change after Intravesical Electrical Stimulation in Spinailized Rat

The clinical benefits of intravesical electrical stimulation (IVES) in patients with increased residual urine or reduced bladder capacity have been reported. However, studies on the underlying mechanism of IVES has been limited to the A delta afferent and parasympathetic neurons. This study investigated the changes in the calcitonin gene-related peptide (CGRP), substance P (SP), and nitric oxide synthase (NOS) expression in the thoracolumbar and lumbosacral dorsal root ganglia (DRG) of spinalized rats to determine the effect of IVES on the C fiber afferent nerve. Forty Sprague-Dawley rats were divided into normal controls (n=10); IVES treated normal rats (n=10), spinalized rats (n=10), and IVES treated spinalized rats (n=10). IVES was performed for 2 weeks (5 days a week). IVES was started 3 weeks after spinalization in the spinalized animals. All animals had the DRG removed at the thoracolumbar (T13-L2) and lumbosacral (L5-S1) level. Changes in the CGRP, SP and n-NOS levels at the DRG were measured by western-blot analysis. The relative density of the CGRP and SP following spinalization was significantly higher compared to the controls in both the T13-L2 and L5-S1 DRG. However, IVES in the spinalized rat significantly decreased the relative density of the CGRP and SP compared to the rats with spinalization alone. A significant increase in the relative density of n-NOS was detected in the L5-S1 DRG following spinalization. However, the density of n-NOS was significantly lower after IVES in both the T13-L2 and L5-S1 DRGs. In conclusion, IVES significantly reduced the CGRP, SP and n-NOS levels in the DRG of spinalized rats. CGRP, SP and n-NOS are the main factors that contribute to the hyperexcitability of the micturition reflex after spinal cord injury. These results suggest that the bladder C fiber afferent is also involved in modulating the micturition reflex by IVES.

Studies on the changes of c-fos protein in spinal cord and neurotransmitter in dorsal root ganglion of the rat with an experimental peripheral neuropathy

Animal models for human chronic pain syndromes have been developed and widely used for pain research. One of these neuropathic pain models by Kim and Chung (1992) has many advantages for operation and pain elicitation. In this neuropathic model we have examined the c-fos protein, substance P, CGRP immunoreactivity in dorsal root ganglia and dorsal horn. 50 Sprague-Dawley rats were used for this study. L5 and L6 spinal nerves were ligated tightly to produce the neuropathic pain model. After 2, 4, 8, 16, and 24 hours and 1 week of surgery, rats were anesthetized and sacrificed by perfusion. After confirmation of the roots transected by the surgery, the L5 and L6 dorsal root ganglions and spinal cord were removed and processed for immunohistochemistry. All tissue sections were immunohistochemically stained for substance P, CGRP and c-fos using the peroxidase-antiperoxidase (PAP) method. The number of immunostained substance P and CGRP dorsal root ganglion cells and c-fos immunoreactive dorsal horn cells were counted and analyzed statistically with Mann-Whitney U test. The results are as follows. The number of c-fos protein immunoreactive neurons in the superficial layer of dorsal horn were increased markedly 2 hours after operation, and gradually decreased to normal level 1 week after operation. The number of c-fos protein immunoreactive neurons in the deep layer of the dorsal horn gradually increased to a peak 24 hours after operation, then decreased to the normal level 1 week after operation. The number of substance P and CGRP immunoreactive L5 and L6 dorsal root ganglion neurons were decreased markedly 1 week after the pain model operation. In conclusion, after neuropathic pain model operation, c-fos proteins were immediately expressed in the superficial layer of spinal dorsal horn, thereafter c-fos proteins in the deep layer of spinal dorsal horn were expressed. CGRP and substance P immunoreactive neurons in DRG were decreased markedly 1 week after neuropathic pain model operation. These decrements do not coincide with the other chronic pain models, which show great increases in these pain transmitting substances. Therefore, the relationship between pain and c-fos, SP and CGRP should be investigated further.