This study investigated the neuroprotective effects and underlying mechanism of Liujing Toutong Tablets(LJTT) on a rat model of permanent middle cerebral artery occlusion(pMCAO). The pMCAO model was established using the suture method. Eighty-four male SPF-grade SD rats were randomly divided into a sham operation group, a model group, a nimodipine group(0.020 g·kg~(-1)), and high-, medium-, and low-dose LJTT groups(2.8, 1.4, and 0.7 g·kg~(-1)). The Longa score, adhesive removal test and laser speckle contrast imaging technique were used to evaluate the degree of neurological functional impairment and changes in local cerebral blood flow. The survival and mortality of rats in each group were recorded daily. After seven days of continuous administration following the model induction, the rats in each group were euthanized, and brain tissue and blood samples were collected for corresponding parameter measurements. Nissl staining was used to examine pathological changes in brain tissue neurons. The levels of tumor necrosis factor-alpha(TNF-α), interleukin-6(IL-6), IL-1β, vascular endothelial growth factor(VEGF), calcitonin gene-related peptide(CGRP), beta-endorphin(β-EP), and endogenous nitric oxide(NO) in rat serum were measured using specific assay kits. The entropy weight method was used to analyze the weights of various indicators. The protein expression levels of nuclear factor kappa-B(NF-κB), inhibitor kappaB alpha(IκBα), phosphorylated IκBα(p-IκBα), and phosphorylated inhibitor of NF-κB kinase alpha(p-IKKα) in brain tissue were determined using Western blot. Immunohistochemistry was used to detect the protein expression of chemokine-like factor 1(CKLF1) and C-C chemokine receptor 5(CCR5) in rat brain tissue. Compared with the sham operation group, the model group showed significantly higher neurological functional impairment scores, prolonged adhesive removal time, decreased cerebral blood flow, increased neuronal damage, reduced survival rate, significantly increased levels of TNF-α, IL-1β, IL-6, CGRP, and NO in serum, significantly decreased levels of VEGF and β-EP, significantly increased expression levels of NF-κB p65, p-IκBα/IκBα, and p-IKKα in rat brain tissue, and significantly upregulated protein expression of CKLF1 and CCR5. Compared with the model group, the high-dose LJTT group significantly improved the neurological functional score of pMCAO rats after oral administration for 7 days. LJTT at all doses significantly reduced adhesive removal time and restored cerebral blood flow. The high-and medium-dose LJTT groups significantly improved neuronal damage. The LJTT groups at all doses showed reduced levels of TNF-α, IL-1β, IL-6, CGRP, and NO in rat serum, increased VEGF and β-EP levels, and significantly decreased expression levels of NF-κB p65, p-IκBα/IκBα, p-IKKα, and CCR5 protein in rat brain tissue. The entropy weight analysis revealed that CGRP and β-EP were significantly affected during the model induction, and LJTT exhibited a strong effect in reducing the release of inflammatory factors such as TNF-α and IL-1β. LJTT may exert a neuroprotective effect on rats with permanent cerebral ischemia by reducing neuroinflammatory damage, and its mechanism may be related to the inhibition of the NF-κB signaling pathway and the regulation of the CKLF1/CCR5 axis. Additionally, LJTT may exert certain analgesic effects by reducing CGRP and NO levels and increasing β-EP levels.
Rats ; Male ; Animals ; NF-kappa B/metabolism* ; NF-KappaB Inhibitor alpha/metabolism* ; Vascular Endothelial Growth Factor A/genetics* ; I-kappa B Kinase/pharmacology* ; Tumor Necrosis Factor-alpha/pharmacology* ; Interleukin-6/genetics* ; Calcitonin Gene-Related Peptide/pharmacology* ; Rats, Sprague-Dawley ; Signal Transduction ; Brain Ischemia/drug therapy* ; Tablets

OBJECTIVE: To investigate the changes of skin temperature, blood infusion and inflammatory cytokines of cutaneous tissue in the sensitized area of colitis model rats, as well as the relationship between sensory and sympathetic nerves and the formation of sensitized area, and to initially reveal the partial physical-chemical characteristics of the sensitized area in the colitis model rats. METHODS: Thirty-five male SD rats were randomly divided into a control group (n=10), a model group (n=18) and a guanethidine group (n=7). 5% dextran sulfate sodium (DSS) was adopted for 6-day free drinking to establish colitis model in the model group and the guanethidine group. On day 6 and 7, in the guanethidine group, guanethidine solution (30 mg/kg) was injected intraperitoneally for sympathetic block. On day 7, after injection of evans blue (EB) solution, the EB extravasation areas on the body surface were observed to investigate the distribution and physical-chemical characteristics of the sensitized area. The control area was set up, 0.5 cm away from the sensitized area, and with the same nerve segment innervation. Disease activity index (DAI) score of rats was compared between the normal group and the model group, and the morphological changes in the colon tissue were investigated with HE method. Using infrared thermal imaging technology and laser speckle flow imaging technology, skin temperature and blood infusion were determined in the sensitized area and the control area of the rats in the model group. Immunofluorescence technique was adopted to observe the expression levels of the positive nerve fibers of substance P (SP), calcitonin gene-related peptide (CGRP) and tyrosine hydroxylase (TH), and the correlation with blood vessels; as well as the expression levels of SP positive nerve fibers/tryptase⁺ mast cells, and tryptase⁺ mast cells/5-hydroxytryptamine (5-HT) in skin tissue in the sensitized area and the control area of the rats in the model group. MSD multi-level factorial method and ELISA were applied to determine the contents of pro-inflammatory and anti-inflammatory cytokines (e.g. TNF-α, IL-1β, IL-6, IL-4 and IL-10) and anti-inflammatory substance corticosterone (CORT). RESULTS: Sensitization occurred at the T₁₂-S₁ segments of the colitis model rats, especially at L₂-L₅ segments. Compared with the normal group, DAI score was increased in the rats of the model group (P<0.05), and the colonic mucosal damage was obvious, with the epithelial cells disordered, even disappeared, crypt destructed, submucosal edema and a large number of inflammatory cells infiltrated. In comparison with the control area, the skin temperature and blood infusion were increased in the sensitized area of the model group (P<0.05, P<0.01); as well as the expression levels of the positive nerve fibers of SP, CGRP and TH of skin tissue (P<0.05), which was specially distributed in peripheral vessels, the expression levels of SP positive nerve fibers/tryptase⁺ mast cells, and tryptase⁺ mast cells/5-HT of the skin tissue were all expanded (P<0.05) in the sensitized area of the model group. Compared with the model group, the number of sensitized areas was reduced in the guanethidine group (P<0.05). In comparison with the control area of the model group, in the sensitized area, the contents of pro-inflammatory cytokines, e.g. TNF-α, IL-1β and IL-6, and the anti-inflammatory substance CORT of skin tissue were all increased (P<0.05); and the contents of IL-6 and TNF-α were negatively correlated with CORT (P<0.05). CONCLUSION The sensitized areas on the body surface of colitis rats are mainly distributed in the L₂-L₅ segments. Sensory and sympathetic nerves are involved in the acupoint sensitization, and the sensitized areas may have the dynamic changes in pro-inflammatory and anti-inflammatory substances.
Animals ; Anti-Inflammatory Agents ; Calcitonin Gene-Related Peptide/metabolism* ; Colitis/metabolism* ; Cytokines/metabolism* ; Guanethidine ; Interleukin-6 ; Male ; Rats ; Rats, Sprague-Dawley ; Serotonin ; Skin Temperature ; Substance P/genetics* ; Tryptases ; Tumor Necrosis Factor-alpha

OBJECTIVE: To observe the analgesic effect of manipulation loading on chronic low back pain (CLBP) model rats and the expression of inflammatory factors in psoas major muscle tissue, and to explore the improvement of manipulation on local inflammatory microenvironment. METHODS: Thirty two SPF male SD rats weighing 340-360g were randomly divided into blank group, sham operation group, chronic low back pain model group and treatment group, with 8 rats in each group. In the model group, L RESULTS: There was no significant difference in PWT and PWL between the blank group and the sham operation group after modeling ( CONCLUSION Local massage loading has analgesic effect on CLBP rats, at the same time, it can inhibit the content of CGRP and NGF in psoas muscle tissue of CLBP rats, and improve the local inflammatory microenvironment.
Animals ; Calcitonin ; Calcitonin Gene-Related Peptide ; Low Back Pain/therapy* ; Male ; Nerve Growth Factor/genetics* ; Rats ; Rats, Sprague-Dawley

OBJECTIVE: To observe the effect of moxibustion at "Zusanli" (ST 36) on distal, middle and proximal colonic mucosal injury and expression of calcitonin gene-related peptide (CGRP) positive nerve fibers of distal colonic mucosa in ulcerative colitis (UC) mice at different time points. METHODS: A total of 51 C57BL/6N mice were randomized into a 7-day control group ( RESULTS: Mucosal injury can be observed in mice after modeling, displaying epithelial layer disappearance, abnormal crypt structure or crypt disappearance. Compared with the 7-day control group, colon length was shortened ( CONCLUSION Moxibustion at "Zusanli" (ST 36) can reduce the expressions of positive nerve fibers of colonic mucosa and CGRP positive nerve fibers of distal colonic mucosa, thus, improve the colonic mucosal injury.
Animals ; Calcitonin ; Calcitonin Gene-Related Peptide/genetics* ; Colitis, Ulcerative/therapy* ; Intestinal Mucosa ; Mice ; Mice, Inbred C57BL ; Moxibustion ; Nerve Fibers

OBJECTIVEPrevious studies have clarified that calcitonin gene-related peptide (CGRP) can promote the biologi- cal activity of osteoblasts. To further reveal the role of CGRP in bone repair, we studied its influence on osteogenic differentia- tion of mouse bone marrow stromal cells (BMSCs) and initially explored the effect of the Hippo signaling pathway with this process.

METHODSBMSCs were induced to osteogenic differentiate osteoblasts by different concentrations of CGRP for a screening of the optimal concentration. CGRP was added in BMSCs, then the activity of alkaline phosphatase (ALP) and the number of mineralized nodules were examined by specific ALP kits after 48 hours and alizarin red staining fluid after 7 days, respectively. The protein expression of p-Mst1/2 was measured by Western blot. Verteporfin was used to block the downstream Yap signaling. The mRNA expression of collagen type I (Col I) and runt-related transcription factor 2 (Runx2) were detected by reverse transcription-polymerase chain reaction.

RESULTSCompared to the blank group, different concentrations of CGRP (10⁻⁹, 10⁻⁸, 10⁻⁷ mol · L⁻¹), especially 10⁻⁸ mol · L⁻¹, significantly increased the ALP activity of BMSCs (P < 0.05). Alizarin red staining also showed more mineralized nodules in 10⁻⁸ mol · L⁻¹ group. The expression of p-Mst1/2 increased in the CGRP group (P < 0.05). Verteporfin treatment effectively decreased the mRNA expression of Runx2 and Col I (P < 0.05).

CONCLUSIONThe Hippo signaling pathway plays a role in CGRP-induced osteogenic differentiation in mouse BMSCs.

Alkaline Phosphatase ; Animals ; Calcitonin ; genetics ; metabolism ; Calcitonin Gene-Related Peptide ; metabolism ; Cell Differentiation ; Cells, Cultured ; Collagen Type I ; Core Binding Factor Alpha 1 Subunit ; Mesenchymal Stromal Cells ; physiology ; Mice ; Osteoblasts ; Osteogenesis ; physiology ; Signal Transduction

OBJECTIVETo observe the neurological protection effects of "paraplegia-triple-needling method" on rats with incomplete spinal cord injury (SCI), so as to make a preliminary exploration on its mechanism.

METHODSA total of 45 SD rats were randomly divided into a paraplegia-triple-needling method group (group A), a regular acupuncture group (group B) and a model group (group C), 15 rats in each one. The rats model of incomplete spinal cord injury was established by modified Allen's method. The acupoints of governor vessel and back-shu points next to the vertebras of upper end and lower end of injured segment as well as motor points in key muscle of lower extremities were treated with acupuncture in the group A; the acupoints of governor vessel and back-shu points next to the vertebras of upper end and lower end of injured segment as well as "Huantiao" (GB 30), "Housanli" (ST 36), "Yanglingquan" (GB 34) and "Genduan"(Extra) were treated with acupuncture in the group B; rats in the group C received no treatment after model establishment but grabbing and immobilization. The needles were retained for 15 min in the group A and group B, once a day for 14 times. 1 d, 7 d and 14 d after model establishment, Basso Beattie Bresnahan (BBB) scores were observed in each group; the morphologic change of injured spinal cord and expression of positive cells of calcitonin gene-related peptide (CGRP) were observed. Results (1) One day after SCI, there was no significant difference of BBB scores among three groups (P> 0. 05); 7 days and 14 days after SCI, BBB scores in the group A and group B were significantly superior to those in the group C (all P<0. 05), and the BBB scores in the group A were superior to those in the group B ( both P<0. 05). (2) There was expression of CGRP positive cells in all three groups, and that in the group A and group B was significantly higher than that in group C (both P<0. 05); 14 days after treatment, the expression in the group A was higher than that in the group B (P<0. 05).

CONCLUSIONThe "paraplegia-triple-needling method" could obviously! improve the motor function of rats with SCI, especially the expression of neuroprotective factor CGRP, which is likely to be one of the mechanisms of neurological protection effect.

Acupuncture Points ; Acupuncture Therapy ; instrumentation ; methods ; Animals ; Calcitonin Gene-Related Peptide ; genetics ; metabolism ; Disease Models, Animal ; Humans ; Male ; Rats ; Rats, Sprague-Dawley ; Spinal Cord Injuries ; genetics ; metabolism ; therapy

OBJECTIVE: To explore the inhibitory effect of calcitonin gene-related peptide (CGRP) on cardiac fibroblast proliferation and collagen synthesis induced by isoprenaline and the underlying mechanism. METHODS: The primary cultured cardiac fibroblasts were incubated with isoprenaline (10(-5) mol/L) for 48 h after pretreatment with CGRP (10(-8) or 10(-7) mol/L) for 1 h. Cell activity was detected by MTT. The mRNA expression of collagen (types I and III) and connective tissue growth factor (CTGF) was determined by RT-PCR, and the levels of intracellular ROS were determined by DCFH-DA fluorescent probe. RESULTS: Isoprenaline significantly promoted fibroblast proliferation and up-regulated collagen (types I and III) and CTGF mRNA expression concomitantly with an increase in ROS production, which were attenuated by CGRP. The effect of CGRP on cardiac fibroblasts was inhibited by CGRP8-37, a selective antagonist of CGRP receptor. CONCLUSION CGRP is able to protect cardiac fibroblasts against isoprenaline-induced proliferation and collagen expression, which might be related to the down-regulation of CTGF expression through inhibition of ROS production.
Animals ; Animals, Newborn ; Calcitonin Gene-Related Peptide ; pharmacology ; Cell Proliferation ; drug effects ; Cells, Cultured ; Collagen Type I ; genetics ; metabolism ; Collagen Type III ; genetics ; metabolism ; Connective Tissue Growth Factor ; genetics ; metabolism ; Fibroblasts ; cytology ; Isoproterenol ; antagonists & inhibitors ; pharmacology ; Myocytes, Cardiac ; cytology ; Primary Cell Culture ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Reactive Oxygen Species ; metabolism

OBJECTIVETo discuss the effect of calcitonin gene-related peptides (CGRP) on epithelial cadherin (E-cd) expression in human bronchial epithelial cells (HBECs) in vitro.

METHODSThe effect of CGRP on E-cd protein and mRNA expression in both normal and O3-challenged HBECs were determined by immunocytochemistry and RT-PCR. The signal transduction pathways of CGRP were observed by using protein kinase C(PKC) inhibitor (H-7), calmodulin(CaM) inhibitor (W-7) and PKA inhibitor (H-89).

RESULTSCGRP increased E-cd mRNA and protein expressions of normal and O3-challenged HBECs in a dose-dependent manner. CGRP had no effect on cytoplasm E-cd expression. Pre-treatment with H-89, H-7 and W-7, the up-regulatory effect of CGRP on E-cd expression was partly abolished.

CONCLUSIONCGRP increased in cytomembrane E-cd expression of normal and O3-challenged HBECs in a dose-dependent manner. E-cd expression on HBECs was strengthened by CGRP via PKA, PKC and CaM pathways.

Bronchi ; cytology ; Cadherins ; metabolism ; Calcitonin Gene-Related Peptide ; administration & dosage ; pharmacology ; Cell Line ; Epithelial Cells ; drug effects ; metabolism ; Humans ; Ozone ; RNA, Messenger ; genetics

OBJECTIVETo investigate the influnce of receptor activity modifying protein 1(RAMP-1) overexpression on enhancing effect of calcitonin gene-related peptide on MG-63 cells proliferation.

METHODSCultured MG-63 osteoblasts in exponential phase of growth were randomly divided into three groups: RAMP-1 overexpression group, empty vector control group and negative control group. RAMP-1 eukaryotic expression vector was constructed and stably transfected into MG-63 cells. Realtime-polymerase chain reaction, Western blotting and immunofluroescence were used respectively to detect the expression of calcitonin receptor-like receptor (CRLR) in the cells and its distribution on cell membrane. The status of proliferation was detected respectively at 0, 24, 48, 72, 96 h by cell counting kit-8 (CCK-8) and cells were collected to analyze their cycle respectively at 0, 8, 16, 24 h by flow cytometry.

RESULTSCRLR protein and mRNA expression levels of MG-63 cells in RAMP-1 overexpression group were significantly higher than the other two groups (P < 0.05). The A value of RAMP-1 overexpression group at 24, 48, 72, 96 h were 0.628 ± 0.175, 0.896 ± 0.592, 1.055 ± 0.004, 1.179 ± 0.618, respectively, which were significantly higher than that of the other two groups (P < 0.05). The difference was most pronounced at 72 h. S-phase fraction of RAMP-1 overexpression group was (1.25 ± 0.13)%, (68.79 ± 0.56)%, (64.49 ± 1.59)%, (57.82 ± 0.75)%, respectively, which were significantly higher than the other two groups (P < 0.05). The difference was most pronounced at 8 h.

CONCLUSIONSRAMP-1 overexpression can promote CRLR distribution on MG-63 cell membrane and enhance CGRP's promotion effect on MG-63 cell proliferation.

Bone Neoplasms ; metabolism ; pathology ; Calcitonin Gene-Related Peptide ; genetics ; metabolism ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; Genetic Vectors ; Humans ; Osteosarcoma ; metabolism ; pathology ; RNA, Messenger ; metabolism ; Random Allocation ; Receptor Activity-Modifying Protein 1 ; genetics ; metabolism ; Transfection

OBJECTIVETo explore the effect of the calcitonin gene related peptide (CGRP) on the phenotypic transformation of corpus cavernosum smooth muscle cells (CCSM) in diabetic rats with erectile dysfunction (ED).

METHODSModels of diabetes and diabetic ED were established in male Sprague-Dawley rats by administration of streptozotocin, and CCSMs were primarily cultured and subjected to immunocytochemical assay. The cells were divided into a diabetic ED and a normal control group, and exposed to 0, 10, 60 and 100 nmol/L of CGRP for 24 hours. Then the relative expressions of calponin 1 (Cnn1) and osteopontin (OPN) mRNA were determined by real-time fluorescence quantitative RT-PCR (qRT-PCR).

RESULTSThe rate of SMalpha-actin positive cells in the CCSMs was (95.94 +/- 0.03) %. The expression of Cnn1 mRNA was significantly lower while that of OPN mRNA remarkably higher in the diabetic ED rats (4.41 +/- 0.29 and 5.28 +/- 0.32) than in the normal controls (10.35 +/- 0.62 and 1.32 +/- 0.24) (P < 0.01). Exposure to 100 nmol/L of CGRP significantly upregulated the expression of Cnn1 mRNA and downregulated that of OPN mRNA as compared with the unexposed rats (6.9 +/- 0.22 vs 4.41 +/- 0.29 and 3.26 +/- 0.31 vs 5.28 +/- 0.32, P < 0.01).

CONCLUSIONCGRP can transform the phenotype of CCSMs in diabetic ED rats from contractile to synthetic type.

Animals ; Calcitonin Gene-Related Peptide ; pharmacology ; Calcium-Binding Proteins ; metabolism ; Cells, Cultured ; Diabetes Mellitus, Experimental ; complications ; genetics ; metabolism ; Erectile Dysfunction ; etiology ; genetics ; metabolism ; Male ; Microfilament Proteins ; metabolism ; Muscle, Smooth ; cytology ; metabolism ; Osteopontin ; metabolism ; Penis ; cytology ; drug effects ; metabolism ; Phenotype ; Rats ; Rats, Sprague-Dawley