The Study On The Anti-inflammatory Effect And Material Basis Of Moxibustion In Radiculitis Model Rats Based On MAPK Signaling Pathway

ObjectiveLumbar Disc Herniation(LDH)is a common disease of orthopedics.Along with the change of people’s life and work style,the incidence of the disease shows a year-by-year upward trend.The main age of onset is about 20-40 years old,the major working population.And it gradually turned to the trend of younger age,causing enormous losses and heavy burdens on individuals and society.The side effects of western medicine and surgical treatment of LDH are getting more attention.Moxibustion as a specific therapy of Chinese medicine treatment for LDH,it is favored by patients and clinicians of all ages for its advantages of convenient,warm and comfortable operation and small side effects.LDH is a disease caused by annulus fibrosus rupture,nucleus pulposus prominent nerve fibers oppression due to disc degeneration,causing nerve root inflammation,then manifested as low back pain,lower extremity dysfunction and other major symptoms.It is all agreed to investigate the pathogenesis of LDH we should focus on inflammation.Most clinical research has shown that moxibustion has a significant effect on the treatment of LDH,but few studies on the mechanism of moxibustion in treating LDH.This study based on the MAPK signaling pathway,to explore the anti-inflammatory mechanism of moxibustion in the treatment of LDH in nerve root inflammation model rats.From the overall animal level and in vitro cell level,a comprehensive and systematic exposition of moxibustion on LDH anti-inflammatory mechanism has been provided,as well as the experimental basis for the clinical application of moxibustion.Methods1.The anti-inflammatory effects and mechanism of moxibustion in radiculitis model ratsSurgery to establish radiculitis model rats and caused LDH according to reference literature.During the experiment,the lower limb function of rats was monitored daily and the Siegal neurological function score was recorded at the same time.Blood samples were taken from the orbital of rats at 2 days before and after modeling and 7 days after intervention.Blood samples were also collected from the abdominal aorta of rats after intervention for 14 days.Serum levels of inflammatory cytokines were measured by ELISA.Real-time RCR method was adopted to detect the m RNA expression of inflammatory cytokines.The histopathological changes in lesions were observed by HE staining.Immunohistochemical method and western blot was employed to detect the expression level of the MAPK pathway related phosphorylated proteins.2.The anti-inflammatory effect and mechanism of Moxa smoke extract on LPS induced RAW264.7 cellsInflammatory cell model was constructed by LPS inducing RAW264.7 cell.By using a gas absorption device,Moxa smoke was dissolved in solvents of different polarities to obtain water soluble extraction(WS),fat soluble extraction(FS)and N-butanol absorption part(NBA).LPS-induced RAW264.7 cells produce inflammatory response to establish inflammatory cell model.MTT assay was used to measure cell viability.The content of inflammatory cytokines in supernatant was detected by ELISA.Real-time RCR was used to detect the m RNA expression of inflammatory cytokines.Western blot was adopted to detect the phosphorylation level of MAPK signaling pathway related proteins.Results1.The anti-inflammatory effect of Moxibustion in radiculitis model ratsMoxibustion intervention could reduce the Siegal neurological function score and improve the lower limb function on rats.After 7 days of Moxibustion intervention,the serum inflammatory cytokines TNF-α levels in the radiculitis model rats was significantly reduced,and in 14 days,the serum inflammatory cytokines IL-1β levels and TNF-α were significantly reduced as well.The level of serum inflammatory cytokines IL-1β and IL-6m RNA the model rats were also found decreasing significantly after 14 days of moxibustion intervention.Histopathological results showed that moxibustion can reduce tissue inflammatory cell infiltration in the model rats,repair nerve and muscle tissue fiber arrangement,and improve neural edema.2.The effect of moxibustion on the phosphorylation level of the MAPK pathway protein in radiculitis ratsThe expression level of the MAPK pathway phosphorylated proteins(p-P38,p-JNK,p-ERK)in radiculitis rats were significantly increased,comparing with sham operation group.Moxibustion intervention could inhibit the MAPK signal pathway and significantly reduce the expression level of p-P38,p-JNK,p-ERK protein.3.The anti-inflammatory effect of Moxa smoke extracts on LPS-induced RAW264.7cellsMoxa smoke extract(WS,FS,NBA)could significantly reduce the level of the supernatant inflammatory cytokines IL-1β and down-regulated the expression of the inflammatory cytokines IL-1β,PGES and IL-6 m RNA in model cells.4.The effect of moxa smoke extract on the phosphorylation level of the MAPK pathway proteins in LPS-induced RAW264.7 cellsThe expression of MAPK pathway related proteins(p-P38,p-JNK,p-ERK)in LPS-induced RAW264.7 cells were significantly increased.WS at 5 μg/m L could downregulate the expression of the p-P38 protein.WS(5～20 μg/m L)could significantly down-regulate the expression of the p-JNK and p-ERK protein.FS at 5 μg/m L could up-regulate the expression of the p-P38 protein,however p-P38 protein did not change significantly while the FS concentration was increased to 10～20 μg/m L.FS(5～20 μg/m L)could significantly down-regulate the expression of the p-JNK,p-ERK protein.The expression levels of the p-P38,p-JNK and p-ERK protein were significantly down-regulated in NBA(5～20 μg/m L).ConclusionMoxibustion has an anti-inflammatory effect on radiculitis model rats,which might related to the regulation of MAPK signaling pathway.In the radiculitis model rats,MAPK signaling pathway’s activation would lead to the upregulation of the inflammatory cytokines,thereby inducing nerve root inflammation and causing lower limb disfunction,and then resulting in higher Siegal neurological function score.Moxibustion treatment could inhibit the MAPK signaling pathway and reduce the level of inflammatory cytokines,then promote tissue repair and reduce lower limb pain with lower Siegal neurological function score.The in vitro experiments showed that the smoke extract(WS,FS,NBA)had a significant anti-inflammatory effect on LPS-induced RAW264.7 cells.This effect was also related to the regulation of MAPK signaling pathway,indicating that Moxa smoke plays an important role in the moxibustion treatment in LDH.