| Objective:The bilateral ovariectomized（OVX）rat model was used to simulate the abnormal state of bone and lipid metabolism in postmenopausal women.Consequently,we employed it to explore the effect and mechanisms of the water extraction of Rehmanniae Radix Praeparata（RR）in bone and lipid metabolism disorders,which were based on the differential mi RNAs in serum exosomes（Exo）and the target gene and the relative signaling pathway between Sham rats and OVX rats.Methods:1.40 3-month-old SPF SD female rats were randomly divided into 5 groups after 1 week of adaptive feeding with water and food ad libitum,namely the sham operation（Sham）group,the model（OVX）group,the low-dose water extraction of RR（RR-L）group,the medium-dose water extraction of RR（RR-M）group,and the high-dose water extraction of RR（RR-H）group.Only part of fat around the ovary was removed from the rats in the Sham group and OVX surgery was applied on the rats in the other groups.After 12 weeks of drug gavage,the serum was collected to measure the level of estrogen（E2）by ELISA to explore the estrogenlike effect of the RR water extraction;uterus were taken for weighing and photographing,livers and kidneys were taken for HE staining to judge the liver and kidney function.2.The femurs of rats were taken for Micro-CT scanning,3D reconstruction and HE staining to observe the changes of bone microstructure and bone mineral density,based on which the optimal dose of the water extraction of RR were determined.Automatic biochemical analyzer and ELISA were used to detect the serum levels of bone metabolism factors including Ca2+,P5+,osteocalcin（OCN）,tartrateresistant acid phosphatase（TRAP）;RT-q PCR was used to detect the femoral alkaline phosphatase（ALP）,OCN,collagen type I alpha 1 chain（COL1A1）and Runt-related transcription factor 2（RUNX2）m RNA levels;Immunohistochemistry（IHC）was used to detect the protein location of COL1A1 and RUNX2 in femur;Western blot was used to detect the protein expression of femoral ALP,COL1A1 and RUNX2,which reflected the impact of bone metabolism by the water extraction of RR.3.The weight and abdominal circumference of rats were measured and the serum lipid metabolism factors including insulin（INS）,total cholesterol（TC）,triglyceride（TG）,low-density lipoprotein cholesterol（LDL-C）and high-density lipoprotein cholesterol（HDL-C）were detected by ELISA and automatic biochemical analyzer;the livers were stained with Oil Red O,all of which were utilized to observe the effect of the water extraction of RR on serum and liver lipid metabolism in OVX rats.HE staining was performed on abdominal adipose to measure the size of adipose cells;the m RNA levels of peroxisome proliferatoractivated receptor γ（PPARγ）,glycoprotein non-metastatic melanoma protein B（GPNMB）,tumor necrosis factor-α（TNF-α）and interleukin-1β（IL-1β）were detected by RT-q PCR;Western blot was used to identify the protein expression of PPARγ,TNF-α and IL-1β in abdominal adipose,all of which were used to observe the effect of the water extraction of RR on lipid metabolism in abdominal adipose.4.Serum Exo was extracted by Exo Quick TM kit and transmission electron microscope,laser nanoparticle size analyzer and Western blot were used to Exo identification.Mi RNAs in Exo were extracted and the level of mi R-29a-3p was detected by RT-q PCR.The m RNA levels of mi R-29a-3p,nuclear factor I/A（NFIA）and canonical Wnt signaling pathway inhibitor-secreted frizzled-related protein 1（SFRP1）in femur and abdominal adipose were detected by RT-q PCR;Western blot was used to detect the key factors of canonical Wnt signaling-SFRP1,low-density lipoprotein receptor-related protein 5（LRP5）,phosphorylated glycogen synthase kinase-3β（P-GSK-3β）（Ser 9）,and glycogen synthesis kinase-3β（GSK-3β）in femur and abdominal adipose,which was beneficial to explore the mechanism of the water extraction of RR on bone and lipid metabolism.Results:1.Low,medium and high doses of the water extraction of RR could increase the serum E2 level of OVX rats（P<0.05）,and could not change their uterine morphology and index（P>0.05）;all of them could not cause obvious pathological changes in liver and kidney of OVX rats either.2.The bone microstructure of femur in OVX rats was severely damaged,and the BMD of OVX rats was also significantly reduced（P < 0.05）,but the bone microstructure of femur could be significantly improved in RR-M group.The level of bone metabolism factors in serum showed that the levels of serum Ca2+,P5+ and TRAP were higher than those of Sham rats（P<0.05）,while the level of serum OCN in OVX rats was lower than that of Sham rats（P<0.05）.The level of serum P5+ could be reduced after treating by RR water extraction（P < 0.05）.Correspondingly,the m RNA expressions of ALP,OCN,COL1A1 and RUNX2 were decreased（P<0.05）in femur of OVX rats,and the protein expressions of ALP,COL1A1 and RUNX2 were also decreased（P<0.05）,both of which could be increased after the treatment of RR water extraction（P<0.05）.Thereby,bone metabolism in OVX rats was improved.3.The weight of OVX rats was significantly higher than that of Sham rats from the 2nd week,and there was no significant decrease after the intervention of RR water extraction（P>0.05）.However,the middle dose of the water extraction of RR could reverse the increasing abdominal circumference of OVX rats（P<0.05）,which could also reduce the levels of INS,LDL-C and liver lipid accumulation in them（P<0.05）.Both the adipose formation-related genes PPARγ and GPNMB and the inflammation factors TNF-α and IL-1β were inhibited by the water extraction of RR（P<0.05）.Thereby,lipid metabolism in OVX rats was improved.4.The levels of mi R-29a-3p in serum Exo,femur and abdominal adipose of OVX rats were significantly lower than those of Sham rats（P<0.05）,but all of them were increased after the intervention of RR water extraction（P<0.05）.Simultaneously,the levels of NFIA in femur and abdominal adipose of OVX rats were significantly increased（P < 0.05）but decreased after intervention（P<0.05）,which was predicted as a target gene of mi R-29a-3p.NFIA regulates the Wnt signaling pathway by targeting SFRP1.The Wnt signaling pathway in both the femur and the abdominal adipose were inhibited in OVX rats（P<0.05）,but were significantly activated after intervention（P<0.05）.As a result,the bone and the lipid metabolism were improved.Conclusion:1.Gavage for 12 weeks by low,medium and high doses of RR water extraction increased the serum E2 level of OVX rats without changing the uterine morphology and index,and could not cause toxic damage to the liver and kidney of them.2.The bone microstructure and BMD of femur were severely damaged.But the RRM could significantly improve the bone microstructure.The improvement of bone metabolism in OVX rats was attributed to the fact that the water extraction of RR could inhibit the bone resorption and promote the formation of femur.3.The weight and abdominal circumference of OVX rats were significantly higher than those of Sham rats,while the water extraction of RR could reduce the abdominal circumference of OVX rats but did not change their weight.The water extraction of RR reduced the levels of INS and LDL-C in rat serum and the lipid accumulation in liver.Besides,the expression of adipose formation-related genes and inflammatory factors in abdominal adipose were inhibited,resulting in the improvement of lipid metabolism in OVX rats.4.The water extraction of RR improved the bone and lipid metabolism in OVX rats by increasing the level of mi R-29a-3p in serum Exo,femur and abdominal adipose,which inhibited the expression of NFIA and activated the Wnt signaling pathway in femur and abdominal adipose.As a result,bone and lipid metabolism were ameliorated. |
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