| Purpose: Established an animal model of Spleen phlegm in Bama miniature pigs,from miR-33 and its co-transcription host genes SREBP-1c,SREBP-2 and downstream pathways ABCA1,ABCG1,NPC1,etc.In order to Invigorate the striking,remove phlegm and remove blood stasis legislation,Respectively discuss with strengthening the striking,striking and eliminating phlegm and eliminating phlegm blood and Invigorate striking,remove phlegm and remove blood stasis of TCM compound for striking phlegm syndrome Obama small liver of pig effect and its mechanism of action of cholesterol metabolism,further interpretations of Invigorate striking,remove phlegm and remove blood stasis method effect and its mechanism of regulating the cholesterol metabolism.Methods: The SPF grade guangxi bama miniature pig was selected as the experimental animal,which was ethically in accordance with the regulations of the ministry of health of the People's Republic of China on the administration of animal experiments(No.55,2001)and the regulations of liaoning university of traditional Chinese medicine on the administration of experimental animals.After 2 weeks of adaptive feeding,48 small pigs were randomly divided into two groups by random number table method: 5 blank control groups,43 models,sprint running training combined with high fat single cage feeding combined with balloon straining method.Modeling Spleen phlegm syndrome animal model.The successful Bama miniature pigs were divided into 6 groups: Model control group(n=7),strengthening the spleen group(n=7),Spleen and elimination phlegm group(n=7),Eliminating phlegm blood group(n=7),Invigorate spleen,remove phlegm and remove blood stasis group(n=7),In addition to the model control group,the traditional Chinese medicine compound was used to intervene.The strengthening of the spleen group was added to the spleen side,the Spleen and eliminating phlegm group was added to the Spleen and eliminated phlegm side,and the Eliminating phlegm blood group was added to the Eliminating phlegm blood group.Invigorate spleen,remove phlegm and remove blood stasis were added to Invigorate spleen,remove phlegm and remove blood stasis for 24 weeks.The indicators are detected as follows: 1.The levels of TC,TG,LDL-C and HDL-C were detected by automatic biochemical analyzer at 0,4,12 and 24 weeks of the experiment;2.HE staining microscopic observation of liver histopathological changes;3.Detection of miR-33 expression in liver tissue of Bama miniature pigs by real-time PCR;4.RT-PCR was used to detect the expression of SREBP-1c mRNA,SREBP-2 mRNA,ABCA1 mRNA,ABCG1 mRNA and NPC1 mRNA in the liver of Bama miniature pigs.5.Western blotting(Western-Blot)was used to detect the expression of SREBP-1c,SREBP-2,ABCA1,ABCG1 and NPC1 in liver tissue.6.All data in this experiment were statistically analyzed using SPSS17.0 software.The results were expressed as meanąstandard deviation((?)ąs),and one-way ANOVA was used for comparison between groups.(P<0.05 indicates statistical significance,P<0.01 indicates significant difference.)Results: 1.Spleen phlegm syndrome Bama miniature pig model serum TC,TG,LDL-C,HDL-C level changes: in the 0th week,4 weeks,12 weeks and 24 weeks of the experiment,Bama miniature pig serum TC,TG and The level of LDL-C increased,compared with the blank control group,there was a significant difference(P<0.01);In the 0th week and the 4th week of the experiment,the serum HDL-C level of Bama miniature pigs increased,compared with the blank control group,there was a significant difference(P<0.01);At week 12,HDL-C levels increased,compared with the blank control group,there was a statistical difference(P <0.05);At week 24,there was no statistical difference in HDL-C levels compared with the blank control group.2.After the intervention of traditional Chinese medicine compound,the serum TC level of Bama miniature pigs changes: Compared with the model control group,serum TC levels in the Spleen and eliminated phlegm group and the Invigorate spleen,remove phlegm and remove blood stasis group decreased after 4 weeks of intervention,with statistical difference(P<0.05).After 12 weeks of intervention,serum TC levels in the Spleen and eliminated phlegm group and Invigorate spleen,remove phlegm and remove blood stasis group decreased,with statistical difference(P<0.05);After 24 weeks of intervention,serum TC levels in the spleen group,the Spleen and eliminated phlegm group,the Eliminating phlegm blood group,and the Invigorate spleen,remove phlegm and remove blood stasis group decreased,with significant differences(P<0.01).After 24 weeks of intervention,the treatment groups were compared: compared with the strengthening the spleen group,serum TC levels in the Spleen and eliminated phlegm group were significantly lower(P<0.05).Compared with the Eliminating phlegm blood group,serum TC levels in the Spleen and eliminated phlegm group were significantly lower(P<0.05).3.After the intervention of Chinese herbal compound,the serum TG level of Bama miniature pigs changes: Compared with the model control group,the serum TG levels of the Eliminating phlegm blood group and the Invigorate spleen,remove phlegm and remove blood stasis group decreased after 4 weeks of intervention,with statistical difference(P<0.05).After 12 weeks of intervention,serum TG levels in the Spleen and eliminated phlegm group,the Eliminating phlegm blood group,and the Invigorate spleen,remove phlegm and remove blood stasis group were decreased,with statistical difference(P<0.05).After 24 weeks of intervention,serum TG levels in the spleen group,the Spleen and elimination phlegm group,the Eliminating phlegm blood group,and the Invigorate spleen,remove phlegm and remove blood stasis group decreased,with significant differences(P<0.01).After 24 weeks of intervention,the treatment groups were compared: compared with the strengthening the spleen group,the serum TG levels of the Invigorate spleen,remove phlegm and remove blood stasis group were significantly lower(P<0.01);Compared with the Eliminating phlegm blood group,serum TG levels in the Invigorate spleen,remove phlegm and remove blood stasis group were significantly lower(P<0.05).4.After the intervention of Chinese herbal compound,the serum LDL-C level of Bama miniature pigs was changed: compared with the model,the serum LDL-C level of the Bama mini-pigs in the strengthening the spleen group decreased at the 4th week of the experiment,which was statistically significant(P<0.05).Invigorate spleen,remove phlegm and remove blood stasis group Bama miniature pig serum LDL-C levels decreased,there was a significant difference(P <0.01).At the 12 th week of the experiment,serum LDL-C levels in the spleen group,the Spleen and elimination phlegm group,and the Invigorate spleen,remove phlegm and remove blood stasis group were decreased,and there was a statistically significant difference between the groups(P<0.05).At the 24 th week of the experiment,serum LDL-C levels were decreased in the spleen group,the Spleen and eliminated phlegm group,the Eliminating phlegm blood group,and the Invigorate spleen,remove phlegm and remove blood stasis group,and there was a significant difference between the groups(P<0.01).In the Invigorate spleen,remove phlegm and remove blood stasis intervention,the serum LDL-C levels in the Spleen and eliminated phlegm group were compared with the Invigorate spleen,remove phlegm and remove blood stasis group and the Eliminating phlegm blood group after 24 weeks of intervention.There was a significant difference(P<0.01)compared with the strengthening the spleen group(P<0.05).5.Changes in serum HDL-C levels in Bama miniature pigs after intervention with Chinese herbal compound: compared with the model control group,after Invigorate spleen,remove phlegm and remove blood stasis intervention,at 4 weeks,12 weeks and 24 weeks,each There was no statistical difference in serum HDL-C levels between the treatment group and the Bama mini-pig.6.HE staining results,the model control group Bama mini-pig liver cells contained larger lipid droplets,pushing the nucleus to one side,hepatocytes swollen and rounded,the central area of the lobules was affected,and the hepatocytes were arranged disorderly.After 24 weeks of intervention,compared with the model control group,the lipid droplets in the liver cells of the strengthening the spleen group,the Spleen and elimination phlegm group,the Eliminating phlegm blood group,and the Invigorate spleen,remove phlegm and remove blood stasis groups gradually decreased,and the liver cell morphology gradually decreased. Recovery,in which the hepatocyte changes were particularly evident in the Invigorate spleen,remove phlegm and remove blood stasis groups.7.Compared with the blank control group,the expression of SREBP-1c gene and protein in the Spleen phlegm model group was significantly increased(P<0.01),but after 24 weeks of intervention with the Chinese herbal compound,the spleen group,the Spleen and eliminating phlegm group.The expression of SREBP-1c gene and protein in the Eliminating phlegm blood group and the Invigorate spleen,remove phlegm and remove blood stasis group was significantly lower than that in the model control group(P<0.01).Comparing the groups,the SREBP-1c gene and protein expression in the Spleen and eliminated phlegm group,the Eliminating phlegm blood group,and the Invigorate spleen,remove phlegm and remove blood stasis group were significantly lower than those in the strengthening spleen group.P<0.01).Compared with the Spleen and eliminated phlegm group,the expression of SREBP-1c gene and protein in Invigorate spleen,remove phlegm and remove blood stasis group was significantly lower(P<0.01).8.Compared with the blank control group,the SREBP-2 gene and protein expression in the Spleen phlegm model control group increased significantly,with significant difference(P<0.01),and the Chinese medicinal compound intervention for 24 weeks,the spleen group,Spleen and eliminate The expression of SREBP-2 gene and protein in the phlegm group,the Eliminating phlegm blood group and the Invigorate spleen,remove phlegm and remove blood stasis group was significantly lower than that of the model control group(P<0.01).Comparing the groups,the expression of SREBP-2 gene and protein in the Eliminating phlegm blood group and the Invigorate spleen,remove phlegm and remove blood stasis group were significantly lower than those in the strengthening spleen group(P<0.01).Compared with the Spleen and eliminated phlegm group,the expression of SREBP-2 gene and protein in the Eliminating phlegm blood group was statistically different(P<0.05),and the expression of SREBP-2 gene and protein in the Invigorate spleen,remove phlegm and remove blood stasis group decreased.There was a significant difference(P<0.01),and there was no statistical difference in the Eliminating phlegm blood group. 9.Expression of miR-33 in liver tissue: Compared with the blank control group,the expression of miR-33 was increased in the model control group,with significant difference(P<0.01).After 24 weeks of intervention with Invigorate spleen,remove phlegm and remove blood stasis method.,the strengthening of the spleen group,the Spleen and eliminated phlegm group,the Eliminating phlegm blood group and the Invigorate spleen,remove phlegm and remove blood stasis group miR-33 gene and protein expression decreased,compared with the model control group,statistically significant(P <0.05,P<0.01).Comparing the groups,the expression of miR-33 gene in the Limimating phlegm blood group and the Invigorate spleen,remove phlegm and remove blood stasis group was significantly lower than that of the strengthening spleen group(P<0.01).Spleen and elimination phlegm The expression of miR-33 was significantly lower in the group(P<0.05).Compared with the Spleen and eliminating phlegm group,the expression of miR-33 was decreased in the Invigorate spleen,remove phlegm and remove blood stasis groups,with significant difference(P<0.01).10.Compared with the blank control group,the expression of ABCA1 gene and protein in the Spleen phlegm model group was significantly lower(P<0.01).After 24 weeks of intervention with traditional Chinese medicine,the spleen group,Spleen and elimination phlegm group,Eliminating The expression of ABCA1 gene and protein in phlegm blood group and Invigorate spleen,remove phlegm and remove blood stasis group was higher than that in the model control group(P<0.01).Comparing the groups,the expression of ABCA1 gene and protein in the Eliminating phlegm blood group and the Invigorate spleen,remove phlegm and remove blood stasis group were significantly higher than those in the strengthening spleen group(P<0.01),Spleen and eliminated.There was no significant difference in ABCA1 gene between phlegm group and ABCA1 protein expression in Spleen and eliminated phlegm group,which was statistically significant(P<0.05).Compared with the Spleen and eliminated phlegm group,the expression of ABCA1 gene and protein in Invigorate spleen,remove phlegm and remove blood stasis group was significantly higher(P<0.05).There was no statistical difference in ABCA1 gene and protein expression between the Invigorate spleen,remove phlegm and remove blood stasis groups compared with the Eliminating phlegm blood group.11.Compared with the blank control group,the expression of ABCG1 gene and protein in the control group of Spleen phlegm model decreased,and there was significant difference(P<0.01).After 24 weeks of intervention with Invigorate spleen,remove phlegm and remove blood stasis,the expression of ABCG1 gene and protein was increased in the spleen group,the Spleen and eliminated phlegm group,the Eliminating phlegm blood group and the Invigorate spleen,remove phlegm and remove blood stasis groups.High,compared with the model control group,there was a significant difference(P <0.01).Comparing the groups,the expression of ABCG1 gene and protein in Invigorate spleen,remove phlegm and remove blood stasis group was significantly higher than that of strengthening spleen group(P<0.01),and ABGL1 gene in Spleen and eliminated phlegm group.The protein expression was increased(P<0.05),and the expression of ABCG1 gene was increased in the Eliminating phlegm blood group(P<0.05).ncreased protein expression,significant difference(P<0.01).Compared with the Spleen and eliminated phlegm group,the expression of ABCG1 gene in the Eliminating phlegm blood group and the Invigorate spleen,remove phlegm and remove blood stasis group was not statistically different,and the protein expression was increased,which was statistically significant(P<0.05).12.Compared with the blank control group,the expression of NPC1 gene and protein in the Spleen phlegm model control group was significantly lower(P<0.01),but after the intervention of Invigorate spleen,remove phlegm and remove blood stasis method for 24 weeks,strengthen the spleen The NPC1 gene and protein expression in the Spleen and eliminated phlegm group,the Eliminating phlegm blood group and the Invigorate spleen,remove phlegm and remove blood stasis group were significantly higher than those in the model control group(P<0.01).Comparing the groups,the expression of NPC1 gene and protein in the Eliminating phlegm blood group and the Invigorate spleen,remove phlegm and remove blood stasis group were significantly higher than those in the strengthening spleen group(P<0.01),Spleen and eliminated.The expression of NPC1 gene in phlegm group was significantly higher(P<0.05),but there was no statistical difference in NPC1 protein expression between Spleen and eliminated phlegm group.Compared with the Spleen and eliminated phlegm group,the expression of NPC1 gene and protein in Invigorate spleen,remove phlegm and remove blood stasis group was significantly increased(P<0.01),and the expression of NPC1 gene and protein in Eliminating phlegm blood group was increased.Learning differences(P<0.05);There was no statistical difference in NPC1 gene and protein expression levels between the Eliminating phlegm blood group and the Invigorate spleen,remove phlegm and remove blood stasis groups.Conclusion: 1.The sprint running training combined with high-fat single-cage feeding combined with balloon injury method can successfully model the Spleen phlegm syndrome of Bama miniature pigs.The serum TC,TG,LDL-C and HDL-C levels are elevated.The level of miR-33 in liver tissue increased,the expression of SREBP-1c and SREBP-2 increased,and the expression of ABCA1,ABCG1 and NPC1 genes and protein decreased,suggesting that there is lipid metabolism disorder in the Spleen phlegm syndrome model.2.The traditional Chinese medicine compound based on Invigorate spleen,remove phlegm and remove blood stasis method can reduce the levels of TC,TG and LDL-C in Spleen phlegm syndrome Bama pigs,and improve the liver tissue morphology of Spleen phlegm syndrome Bama mini-pig,but for HDL There was no significant difference.3.The traditional Chinese medicine compound based on Invigorate spleen,remove phlegm and remove blood stasis method can inhibit the expression of ABCA1,ABCG1,NPC1 gene and protein,and regulate liver cholesterol metabolism and blood lipid level by inhibiting the expression of SREBP-1c and SREBP-2 genes and proteins in liver tissue.This may be one of the mechanisms for its lipid regulation.4.The Chinese herbal compound based on Invigorate spleen,remove phlegm and remove blood stasis can affect the expression of miR-33,thereby affecting its downstream pathways ABCA1,ABCG1 and NPC1,affecting cholesterol metabolism. |
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