| Rehmannia is a traditional Chinese traditional medicine in China.According to the different processing methods of Rehmannia glutinosa,the effect of Rehmannia glutinosa is quite different,Modern pharmacology studies have found that Rehmannia has many pharmacological effects,such as anti Alzheimer's disease,protection of gastric mucosa,protection of cardiovascular system and anti-tumor.There are a lot of studies have shown that the black rehmannia pill containing rehmannia and six flavored Rehmannia pills can play an active role in the treatment of renal fibrosis.Tang Xuedong's study has shown that Rehmannia can reduce the expression of T beta R I and Smad2 mRNA and increase the expression of Smad7 mRNA.In traditional Chinese medicine,gold is the mother of water and.It means that lungs and kidneys are closely related.Therefore,it is worth exploring whether Rehmannia Rehmannia can inhibit interstitial fibrosis.The content of this study includes three parts,1)Determination of Catalpol,leonuride and verbascoside by HPLC method in ultrasonic and reflux Rehmannia extract of different concentration of methanol,optimize the extraction conditions of drugs.2)The optimum injection dose and time of the BLM model of pulmonary fibrosis in mice were obtained by pathological observation.3)Comparison of inflammation and fibrosis in each group by observing the pathological section of lung tissue in mice which had given Rehmannia extract.We compared the effects of different products of Rehmannia glutinosa on pulmonary interstitial fibrosis in mice,and explored the mechanism of its action by comparing the characteristics of collagen,the indicators of oxidative damage in lung and the expression of protein in fibrotic signaling pathway.Test 1 Determination of the composition of Rehmannia extract by HPLC Three kinds of Radix Rehmanniae are extracted with 20%,60%and 100%methanol solutions by two extraction methods of reflux and ultrasound.We detect the content of Catalpol,leonuride and acteoside in Rehmannia extract by HPLC.And we check the stability of the target component in the extract within 24h.Result show that the content of catalpol,leonuride and verbascoside decreased with the increase of the concentration of methanol in the same preparation method,and the 20%is the best concentration of methanol.In Radix Rehmanniae Recens the extraction rate of Catalpol was 15.4mg/g,the extraction rate of Leonurus glucoside was 2.1mg/g and the extraction rate of glucoside is 0.4mg/g.In Rehmannia glutinosa Libosch the extraction rate of Catalpol was 6.6mg/g,the extraction rate of Leonurus glucoside was 0.7mg/g and the extraction rate of glucoside is 0.3mg/g.In Prepared rehmannia root the extraction rate of Catalpol was 0.3mg/g,the extraction rate of Leonurus glucoside was 0.1mg/g and the extraction rate of glucoside is 0.2mg/g.When 20%methanol is used as a solvent,ultrasonic and reflux have little effect on the extraction rate of target components.Three target components are stable in 20%methanol extract within 24h.With the deep processing of rehmannia,Catalpol,leonuride and verbascoside content were significantly lower.Test 2 Effect of BLM dose on pulmonary fibrosis in mice The experimental mice were divided into group O,group L,group M and group H.It is 30 rats in each group.The model was made by intraperitoneal injection of BLM 40mg/kg·2d.It is group L which injected 4 times,group M was injected 5 times and group H was injected 6 times.Group O is treated with 0.9%NaCl.It is nessary to observe mental state,fur state,respiratory sound,exercise and death of mouse from the first day.The mice were randomly selected and killed by removing from the eyeball at 21st day,28th day and 35th day.Lungs were collected to make pathological sections.Szapiel method is used to evaluate pulmonary inflammation and fibrosis.The results show that the model mice died between the experimental 14-27d,And the mortality rate was 0%in group L,30%in group M and 50%in group H.The pathological section of mice showed that the level of pulmonary inflammation was positively correlated with the number of BLM injections,and the inflammation increased with time.At 21d,only a small amount of pathological collagen deposition was observed in group H and M.Collagen deposition was observed on the twenty-eighth day of the model mice,but only a few scattered collagen deposits were found in the L group.The collagen in the lung tissue of M and H groups was large and sticky.To thirty-fifth days,pulmonary fibrosis in group L was still aggravated,and the pulmonary fibrosis in M and H groups was significantly reduced.It was suggested that a large amount of collagen deposition in the lungs of mouse 28d was found in the 5 consecutive injection of BLM40mg/kg.48h.In addition that only 30%of the mice died in this scheme.So this method can be used as a recommended method for the model of pulmonary interstitial fibrosis in mice.Test 2 Effect and mechanism of Rehmannia glutinosa on pulmonary fibrosisy Preparaing the tested drugs and mouse models based on the results of test 1 and test 2.We set up the mold and give the Rehmannia extract from the first day.And we stop giving the Rehmannia extract at the 23rd day.At the 28th day,we kill the mice by removing the eyeball and collecting the lungs.And then,The lung pathological sections should be made to evaluate the level of inflammation and fibrosis.Lung tislOsue homogenate was used to detect the hydroxyproline,the MDA,the SOD and the GSH-px.Western blot was used to detect the protein in the TGF-beta 1/Smads pathway.Results show that all the extract of Rehmannia glutinosa can reduce the pulmonary fibrosis and inflammation in the model mice to some extent,and reduce the collagen level in the lung tissue.But the effect of Radix Rehmanniae Recens on pulmonary fibrosis is the weaker than Rehmannia glutinosa Libosch and Prepared rehmannia root.Compared with the model group,the MDA in the lung tissue of all the control groups was significantly higher(P<0.01).At the same time,the activity of GSH-px increases significantly(P<0.05),and the activity of SOD increases slightly(P>0.05).This shows that the three Rehmannia extract can enhance the antioxidant capacity of lung tissue by improving the activity of SOD and GSH-px.The results of TGF-beta 1/Smads showed that the expression level of TGF-beta 1 in the lungs of the Rehmannia glutinosa Libosch group and the Prepared rehmannia root group was significantly lower than that of the model group(P<0.01),and the expression of Smad2/3 protein was also significantly inhibited(P<0.05).The expression of TGF-?1 and Smad2/3 in the treatment group of Radix Rehmanniae Recens was also inhibited slightly(P>0.05).There was no significant difference in the expression of T?R ?,T?R ?,Smad4,Smad6 and Smad7 in all the mice.The results showed that extracts from the Rehmannia glutinosa Libosch and the Prepared rehmannia root could effectively prevent and control lung fibers,reduce lung injury and collagen deposition,and inhibit the development of pulmonary fibrosis in mice.The results showed that extracts from Rehmannia glutinosa and Rehmannia glutinosa could effectively reduce the lung injury and collagen deposition in mice,and play a role in inhibiting the development of pulmonary fibrosis.Conclusion:Refluxing with20%methanol is the best extraction method for making the extraction of Rehmannia glutinosa.The pulmonary interstitial fibrosis model of mice can be successfully prepared by intraperitoneal injection of BLM40mg/kg 2D for 5 consecutive times.And 28d is the best time to observe the pulmonary fibrosis.The level of pneumonia and pulmonary fibrosis in the lungs of the mice in control group of Rehmannia glutinosa Libosch and Prepared rehmannia root was obviously reduced,after continuous 23 days of treatment with Rehmannia extract.Rehmannia can reduce lung oxidative damage by enhancing SOD and GSH-px activity,and it lessen the fibrotic effect of TGF-beta 1/Smads by inhibiting the expression of TGF-beta 1 and Smad2/3. |
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