Effects Of Total Glycosides Of Paeony On Th17/Treg-related Cytokine Regulation And Inhibitory Mechanism For MRC-5 Proliferation

Objective:To study the effect of TGP on the proliferation of human embryonic lung fibroblasts MRC-5;to observe the effects of total glycosides of paeony（TGP）on Th17/Treg-related cytokines in mice with bleomycin-induced pulmonary fibrosis;and to preliminarily investigate their possible mechanism for pulmonary fibrosis inhibition,in order to provide preliminary theoretical and animal experimental bases for the clinical prevention and treatment of pulmonary fibrosis.Methods:1.MRC-5 cells were treated with different doses of TGP(0.5mg·L^-1,2.5 mg·L^-1,12.5 mg·L^-1,62.5 mg·L^-1,125 mg·L^-1)for 24 h,48 h and 72h.MTT assay was used to detect the effect of TGP on MRC-5 cell proliferation,and the time-concentration inhibition curves were plotted.TGP(62.5 mg·L^-1,125 mg·L^-1)were screened to treat MRC-5 cells for 48h.Cell cycle distribution was detected with flow cytometer.The apoptosis of MRC-5 cells was detected by TUNEL assay and Annexin V-FITC/PI double staining.The expressions of TGF-β₁ in various groups were observed by Western blotting.2.Forty-nine female C57BL/6J mice were randomly divided into 7 groups.Mice in the pulmonary fibrosis model group（n=7）were given 2.5 g·L^-1aerosol bleomycin dilution continuously for a total of 8 h.Mice in the blank control group（n=7）were given an equal volume of normal saline aerosol for 8 h.For the TGP low-,medium-and high-dose groups（n=7in each group）,aerosol bleomycin was given in the same manner as the model group.These three groups were also intragastrically treated with TGP(0.27 mg·g^-1·d,0.81 mg·g^-1·d,2.43 mg·g^-1·d)diluted with 0.5%carboxymethyl cellulose sodium,respectively.Mice in the pirfenidone group（n=7）were given aerosol bleomycin in the same manner as the model group,which were also intragastrically administered with pirfenidone(0.09 mg·g^-1·d)solution.In addition to the aerosol bleomycin administration in the same manner as the model group,mice in the TGP+pirfenidone group（n=7）were also intragastrically administered with a mixed solution of pirfenidone(0.09mg·g^-1·d)and TGP(0.81 mg·g^-1·d).All medications were given once daily for 30 consecutive days.At the end of the experiment,mice were weighed,and eyeball blood was collected for ELISA assay of serum IL-17,TGF-β,IL-6 and IL-10 levels.Pathomorphological changes and fibrotic degree of mouse lung tissues were observed by HE and Masson staining.The IL-17A and TGF-β₁levels in mouse lung tissues were detected by IHC.RT-PCR was used to detect the lung tissue levels of IL-17A,TGF-β₁,Ro Rγt and Foxp3 in the mice.Results:1.Proliferation inhibiting rate of MRC-5 cell was increased gradually with elevation in TGP concentration and extension in treatment duration.Number of cells at G1 stage,apoptosis index and apoptosis rate increased in TGP 62.5mg·L^-11 and 125mg·L^-11 treatment groups;while number of cells at G2 and S stages,expression levels of CyclinD1 and CDK4 mRNA,as well as TGF-β₁ protein decreased.Differences were of statistically significance（P<0.05）compared with those in Control group;in addition,TGP 125mg·L-1 had more remarkable effects than TGP62.5mg·L^-1（P<0.05）.2.Compared with blank group,lung coefficient and hydroxyproline content in lung tissue were increased in C57BL/6J mouse model group;Ashcroft score was increased;expression levels of TGF-β₁ and IL-17A proteins were elevated;plasma IL-17,TGF-β₁ and IL-6 levels were elevated;IL-10 level was decreased;expression levels of IL-17A,RORγt and TGF-β₁ mRNA in lung tissue were increased;and expression level of Foxp3 mRNA was decreased（P<0.05）.Compared with model group,lung coefficient and hydroxyproline content in lung tissue were lessened in all TGP treatment groups,pirfenidone group and TGP with pirfenidone group（P<0.05）;while those showed no statistically significant differences among all TGP treatment groups（P>0.05）;compared with pirfenidone group,those in TGP with pirfenidone group were decreased（P<0.05）.Compared with model group,pulmonary fibrosis Ashcroft score in all treatment groups was reduced;when compared with high dose TGP group,that in pirfenidone group was decreased（P<0.05）;compared with pirfenidone group,that in TGP with pirfenidone group was further reduced（P<0.05）.Compared with model group,expression levels of TGF-β₁ and IL-17A proteins in all TGP treatment groups,pirfenidone group and TGP with pirfenidone group showed a decreasing trend;and compared with pirfenidone group,those in TGP with pirfenidone group were further reduced（P<0.05）.Plasma IL-17 and IL-6levels were decreased in median and high dose TGP groups relative to low dose TGP group,while IL-10 level was elevated（P<0.05）.TGF-β₁and IL-6 levels were reduced in pirfenidone group when compared among all TGP treatment groups;plasma IL-10 concentration in pirfenidone group was elevated when compared with median and high dose TGP groups（P<0.05）.Compared with pirfenidone group,plasma IL-17,IL-6 and TGF-β₁ levels in TGP with pirfenidone group were further decreased,while IL-10 concentration was further elevated（P<0.05）.Compared with blank group,expression of IL-17,TGF-β₁ and RORγt mRNA in lung tissue of model group was up-regulated,while that of Foxp3 mRNA was down-regulated（P<0.05）.Compared with model group,expression levels of IL-17,TGF-β₁ and RORγtmRNA in all TGP treatment groups,pirfenidone group and TGP with pirfenidone group were decreased to various degrees,while expression of Foxp3mRNA was up-regulated.TGP with pirfenidone group had stronger effects than pirfenidone group（P<0.05）.Conclusion:1.TGP can inhibit proliferation of MRC-5 cell,the mechanism of action of which may be related to blocking cell cycle progression,inhibition of cell proliferation and promotion of cell apoptosis.2.Atomization inhalation of bleomycin can successfully construct the pulmonary fibrosis model.TGP combined with pirfenidone can partly improve bleomycin-induced pulmonary fibrosis model,which may be related to the regulation of Th17/Treg associated cytokines and specific upstream key transcription factors.Inhibiting expression of Th17/Treg associated cytokines（such as IL-17,TGF-β₁ and IL-6）,increasing that of IL-10,suppressing that of its upstream transcription factor RORγt mRNA,and elevating that of Foxp3 mRNA may contribute to preventing pulmonary fibrosis and delaying pulmonary fibrosis process.