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ObjectiveTo investigate the mechanism of Xiaonang Tiaojing decoction（XNTJD）in improving polycystic ovary syndrome with insulin resistance（PCOS-IR）model rats based on anti-Müllerian hormone（AMH）/AMH type Ⅱ receptor（AMHRⅡ）signaling pathway. MethodForty-eight adult female SD rats were randomly divided into the blank group， model group， XNTJD group（11.4 g·kg-1·d-1） and Diane-35 group（0.21 g·kg-1·d-1）， PCOS-IR model was established by high-fat and high-sugar diet combined with letrozole in rats of all groups except the blank group， rats in the administration groups were given the corresponding dose of drugs by gavage for 15 days with an interval of 1 d every 4 d， normal saline of the same volume was given to the blank group and the model group. Estrous cycle was recorded daily during treatment. At the end of treatment， body weight and Lee's index were recorded， AMH， luteinizing hormone（LH）， LH/follicle stimulating hormone（FSH）， testosterone（T）and estradiol（E2）levels were measured by enzyme-linked immunosorbent assay（ELISA）， fasting plasma glucose（FPG）was measured by glucometer， fasting insulin（FINS） level was measured by radioimmunoassay（RIA）， and the insulin resistance index（HOMA-IR） and insulin sensitivity index（QUICKI）were calculated， triglyceride（TG）and total cholesterol（TC）levels were measured by automatic biochemical analyzer， hematoxylin-eosin（HE）staining was used to observe the morphological changes of the ovary， the levels of AMHRⅡ， bone morphogenetic protein-15（BMP-15）and Smad5 in ovarian tissues were detected by immunohistochemistry（IHC），Western blot was used to analyze the protein expression levels of AMHRⅡ， BMP-15 and Smad5. ResultCompared with the blank group， a large number of leukocytes were observed in the vaginal exfoliated cells of rats in the model group， mainly in diestrus， the levels of body weight， Lee's index， glucose-lipid metabolism indexes（FPG， FINS， HOMA-IR， TG， TC）， AMH and sex hormones（LH， LH/FSH， T）were significantly increased（P<0.01）， and QUICKI and E2 levels were significantly decreased（P<0.01）， there were more cystic bulges on the ovarian surface， more wet weight， more atretic and cystic dilated follicles in the ovarian tissues， and the thickness of granulosa cell layer was reduced without oocytes， the expression level of AMHRⅡ protein in ovarian tissues was significantly increased（P<0.01）， and the expression levels of BMP-15 and Smad5 proteins were significantly decreased（P<0.01）. Compared with the model group， the exfoliated cells in the vagina of rats treated with XNTJD group showed keratinocytes from the 5th to 6th day of treatment， and a stable estrous cycle gradually appeared， body weight， Lee's index， glucose-lipid metabolism indexes（FPG， FINS， HOMA-IR， TG， TC）， AMH and sex hormones（LH， LH/FSH， T）levels were significantly decreased（P<0.05， P<0.01）， QUICKI and E2 levels were significantly increased（P<0.01）， ovarian surface was smoother and lighter in wet weight， oocytes and mature follicles were observed in ovarian tissues， the thickness of granulosa cell layer increased and the morphology was intact， the expression levels of BMP-15 and Smad5 proteins were significantly increased（P<0.01）and the expression level of AMHRⅡ protein was significantly decreased（P<0.01）in ovarian tissues. ConclusionXNTJD may mediate the up-regulation of BMP-15 and Smad5 in ovarian tissues of PCOS-IR rats by down-regulating AMH/AMHRⅡ， thereby improving ovarian function， sex hormones and glucose-lipid metabolism levels in PCOS-IR rats.
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OBJECTIVE: To observe the effect of guhong injection(GHI) on tibial fracture healing in rats and to explore the mechanism of the action of GHI. METHODS: One hundred and eighty male SD rats were randomly divided into 6 groups with 30 rats in each group: sham operation group, model group, positive drug group(compound ossotide injection, 5 mL•kg-1), low, medium and high dose of GHI groups(2.5, 5, 10 mL•kg-1). In addition to the sham operation group, the other groups established the rat model of tibial fracture. All were given once daily intraperitoneal injections and samples were taken at 1st, 2nd, 4th and 6th week. Blood biochemical analysis and Elisa kit detection were performed on blood samples. X-rays, biomechanical tests, immunohistochemistry and RT-PCR were performed on the tibial samples. RESULTS: ①After administration for one, two and four weeks, the levels of serum calcium(Ca) and phosphorus(P) in medium and high dose of GHI groups were higher than those in model group(P<0.05). ②X-ray showed that the outer callus growth and the disappearance of fracture line in all dose groups of GHI were faster than those in model group. ③Compared with model group, the maximum load and rigidity of medium and high dose of GHI groups were increased at each time point(P<0.05), and the trend of stress line graph were improved obviously. The content of alkaline phosphatase(ALP) in medium and high dose of GHI groups were higher than that in model group at each time point(P<0.05). Compared with model group, the serum levels of PDGF were increased in all dose groups of GHI(P<0.05 or P<0.01). After administration for one, two and four weeks, the serum BMP-2 in all dose groups of GHI were higher than those in model group(P<0.05 or P<0.01). ⑤Compared with model group, the expression of Runx2 mRNA were increased in medium and high dose of GHI groups, as well as Smad5 protein expression(P<0.05 or P<0.01). CONCLUSION: GHI could significantly improve the biomechanical properties of bone in fracture rats. The promotion of fracture healing might be through the upregulation of PDGF and BMP-2 expression in different stages of bone healing, and the regulation of BMP/Smad5/ Runx2 signaling may be one of the mechanisms of promoting fracture healing.
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Objective To investigate the role of miR-155 in diabetic nephropathy(DN)and its mecha-nism. Methods MiR-155 expression level in kidney was detected by real-time PCR and in situ hybridization. The target gene of miR-155 was predicted by bioinformatics and verified by Western Blot and double luciferase reporter activity. Western Blot was used to detect the related marker proteins of mesangial cells proliferation and mesangial matrix. Results (1)The expression of miR-155 increased in DN renal tissue and high glucose-stimulated renal cells.(2)MiR-155 was related to the regulation of Smad5 gene expression.(3)MiR-155 promoted the mesangial cells proliferation and increased extracellular matrix by down-regulating Smad5 expression. Conclusions MiR-155 can promote the mesangial cells proliferation and renal fibrosis by regulating Smad5 gene,providing a basis for further understanding the pathogenesis of DN.
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Objective To investigate the role of miR-155 in diabetic nephropathy(DN)and its mecha-nism. Methods MiR-155 expression level in kidney was detected by real-time PCR and in situ hybridization. The target gene of miR-155 was predicted by bioinformatics and verified by Western Blot and double luciferase reporter activity. Western Blot was used to detect the related marker proteins of mesangial cells proliferation and mesangial matrix. Results (1)The expression of miR-155 increased in DN renal tissue and high glucose-stimulated renal cells.(2)MiR-155 was related to the regulation of Smad5 gene expression.(3)MiR-155 promoted the mesangial cells proliferation and increased extracellular matrix by down-regulating Smad5 expression. Conclusions MiR-155 can promote the mesangial cells proliferation and renal fibrosis by regulating Smad5 gene,providing a basis for further understanding the pathogenesis of DN.
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Objective To observe the Smad5 gene knockout and induced-changes of auditory physiology and cochlea morphology in mouse, to explore whether Smad5 gene is a new gene related to hearing function. Methods Double blind control methods were used to detect the auditory threshold of auditory brainstem responses (ABR) in the mouse of Smad5 (+/+) to Smad5(+/-) and the cochlea morphology (cochlea paraffin-cut section and basal membrane spreading section). The hair cell count was also taken. Results As shown by ABR audiometry, the average hearing of Smad5 (+/-) mouse (aged 24 weeks) is 90.63?17.65 dB (SPL) and that of Smad5(+/+) mouse is 63?19.94 dB(SPL), which are of significant difference (P0.05). Conclusion Smad5(+/-) gene knockout can cause the mouse a auditory threshold decline in moderate or severe extent. Cochlea morphology indicated that hair cells (mainly outer hair cells) in mouse cochlea basal membrane became deficient. A mechanism of Smad5 gene knockout to caused the deafness and the deficiency of hair cells remained to be further studied.
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Objective To investigate the expression of Smad5 gene in the cochlea and to identify its location. Methods Reverse transcription polymerase chain reaction (RT-PCR) was employed to detect the expression of Smad5, the in situ hybridization and the immunohistochemical technique were used to localize the Smad5 messenger RNA and the Smad5 protein in mouse cochlea. Results Smad5 gene expressed in cochlea at a high level. Smad5 expression was concentrated in supporting cell, hair cell, spiral ganglion, epithelium of stria vascularis and basilar membrane. Conclusion The Smad5 proteins exist in the mouse cochlea and it may be involved in the cochlear formation and the differentiation of hair cell. Smad5 might be an essential factor for the development of normal cochlea.