| Objects:To observe the effects of Xuling Jianggu Decoction on Bone Mineral Density,morphology and expression of genes related to BMP/Smads signaling pathway in osteoporosis model rats,in order to explore the molecular mechanism of Xuling Jianggu Decoction in the treatment of postmenopausal osteoporosis.Methods:1.Establishment of animal modelForty 6-month-old clean grade female SD rats were randomly divided into sham operation group,model group and low dose group(hereinafter referred to as low dose group),medium dose group(hereinafter referred to as medium dose group)and high dose group(hereinafter referred to as high dose group)by random number table method,with 8 rats in each group.By ovariectomy,all rats except the sham group were subjected to bilateral ovariectomy to establish an osteoporotic rat model,and the sham group was subjected to the removal of adipose tissue of appropriate size near the ovary.2.Drug InterventionIntragastric administration started 30 days after modeling: The dosage of low,middle and high group is 7.5g/(kg*d),15g/(kg*d),30g/(kg*d)each rat.Each rat in model group and sham operation group was given normal saline 15g/(kg*d),continuous administration for 12 weeks.3.Index detectionAnesthetized rats and draw materials,the specimens of rat lumbar vertebrae and left and right tibia were separated and processed accordingly,and various indexes were detected.Take the right tibia,remove the surrounding soft tissues,and rinse with saline.Tibial bone mineral density of rats in each group was measured by Hologic Discovery W dual-energy X-ray bone mineral density meter.Sections were embedded after fixation and decalcification of the left tibia by the H.E.staining method.Observation of Tibial Trabecular Shape and image acquisition and analysis.Rat lumbar vertebrae were harvested for reverse transcription polymerase chain reaction.Detection of BMP4,Smad1,Smad5,Smad9 mRNA expressionStatistical analysis of the data obtained.Use one-way ANOVA of variance for normal distribution data.If the variance is uniform,LSD method is used,If not,use Dunnett's T3 method.Analysis the data by Kruskal-Wallis one-way ANOVA if the data inconsistent with normal distribution.Take ? = 0.05 as the boundary of statistical significance.Results:1.Weight and BMDThe weight of rats in each group was similar,except that the weight in the sham operation group was higher than that in the model group(P<0.05),and there was no significant difference between the other groups.In terms of BMD,the right tibia of the sham group had the highest BMD and the model group had the lowest BMD.The comparison between groups showed that BMD in sham operation group and medium dose group was higher than other groups,and the difference was statistically significant.2.MorphologyAfter H.E.staining,the trabeculae under epiphyseal line of tibia in each group were as follows:In the sham operation group,there were many trabeculae under epiphyseal line,which were closely arranged,evenly distributed,with small spacing between trabeculae and regular arrangement,and distributed in a network.In the model group,there were fewer trabeculae under epiphyseal line of tibia,sparsely arranged and unevenly distributed.The number of trabeculae under epiphyseal line in the middle dose group was slightly less than that in the sham operation group,but it was significantly more than that in the model group.The arrangement of trabeculae was more uniform and the space between trabeculae was medium.The number of trabeculae in the low and high dose group was significantly less than that in the sham operation group,but still more than that in the model group,with sparse arrangement and large spacing between trabeculae3.Morphological data%Tb.Ar data is the highest in the sham group,and the lowest is the model group.Sham operation group was higher than model group,low and high dose group in %Tb.Ar(P<0.05).The middle dose group was higher than the model group(P<0.05).There were no differences among the other groups(P>0.05).Tb.Th is the highest in the sham group,and the lowest is the model group.Sham operation group was higher than model group and the high dose group(P<0.05).The middle dose group was higher than the model group(P<0.05).There were no differences among the other groups(P>0.05).Tb.Sp in model group was much higher than that in other groups(P<0.05).There was no significant difference between the other groups(P>0.05).The above results further corroborate the results of morphological microscopy observation.The% Tb.Ar and Tb.Th of sham operation group and medium dose group were higher than other groups,and the Tb.Sp of model group was higher than other groups,suggesting that the bone micro-structure of sham operation group and medium dose group was better,and the bone micro-structure of model group was worse.4.RT-PCRThe expression levels of BMP4,Smad1,Smad5 and smad9 mRNA in the model group were the lowest in all groups.BMP4 mRNA expression in sham operation group was significantly higher than that in other groups(P<0.05).The expression of BMP4 mRNA in the medium dose group was higher than that in the model group(P<0.05).There was no significant difference between the other groups(P>0.05).The expression of Smad1,5,9 mRNA in the sham operation group was higher than that in the model group,the low dose group and the high dose group(P<0.05),and there was little difference between the medium dose group and the sham operation group(P>0.05).The expression of Smad1 in the middle dose group was higher than that in the model group(P<0.05).Although the expression of Smad5 and 9 was not significantly different from that of the model group,the relative quantitative values of Smad5 and Smad9 were significantly different,and the nonsense might be related to the excessive standard deviation.Conclusions:Animal experiments confirmed that Xuling Jianggu Decoction could improve the bone density,bone tissue microstructure and the expression of BMP4,Smad1,Smad5,Smad9 mRNA in BMP/Smads pathway in osteoporosis model rats,and the middle dose group had the best effect,which indicated that the molecular mechanism of Xuling Jianggu Decoction inthe treatment of osteoporosis was related to the regulation of BMP/Smads signaling pathway. |
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