Het opreguleren van MicroRNA-410 of het neerwaarts reguleren van Wnt-11 verhoogt osteoblasten en vermindert osteoclasten om osteonecrose van de femurkop te verlichten

Resultaten

Pathologische veranderingen in ONFH-weefsels van klinische monsters

HE-kleuring werd toegepast om de pathologische veranderingen van de femurkop in de femurhalsfractuurgroep (controlegroep) en de ONFH-groep te observeren; de resultaten meldden dat in de controlegroep de trabeculaire botdichtheid goed verdeeld was en dat de structuur integriteit was. In de ONFH-groep waren er veel lege botlacunes in het bot-cerebellum, waarbij botcellen afnamen en bottrabeculae continu veranderden. Bovendien waren er veel andere weefselhyperplasie rond het bot-cerebellum (Fig. 1a).

Pathologische veranderingen in ONFH-weefsels van klinische monsters. een Resultaten van HE-kleuring in elke groep (200 ×). b Resultaten van TRAP-kleuring en het aantal TRAP-positieve cellen in elke groep (200 ×). c Resultaten van ALP-kleuring en ALP-positieve cellen tellen in elke groep (400 x). *P <0,05 vs. de controlegroep

TRAP-kleuring onthulde dat TRAP voornamelijk werd gevonden in osteoclasten en vaak werd gebruikt om volwassen osteoclasten in amarant te identificeren. In vergelijking met de controlegroep steeg het aantal TRAP-positieve osteoclasten in de ONFH-groep, was de morfologie van osteoclasten divers en waren de cellen groot met een multinucleair uiterlijk. Duidelijke botdefecten werden waargenomen in het botcerebellum naast osteoclasten, die botresorptie vertoonden (figuur 1b).

Het resultaat van ALP-kleuring toonde aan dat ALP een markerenzym was voor osteoblastdifferentiatie en rijping, dat betrokken was bij de regulatie van verkalking van botmatrixrijping. Daarom werd ALP-expressie vaak gebruikt om osteoblasten te identificeren. In het cytoplasma van rijpe osteoblasten waren bruine of koffiedeeltjes te zien. Met betrekking tot de controlegroep nam het aantal ALP-positieve osteoblasten af ​​in de ONFH-groep (Fig. 1c).

MiR-410, ALP, BGLAP en Collα1 Degraded en Wnt-11, ACP5, CTSK en MMP9 Enhanced in ONFH-weefsels van de klinische monsters

Western-blot-analyse en RT-qPCR toonden aan dat miR-410-expressie in de ONFH-groep afnam ten opzichte van die in de controlegroep, terwijl Wnt-11-expressie toenam; de expressie van osteoblast-gerelateerde factoren ALP, BGLAP en Collα1 afgebroken; en de osteoclast-gerelateerde factoren ACP5-, CTSK- en MMP9-expressie namen toe (alle P <0,05) (Fig. 2a–c).

MiR-410, ALP, BGLAP en Collα1 namen af ​​en Wnt-11, ACP5, CTSK en MMP9 namen toe in ONFH-weefsels van klinische monsters. een Detectie van miR-410, Wnt-11 en osteoblast- en osteoclast-gerelateerde genen door RT-qPCR. b Wnt-11 en osteoblast- en osteoclast-gerelateerde eiwitten gedetecteerd door western blot-analyse. c Eiwitbanden van Wnt-11 en osteoblast- en osteoclast-gerelateerde eiwitten. d Wnt11-expressie (400 ×) en vergelijking van Wnt11-eiwitpositieve snelheid gedetecteerd door immunohistochemische kleuring. *P <0,05 vs. de controlegroep

Wnt-11-expressie werd getest door middel van immunohistochemie en de resultaten toonden aan dat Wnt-11-eiwit voornamelijk tot expressie werd gebracht in het cytoplasma, en de positieve expressie was in wezen bruinachtig geel of bruin. In tegenstelling tot de controlegroep was de positieve snelheid van Wnt-11-eiwitexpressie in de ONFH-groep verhoogd (P <0,05) (Fig. 2d).

Opgereguleerde miR-410 en gedownreguleerde Wnt-11 Verhoog de BMD en BV/TV van ratten

Micro-CT suggereerde dat in de normale groep het uiterlijk van de femurkop rond was, de dikte van de nek uniform was en de structuur van de bottrabeculae continu en gelijkmatig verdeeld was. De femurkop van de ratten in de ONFH-groep, agomir-NC-groep, siRNA-NC-groep en agomir-miR-410 + OE-Wnt-11-groep zakte geleidelijk in, het botresorptiegebied verscheen geleidelijk, de nek van de dijbeenderen werd dunner en de bottrabeculae werden gebroken en de continuïteit vernietigd. In de agomir-miR-410-groep, Wnt-11-siRNA-groep en agomir-miR-410 + OE-NC-groep bleef het uiterlijk van de ratten intact, er trad geen collaps op, er verscheen geen duidelijk botresorptiegebied, de bottrabeculae waren normaal gerangschikt en de structuur was voltooid (Fig. 3a).

Sterk tot expressie gebrachte miR-410 en slecht tot expressie gebrachte Wnt-11 verhogen BMD en BV/TV van ratten. een Micro-CT-resultaten van ratten in elke groep. b Resultaten van BMD-analyse. c Resultaten van BV/TV-analyse. *P <0,05 vs. de normale groep. ⁺ P <0,05 vs. de agomir-NC-groep. ^# P <0,05 vs. de siRNA-NC-groep. ^& P <0,05 vs. de agomir-miR-410 + OE-NC-groep

De resultaten van botmetrologie meldden dat in tegenstelling tot de normale groep, de BMD en BV/TV in de ONFH-groep depressief waren (beide P <0,05). Met betrekking tot de agomir-NC-groep en de siRNA-NC-groep, waren de BMD en BV/TV verhoogd in de agomir-miR-410-groep en de Wnt-11-siRNA-groep (allemaal P <0,05). In vergelijking met de agomir-miR-410 + OE-NC-groep, werden de BMD en BV/TV in de agomir-miR-410 + OE-Wnt-11-groep geschrapt (beide P <0,05) (Fig. 3b, c).

Overexpressie van miR-410 en slechte expressie van Wnt-11 Verhoog de BMD-niveaus van de femurschacht, femurkop en wervelkolom, en ook het verhoogde serumcalcium en fosforgehalten van ratten

Röntgenfoto's zagen dat het uiterlijk van de femurkop in de normale groep rond was, de dichtheid van de femurkop uniform was en de structuur compleet was. In de ONFH-groep, agomir-NC-groep, siRNA-NC-groep en agomir-miR-410 + OE-Wnt11-groep werd het uiterlijk van de femurkop van ratten dunner en verscheen het botresorptiegebied. Het uiterlijk was gelijk en de dikte van de femurkop van de ratten was compleet in de agomir-miR-410-groep, Wnt11-siRNA-groep en agomir-miR-410-groep + OE-NC-groep (Fig. 4a).

Upregulated miR-410 and downregulated Wnt-11 increase the bone mineral density level of the femoral shaft, femoral head, and spinal column, and also raise the serum calcium and phosphorus levels of rats. een Animal X-ray observation results. b Comparison of the bone mineral density of the femoral shaft, femoral head, and spine in each group. c Comparison of the serum calcium and phosphorus levels in each group. *P <0.05 vs. the normal group. ⁺ P <0.05 vs. the agomir-NC group. ^# P <0.05 vs. the siRNA-NC group. ^& P <0.05 vs. the agomir-miR-410 + OE-NC group

BMD and serum calcium and phosphorus level determination reported that compared to the normal group, the BMD of the femoral shaft, femoral head, and spine, as well as the serum calcium and phosphorus levels, fell in the ONFH group (all P <0,05). In contrast with the agomir-NC group and siRNA-NC group, the BMD of the femoral shaft, femoral head, and spine, as well as serum calcium and phosphorus levels, ascended in the agomir-miR-410 group and Wnt-11-siRNA group (all P <0,05). In relation to the agomir-miR-410 + OE-NC group, the BMD of the femoral shaft, femoral head, and spine, as well as the serum calcium and phosphorus levels in the agomir-miR-410 + OE-Wnt-11 group, was abated (all P <0.05) (Fig. 4b, c).

Silencing Wnt-11 and Upregulating miR-410 Alleviate the Pathological Changes of Rat Tissues and Restrain Apoptosis of Osteocytes

The results of HE staining showed that the bone trabeculae were neat and clear and arranged regularly and tightly. Bone cells filled the bone lacunae, and the calcified zone was well connected to the subcartilage bone trabeculae in the normal group. In the ONFH group, agomir-NC group, siRNA-NC group, and agomir-miR-410 + OE-Wnt-11 group, the bone trabecula was sparse; thinned, and even broken; the structure was disordered; and some fragments appeared. Some osteocytes in the bone lacuna were necrotic and a large number of bone lacuna was in emptiness that no bone cells filled in, and obvious proliferation of granulation tissue was observed in the necrosis bone trabecular space, which was wrapped around the necrotic bone trabeculae. In the agomir-miR-410 group, the Wnt-11-siRNA group, and the agomir-miR-410 + OE-NC group, the appearance of the rats remained intact, no obvious bone resorption area appeared and the bone trabeculae were arranged normally and the structure was complete (Fig. 5a).

Poor expression of Wnt-11 and overexpression of miR-410 alleviate pathological changes of rats tissues and restrain apoptosis of osteocytes. een Morphological observation of the femoral head of rats in each group (200 ×). b Ultrastructural observation of bone cells in rats of each group (8000 ×). c The results of TUNEL staining (200 ×). d Detection of apoptosis rate by TUNEL assay. e Detection of the Bax and Bcl-2 mRNA expression by RT-qPCR. v , g Detection of the Bax and Bcl-2 protein expression by western blot analysis. *P <0.05 vs. the normal group. ⁺ P <0.05 vs. the agomir-NC group. ^# P <0.05 vs. the siRNA-NC group. ^& P <0.05 vs. the agomir-miR-410 + OE-NC group

Electron microscopic observation observed that the shape of the bone cells in the normal group was consistent with the lacunae, and there was a small gap between the cell and the lacunae wall. The organelles were abundant, the cells in the lacunae were normal, the nucleus was egg-shaped, the nuclear membrane was intact, the chromatin did not agglutinate, and the cytoplasmic pseudo-foot stretched to the peripheral bone small tube and was connected with the adjacent bone cells. Osteoblasts were located on the surface of the bone trabecula, the sample was long, and there were many organelles. In the ONFH group, agomir-NC group, siRNA-NC group, and agomir-miR-410 + OE-Wnt-11 group, a large number of lipid deposits were found in the cytoplasm of osteoblasts, the gap between capsule and lacunae well was further widened, edema bright bands appeared between the nuclear membrane and cytoplasm, nuclear margination showed with compression, nuclear membrane was intact, mitochondria in cytoplasm was swollen, and the endoplasmic reticulum and Gore apparatus disappeared. In the agomir-miR-410 group, Wnt-11-siRNA group, and agomir-miR-410 + OE-NC group, the morphology of rat osteoblasts was normal, chromatin aggregation was found in the nucleus of a small number of cells, no obvious lipid droplets were found in the cytoplasm, and the nuclear membrane was intact (Fig. 5b).

The results of TUNEL staining demonstrated that compared to the normal group, the apoptosis rate of osteocytes in the ONFH group was raised (P <0,05). In contrast with the agomir-NC group and the siRNA-NC group, the apoptosis rate of osteocytes abated in the agomir-miR-410 group and the Wnt-11-siRNA group (both P <0,05). In relation to the agomir-miR-410 + OE-NC group, the apoptosis rate of osteocytes enhanced in the agomir-miR-410 + OE-Wnt-11 group (P <0.05) (Fig. 5c, d).

Western blot analysis and RT-qPCR reported that the Bcl-2 expression reduced and Bax expression raised in the ONFH group compared to the normal group (both P <0,05). By comparison with the agomir-NC group and the siRNA-NC group, Bcl-2 expression ascended and Bax expression descended in the agomir-miR-410 group and the Wnt-11-siRNA group (all P <0,05). In contrast with the agomir-miR-410 + OE-NC group, the Bcl-2 expression declined and Bax expression appended in the agomir-miR-410 + OE-Wnt-11 group (both P <0.05) (Fig. 5e–g).

Overexpression of miR-410 and Low Expression of Wnt-11 Increase the Number of Osteoblasts and Decrease the Number of Osteoclasts

TRAP staining revealed that in the normal group, the morphology of osteoclasts with positive TRAP staining was different, most of them were shuttle or fusiform, few large osteoclasts were polygons, and most of them were distributed around the bone cerebellum. In the ONFH group, agomir-NC group, siRNA-NC group, and agomir-miR-410 + OE-Wnt-11 group, TRAP-positive cells were ascended, the morphology of cells was diverse in large polygons and polykaryotes, bone resorption was found in the bone trabeculae adjacent to osteoclasts, and typical resorption lacunae were formed. In the agomir-miR-410 group, Wnt-11-siRNA group, and agomir-miR-410 + OE-NC group, the number of cells positive for TRAP staining was reduced, the cells were in a long strip shape and the morphology was more regular, the polygonous polynuclear osteoclasts were rare, and the bone structure of adjacent bone trabeculae was relatively complete (Fig. 6a).

Overexpression of miR-410 and low expression of Wnt-11 increase the number of osteoblasts and decrease the number of osteoclasts. een Results of TRAP staining in osteoclasts of rats (200 ×). b Count of positive cells in TRAP staining. c Results of ALP staining in osteoblasts of rats in each group (200 ×). d Count of positive cells determined by ALP staining. *P <0.05 vs. the normal group. ⁺ P <0.05 vs. the agomir-NC group. ^# P <0.05 vs. the siRNA-NC group. ^& P <0.05 vs. the agomir-miR-410 + OE-NC group

ALP staining presented that in the normal group, in osteoblasts, which were positive for ALP staining, the morphology was small and round. The cells were distributed in aggregation, most of which were located in the bone trabecular space of bone marrow cavity and on the surface of some bone trabeculae. In the ONFH group, agomir-NC group, siRNA-NC group, and agomir-miR-410 + OE-Wnt-11 group, the number of osteoblasts positive for ALP staining was dispersedly distributed in the trabecular space of the bone marrow cavity. In the agomir-miR-410 group, Wnt-11-siRNA group, and agomir-miR-410 + OE-NC group, the number of osteoblast-positive cells enhanced, and they were distributed in the trabecular space of the bone marrow cavity and the surface of the part of the bone trabecular (Fig. 6c).

By comparison with the normal group, the number of osteoblasts per unit area was suppressed and the number of osteoclasts was heightened in the ONFH group (both P <0,05). In relation to the agomir-NC group and the siRNA-NC group, the number of osteoblasts per unit area was heightened and the number of osteoclasts was declined in the agomir-miR-410 group and Wnt-11-siRNA group (all P <0,05). In contrast to the agomir-miR-410 + OE-NC group, the number of osteoblasts per unit area was declined and the number of osteoclasts was raised in the agomir-miR-410 + OE-Wnt-11 group (both P <0.05) (Fig. 6b, d).

Highly Expressed miR-410 and Lowly Expressed Wnt-11 Elevate the Expression of Osteocalcin (OCN), ALP, BGLAP, and Collα1, as well as Abate C- and N-terminal Telopeptides of Type I collagen (NTX-1, CTX-1), ACP5, CTSK, and MMP9 expression in ONFH Rats

The results of ELISA revealed that in relation to the normal group, the level of osteogenic function index ALP and OCN degraded while the levels of osteoclast function index NTX-1 and CTX-1 heightened in the ONFH group (all P <0,05). In contrast with the agomir-NC group and the siRNA-NC group, ALP and OCN ascended and NTX-1 and CTX-1 descended in the agomir-miR-410 group and the Wnt-11-siRNA group (all P <0,05). By comparison with the agomir-miR-410 + OE-NC group, ALP and OCN dropped and NTX-1 and CTX-1 appended in the agomir-miR-410 + OE-Wnt-11 group (all P <0.05) (Fig. 7a).

Highly expressed miR-410 and lowly expressed Wnt-11 elevate the expression of OCN, ALP, BGLAP, and Collα1 as well as abate NTX-1, CTX-1, ACP5, CTSK, and MMP9 expression in ONFH rats. een Detection of osteogenesis and osteoclast function indices in the serum of rats in each group by ELISA. b Detection of osteoblast- and osteoclast-related genes by RT-qPCR. c , d Detection of osteoblast- and osteoclast-related genes protein expression by western blot analysis. *P <0.05 vs. the normal group. ⁺ P <0.05 vs. the agomir-NC group. ^# P <0.05 vs. the siRNA-NC group. ^& P <0.05 vs. the agomir-miR-410 + OE-NC group

The results of western blot analysis and RT-qPCR revealed that in relation to the normal group, the expression of osteoblast-related factors ALP, BGLAP, and Collα1 degraded, and osteoclast-related factors ACP5, CTSK, and MMP9 expression ascended (all P <0,05). In contrast with the agomir-NC group and the siRNA-NC group, the expression of ALP, BGLAP, and Collα1 elevated, and ACP5, CTSK, and MMP9 expression abated in the agomir-miR-410 group and the Wnt-11-siRNA group (all P <0,05). By comparison with the agomir-miR-410 + OE-NC group, the expression of ALP, BGLAP, and Collα1 depressed, and ACP5, CTSK, and MMP9 expression heightened in the agomir-miR-410 + OE-Wnt-11 group (all P <0.05) (Fig. 7b–d).

Elevated Wnt-11 and Decreased miR-410 are Found in ONFH Tissues of Rats as well as Wnt-11 is the Target Gene of miR-410

The targeting relationship between miR-410 and Wnt-11 gene was analyzed by an online analysis software. It was displayed that a specific binding region existed between Wnt-11 gene sequence and miR-410 sequence, implying that Wnt-11 was the target gene of miR-410 (Fig. 8a). Luciferase activity assay was utilized to verify this relationship (Fig. 8b). The results presented that compared to the NC group, the luciferase activity depressed in the miR-410 mimics group (P <0.05), but there was no significant difference in the luciferase activity of MUT 3′UTR (P> 0.05), indicating that miR-410 could specifically bind to Wnt-11.

Increased Wnt-11 and decreased miR-410 are found in ONFH tissues as well as Wnt-11 is the target gene of miR-410. een Prediction of binding sites of miR-410 on Wnt-11 3′UTR. b Luciferase activity detection for verifying the relationship between miR-410 and Wnt-11. c Detection of miR-410 and Wnt-11 expression by RT-qPCR. d , e Wnt-11 protein expression determined by western blot analysis. v Expression of Wnt-11 in femoral head of rats in each group detected by immunohistochemistry (200 ×). g Comparison of the positive rate of Wnt-11 protein expression in each group. *P <0.05 vs. the normal group. ⁺ P <0.05 vs. the agomir-NC group. ^# P <0.05 vs. the siRNA-NC group. ^& P <0.05 vs. the agomir-miR-410 + OE-NC group

The results of western blot analysis and RT-qPCR revealed that in relation to the normal group, miR-410 expression declined and Wnt-11 expression raised in the ONFH group (both P <0,05). By comparison with the agomir-NC group, miR-410 raised and Wnt-11 reduced in the agomir-miR-410 group (both P <0,05). In contrast with the siRNA-NC group, Wnt-11 declined in the Wnt-11-siRNA group (P <0,05). Wnt-11 enhanced in the agomir-miR-410 + OE-Wnt-11 group relative to that in the agomir-miR-410 + OE-NC group (P <0.05) (Fig. 8c–e).

>Wnt-11 expression was verified by immunohistochemistry. Wnt-11 protein was mainly expressed in the cytoplasm, and the positive expression was mainly brownish yellow or brown. Compared to the normal group, the positive rate of Wnt-11 protein expression in the ONFH group was raised (P <0,05). By comparison with the agomir-NC group and the siRNA-NC group, the positive rate of the Wnt-11 protein expression descended in the agomir-miR-410 group and Wnt-11-siRNA group (both P <0,05). In relation to the agomir-miR-410 + OE-NC group, the positive rate of Wnt-11 protein expression enhanced in the agomir-miR-410 + OE-Wnt-11 group (P <0.05) (Fig. 8f, g).