| Terminalia chebula Retz(TC)is the dried ripe fructus of Terminalia chebula Retz.or Terminalia chebula Retz.var.tomentella Kurt.Rubia cordifolia L.processed Terminalia chebula Retz(RTC)is a calming formula obtained by the Rubia cordifolia L.ash and ale.Now,studies on spleen morphological structure and immune function of Terminalia chebula Retz polysaccharide(TCP)and Rubia cordifolia L.processed Terminalia chebula Retz polysaccharide(RTCP)have not been reported.In this experiment,the RTC and TC was used as the research object,and the active ingredient-polysaccharide was extracted.The 21-day-old Chinese yellow quail were randomly selected and divided into model group,blank group,TCP group and RTCP group.After the age of 31 days,the effects of RTCP and TCP polysaccharides on the morphological structure and immune function of spleen of immunosuppressed Chinese yellow quail were studied by light microscope and RT-qPCR,aiming to expand the application of TC and RTC.The results indicate that:1.The screening index is the on extraction rate of TCP and RTCP.Through the single factor experiment and the orthogonal experiment,the main factors influencing TCP and RTCP in cellulase extraction and soxhlet extraction method are studied.(1)The best extraction process for extracting water-soluble crude polysaccharides from TC by the cellulase method was determined as follows: solid-liquid ratio is 1:50,extraction temperature is 50℃,extraction time is 60 min and the enzyme dosage is 1.8%,the biggest extraction rate of TCP in this term reaches 2.92%.The best extraction process for extracting water-soluble crude polysaccharides from RTC by the cellulase method was determined as follows: solid-liquid ratio is 1:40,extraction temperature is 40℃,extraction time is 90 min and the enzyme dosage is 1.8%.the biggest extraction rate of RTCP in this term reaches 3.29%.(2)The optimal extraction process of soxhlet method for extraction of water-soluble crude polysaccharides from TC was as follows: solid-liquid ratio is 1:40,extraction time is4 h,extraction times is 5 times,the concent of ethanol in the solvent is 0%.the biggest extraction rate of TCP in this term reaches 4.63%.The optimum extraction technology of soxhlet method for extracting water-soluble crude polysaccharides from RTC was as follows: solid-liquid ratio is 1:40,extraction time is 4 h,extraction times is 5 times,and the content of ethanol in solvent is 0%.the biggest extraction rate of RTCP in this term reaches4.76%.The above two extraction methods have confirmed that the RTCP extraction rate is better than TCP under the same extraction method.In order to further explore whether the immune effect of the extracted TCP and RTCP is different under the same extraction method,Soxhlet extraction will be used in animal experiments The TCP and RTCP obtained by the method were used for subsequent experiments.2.Using HE,TUNEL staining,qPCR and others,the immune effects of TCP and RTCP extracted by Soxhlet were compared.To study the structural changes,apoptosis changes and the immune-related genes of T-bet and Gata-3 in spleen tissues from Chinese yellow quail.The results indicate that:(1)Histopathological results showed that in the model group,the spleen atrophied,the number of splenic nodules decreased and the area decreased.The splenic nodules’ s number in the model group was extremely significantly(P<0.01)lower than that in the blank group.Compared with the model group,the spleen volume of the TCP group and the RTCP group increased,and the number and area of spleen nodules increased.Among them,the spleen nodules in the RTCP group were extremely significantly(P<0.01)more positivethan those in the model group,and there was no significantly(P>0.05)difference between the data and the blank group(2)The TUNEL results indicate that: The positive cells of TUNEL in the model group,blank group,TCP group and RTCP group were distributed in white pulp and red pulp,but mainly in white pulp,and the nuclei of the positive cells were brown and yellow.TUNEL positive cells were significantly more distributed in model group than in blank group,TCP group and RTCP group.In the model group the positive cells of TUNEL were extremely significantly(P < 0.01)than those in the blank group.TCP group,RTCP group were extremely significantly(P < 0.01)higher than blank group,but significantly(P < 0.05)lower than model group.(3)RT-qPCR results showed that: The effects of TCP and RTCP on GATA-3 and T-bet in the spleen of Chinese yellow quail were as follows.The expression of GATA-3 gene in RTCP group was extremely significantly(P<0.01)higher than that in negative group and TCP group.the blank group was remarkably(P<0.05)higher than the model group and the TCP group.However,there was no remarkable diffrerece in the expression of T-bet gene among these groups.The results indicate that these TCP and RTCP both can ameliorate the morphology,structure and immunity of the spleen of immunosuppressed Chinese yellow quail caused by CTX,but the processed RTCP could improve the immune function of the spleen of immunosuppressed Chinese yellow quail and Conducive to regulating the immune status of the spleen. |
