Research On The Anti-asthmatic Activities Of Guang-Pheretima And Its Underlying Mechanisms

Objective:Pheretima has been recorded in Chinese herbal medical works for nearly 2000 years for its excellent anti-inflammation,anti-asthma,thrombolysis,anticonvulsants and diuresis activities.Among them,praised as one of the "Top 10 Guangdong-origin Materia Medica",Guang-Pheretima is widely used in the treatment of a variety of inflammatory diseases and is considered as the best variety of Pheretima by numerous previous experts.Although Guang-Pheretima exerts prominent anti-inflammatory efficacy in clinical use,its pharmacological effects and underlying molecular mechanism has not been studied in-depth.As a consequence,we explored the anti-inflammatory activities of Guang-Pheretima decoction(GPD)in the ovalbumin(OVA)induced asthmatic BALB/c mice and the nuclear factors-?B(NF-?B)signaling pathway involved in its therapeutic effects.Besides,by observing the anti-inflammatory effects of Guang-Pheretima decoction and protein-free Guang-Pheretima decoction(PF-GPD)on lipopolysaccharide(LPS)induced Raw 264.7 cells,we investigated their anti-inflammatory activity of inhibiting the NF-?B activation.Finally,we attempted to systematically reveal the anti-asthmatic activities of Guang-Pheretima in both apparent symptoms and molecular mechanism through animals and cells experiments.Methods:In the animal experiments,OVA was used to sensitize and challenge the airway of the mice,and GPD was administrated by gavage.We measured airway hyperresponsiveness(AHR)in the mice 24 hours following a final methacholine challenge with whole-body plethysmography.The bronchoalveolar lavage fluid(BALF),serum and pulmonary tissues were collected 48 hours after the last challenge.The levels of inflammatory factors and the related mRNAs were determined by enzyme-linked immunosorbent assay(ELISA)and real-time polymerase chain reaction(RT-PCR),respectively.The number of differential inflammatory cells in the BALF was counted.Serum total and OVA-specific IgE levels were measured with ELISA.The activation of NF-?B signaling in the lung was detected by Western Blotting.In addition,the lung tissues were stained with hematoxylin and eosin or periodic acid Schiff stain for different histopathological examination.In the cell experiments,LPS was used to stimulate the RAW 264.7 cells to establish the inflammatory model.Observation by thiazole blue(MTT)assay on various concentrations of GPD and PF-GPD for the influence of the cell vitality,and choose the concentrations showed no cytotoxicity to continue the following experiments.After determined the nitric oxide(NO)synthesis activity of cells by Griess reagent,prostaglandin E2(PGE2),tumor necrosis factor-alpha(TNF-?).IL-1? and IL-6 in the cell supernatant were measured by ELISA.After discovering that GPD which is under the no-cytotoxicity concentrations is unable to suppress the inflammatory cytokines listed above,we determined to carry out our research only with PF-GPD.The protein of inducible nitric oxide synthase(iNOS)and cyclooxygenase-2(COX-2),which are the key enzymes of NO and PGE2 synthesis respectively,were determined by Western Blot analysis.Afterwards,the relative concentrations of iNOS and COX-2 mRNA were determined by Real-Time PCR.Finally,by the means of Western Blot,we observed the suppressive effect of PF-GPD on the degradation of ?B inhibitor(I?B-?),which could lead to the translocation of NF-?B,to calculate the mechanism of Guang-Pheretima in anti-asthmatic activities.Results:In the animal experiments,the Penh values were significantly higher in the OVA group compared to the normal control(NC)group at all concentrations of methacholine(P<0.01).Compared with the OVA group,the Penh values following methacholine challenge were significantly reduced in the medium and high groups at all corresponding concentrations(P<0.01).In contrast,the Penh values following methacholine challenge in the low group were lowered significantly within the concentration range of 6.25-25 mg/mL(P<0.01).However,the Penh values at the concentration of 50 mg/mL were not significantly different between the low group and the OVA group(P = 0.292).To assess the effect of GPD on the recruitment of eosinophils in the lungs,cell counting was conducted.Total cell counts,as well as the numbers of eosinophils and other inflammatory cells were markedly higher in the OVA group compared with those in the NC group(P<0.01).All of the GPD-treated groups had significantly lower eosinophil counts compared with that of the OVA group(P<0.01).The low and medium groups also had significantly decreased total cell counts(P<0.01).In the high group,the numbers of all cells measured were decreased,including the numbers of total cells,eosinophils and other inflammatory cells(P<0.01).To evaluate the effects of GPD on cytokines and IgE.the levels of IL-4,IL-5 and 1L-13 in the BALF and the levels of total and OVA-specific IgE in the serum were determined by ELISA 48 hours after the last OVA challenge.The above factors in the OVA group were all significantly higher compared to the corresponding measurements in the NC group(P<0.01).All cytokines examined and total IgE were significantly reduced(P<0.05)in the low group,with a particularly large decrease in IL-5(P<0.01);however.OVA-specific IgE levels were unchanged(P = 0.106).We investigated the mRNA levels of these cytokines in the lung tissues of GPD-treated mice using Real-Time PCR.The results showed that all GPD-treated groups and the DEX group exhibited remarkable reductions in the mRNA levels of all cytokines tested when compared with those of the OVA group(P<0.01).We examined the levels of nuclear and cytoplasmic NF-?B in the lung tissues of GPD-treated mice to elucidate the signaling transduction pathway underlying its anti-inflammatory and anti-asthmatic properties.Nuclear NF-?B levels were significantly higher in the OVA group compared with those in the NC group(P<0.01).In contrast,the level of cytoplasmic NF-?B was significantly lower in the OVA group compared with that in the NC group(P<0.01)and the levels of cytoplasmic NF-?B in the medium and high groups was elevated as compared with that in the OVA group(P<0.01).We evaluated the histopathology of asthmatic mice administered GPD using HE and PAS staining.We observed massive inflammatory cell infiltration in the OVA group compared with the NC group.This effect was attenuated in the medium and high groups as compared with the OVA group(P<0.01).In addition,mucus secretion by the airway epithelial cells was significantly enhanced in the OVA group compared with the NC group(P<0.01).The mucous optical density revealed that mucus secretion in the airway was remarkably alleviated in the asthmatic mice in all the GPD groups(P<0.01).After the mice were sacrificed,the muscle tissue,digestive tract,stomach,intestine,colon,liver,kidneys,spleen,lungs,and lymph nodes of the mice were examined.We did not detect the presence of tumors nor other abnormalities.In the cell experiments,5-20 ?g/mL GPD and 40-320 ?g/mL PF-GPD showed no cytotoxicity.In the observation of NO production in RAW 264.7,we found GPD did not suppress NO secretion under the stimulation of LPS(P>0.05).whereas the PF-GPD under the concentration of 40-320 ?g/mL significantly suppressed the cells to generate NO(P<0.01);In the observation of suppressive effect of Guang-Pheretima on PGE2 and pro-inflammatory cytokines,we found GPD can't suppress LPS-induced PGE2 nor TNF-a,IL-1?,IL-6.In contrast,40-320 ?g/mL PF-GPD significantly inhibited the generation of the inflammatory related proteins(P<0.01);In view of the results above,we continued the following experiments only with the PF-GPD,and found that 40-320 ?g/mL PF-GPD not only significantly reduced iNOS and COX-2 proteins,but also remarkably suppressed the relative levels of their corresponding mRNA(P<0.01);In the observation of the suppressive effect of PF-GPD in NF-?B nuclear translocation,we found that 40-320 ?g/mL PF-GPD inhibited the degradation of I?B-? in cytoplasm.As a result,NF-?B levels were elevated in the cytoplasm and lowered in the nuclear.Conclusion:After investigated the anti-asthmatic activities and mechanism of Guang-Pheretima by the means of both animal and cell experiments,we found Guang-Pheretima can suppress NF-?B nuclear translocation through the inhibition of I?B-adegradation.As a result,the mRNA of some inflammation related molecule(IL-4,IL-5,IL-13,iNOS,COX-2)were restrained under transcriptional levels,and thus reduce the generation of the IgE,NO and PGE2,and suppress lymphocytes infiltration and mucus secretion in the lung tissues,which eventually help to relieve inflammation in asthma and symptoms such as AHR.Therefore,this study not only serve as a scientific basis of healing asthma and other inflammatory diseases with Guang-Pheretima,but also lay a foundation for the further study of anti-inflammatory activities of this prestigious traditional Chinese medicine.