Research On Anti-fibrosis Effects Of Polysaccharide Of Prismatomeris Tetrandra After Extraction And Separation

Objectives:?To extract and separate the polysaccharide of Prismatomeris tetrandra?PSPT?,and to develop the determination method to test the content in the samples.?To investigate the anti-fibrosis effect of PTST on the model-Wistar rats with silicosis from the perspectives of both inhibiting inflammatory cytokines and affecting epithelial-mesenchymal transition?EMT?.Methods:?The PSPT was extracted from Prismatomeris tetrandra?PT?by ultrasonic water heating and was purified by sedimentation of ethyl alcohol.The Savage method was adopted to remove its proteins and ultrafiltration membrane was use to separate it.The content of polysaccharide was detected by HPLC-ELSD.The phenol-sulfuric acid method was used to detect he total content of PSPT at the colorimetric wavelength of 485nm with D-glucose as a control.?The rat model of silicosis was established by oropharyngeal method.120 SPF male Wistar rats were randomly divided into the control group and the silicosis model group.The rats in the control group were instilled with 1.00 ml of normal saline,and those in the silicosis model group were instilled with 1.00 ml of aqueous suspension with 50.0 mg/ml Si O₂to establish the silicosis fibrosis model.After successful modeling,the rats in the silicosis model group were randomly divided into intervention groups receiving low,medium,or high dose of PSPT?125,250,500 mg/g respectively?and the model group,with 24 rats in each group.The rats in the low-,medium-,and high-intervention groups were given the drugs with corresponding doses,respectively,the control group and the model group were given the equal amount of saline.Six rats in each group were sacrificed on day 7,day 14,day 28,and day 56,and their lung tissues were collected.Both the body weight and behavioral changes of rats were oberved;q RT-PCR was adopted to detect the m RNA levels of interleukin 1??IL-1??,interleukin 6?IL-6?,of inflammasome of nucleotide-binding oligomerization domain-like receptor 3?NALP3?and of tumor necrosis factor alpha?TNF-??.Their lung coefficients were calculated and the content of lung hydroxyproline was detected;HE and Masson staining were used to detect the pathological changes in the lung tissues of rats.q RT-PCR was used to detect the m RNA levels of transforming growth factor??TGF-??,of epithelial cadherin?E-cadherin?and of?-smooth muscle actin??-SMA?,and Vimentin in lung tissues.The isotopic labeling quantitative technique?i TRAQ?was used to identify the expressions of differential proteins in lung tissues.Western blotting was used to detect in the protein content expressions of E-cadherin,?-SMA in lung tissues.Results:?560.0 g of PSPT was extracted and purified from 10.0 kg of PT by this method,and the yield was 5.6%.The principal component content of PSPT groups was 64.0%,which was detected by HPLC-ELSD.The absorbance of the solution by phenol-sulfuric acid showed a good linear relationship with glucose concentration at 20.0?120.0?g/ml.The regression equation was A=0.0089C+0.0455?R²=0.9986?.The reproducibility was RSD=0.391%?n=6?.The accuracy was RSD=0.265%?n=6?.The stability within 120 mins was RSD=0.386%?n=7?.The average recovery rate was 99.7±1.01%?n=6?.The total content of polysaccharides in the four batches of samples were 2.13?3.04%.??1?The rats in the control group and the PSPT treated group had normal diet and activity;those in the model group had less movement with arched back symptoms.The lung surface of the rats of the control group was smooth and elastic.The weights of rats in the model group were lighter than those of the other groups,but the difference was not significant?P>0.05?.?2?The model group showed unevenness and atrophy in the lung tissue surface.The color,elasticity,and nodular-like changes of the lung tissues in rats of each intervention group were all improved.?3?Compared with those of the control group,the m RNA expression levels of IL-1?,IL-6,NALP3,and TNF-?in the lung tissues of the model group on d7,d14,d28,and d56 were all increased?P<0.001?.Compared with those of the model group,the m RNA expression levels of IL-1?,IL-6,NALP3,and TNF-?at each detection time in the lung tissues of rats in each intervention group with PSPT were generally reduced?P<0.05?.?4?The quantitative results of the i TRAQ proteome showed that the total number of differential proteins identified in the model group was 214 as compared to the control group,among which 137 were up-regulated proteins and 77 were down-regulated proteins.?5?Compared with the control group,the alveolar inflammation and pulmonary fibrosis in rats of the model group were exacerbated on day 56.Their lung coefficients and the hydroxyprolines were also increased,and the m RNA and protein expression levels of Vimentin and?-SMA were significantly increased.Their E-cadherin m RNA and protein expression levels were significantly reduced and m RNA expression levels of TGF-?were significantly increased.Compared with the model group,the alveolar inflammation and pulmonary fibrosis in rats of the intervention groups with different doses of PSPT decreased on day 56.Both their lung coefficients and the hydroxyprolines were decreased;their m RNA and protein expression levels of vimentin and?-SMA were significantly reduced;the RNA and protein expression levels of E-cadherinm were significantly increased,and the m RNA expression level of TGF-?was significantly decreased.Conclusions:?The ultrasonic extraction and ultrafiltration membrane separation technology for PSPT is simple,fast and efficient,which is suitable for large-scale production.The content of PSPT single components can be measured by the HPLC-ELSD method simply and conveniently,and the total amount of PSPT can be determined by the phenol-sulfuric acid method.?PSPT can reduce the expression level of certain pro-inflammatory cytokines,inhibit the infiltration of inflammatory cells,and alleviate the damage to the lung tissues of experimental rats with silicosis.The epithelial-mesenchymal transition?EMT?plays an important role in the occurrence and development of silicosis fibrosis.PSPT can inhibit the EMT of the lungs of rats with silicosis,reduce the deposition of extracellular matrix,and can effectively interfere with the process of silicosis fibrosis.PSPT is of great significance in the quality control of Zhuang's PT tablets.