17β-Estradiol inhibits intervertebral disc degeneration by down-regulating MMP-3 and MMP-13 and up-regulating type II collagen in a rat model

Figures & data

Figure 1. Ovariectomy (OVX). Chloral hydrate was used for rats to be administered general anesthesia; a line was marked as the surgical incision, about 1.5 cm long on the position of 1 cm below the costal margin and 1.5 cm beside the spine. (A) The skin around the surgical incision was shaved; (B) and (C) incised the skin, entered the peritoneal cavity, found the ovaries along with the adipose tissue exposed in the incision and removed the ovaries; (D) sutured the skin.

Table 1. Histological grading scores (H&E) of AF and NP.

Scores^a	AF	NP
0	Well-organized fibrous lamellae without ruptured or serpentine fibers	Normal cellularity with large vacuoles and stellar-shaped nucleus consistently dispersed in the nucleus
1	Ruptured or serpentine fibers in less than 30% of the annulus	Slight decrease in the number of cells (<50%) with/without cell clustering
2	Ruptured or serpentine fibers in more than 30% of the annulus with reversed contour	Moderate to severe decrease in the number of cells (>50%) with mostly no vacuolization and occupied by proliferative CNT (<50% of nucleus area)
3	Indistinct and disorganized annulus	Severe replacement by proliferative CNT (>50% of nucleus area) with small area of vacuole cells

H&E: hematoxylin and eosin; AF: annulus fibrosus; NP: nucleus pulposus; CNT: connective tissue.

^a Scores range from a normal disc (0 point) to severely degenerated disc (6 points).

Table 2. Sense and antisense sequences of primers.

Gene	Forward primer (5′→3′)	Reverse primer (5′→3′)
COL2a1	5′-TCGGGGCCTTTCTC CCAAC-3′	5′-CACGGATGGTCCCAAAGG-3′
MMP-3	5′-ATGATGAACGATGGACAGATGA-3′	5′-CATTGGCTGAGTGAAAGAGACC-3′
MMP-13	5′-GGCCAGAACTTCCCAACCA-3′	5′-ACCCTCCATAATGTCATACCC-3′
Caspase-3	5′-TGGAATTGATGCGTGATGTT-3′	5′-GTCGGCATACTGTTTCAGCA-3′
GAPDH	5′-AACTTTGGCATCGTGGAAGGG-3′	5′-AGGGATGATGTTCTGGGCTGC-3′

Figure 2. The radiographs were used to measure DHI. On the left is a anteroposterior film and right is lateral film. The average intervertebral disc height of Co5/Co6, Co6/Co7 and Co7/Co8 was defined as DHI. Forty, three-month-old female Sprague Dawley rats were randomly divided into four groups: Sham, OVX, E2 and ICI. Sham group only underwent the resection of a bit of fat; OVX group underwent bilateral ovariectomy; E2 group was treated with 17β-Estradiol based on OVX and ICI group was treated with 17β-Estradiol and were also pretreated with ICI182780 (inhibitor of the estrogen receptor) based on OVX (Mean ± SD; n = 10). DHI: Disc Height Index; E2: 17β-Estradiol. *p < .05.

Figure 3. The sections of rat intervertebral disc tissues were stained with HE. Forty, 3-month-old female Sprague Dawley rats were randomly divided into four groups: Sham, OVX, E2 and ICI. Sham group only underwent the resection of a bit fat; OVX group underwent bilateral ovariectomy; E2 group was treated with 17β-Estradiol based on OVX; ICI group was treated with 17β-Estradiol and were also pretreated with ICI182780 (inhibitor of the estrogen receptor) based on OVX (Mean ± SD; n = 10). HE: hematoxylin and eosin; E2: 17β-Estradiol. * p < .05.

Figure 4. Histological grading scores of rat intervertebral disc tissues were obtained from HE staining. Forty, three-month old female Sprague Dawley rats were randomly divided into four groups: Sham, OVX, E2 and ICI. Sham group only underwent the resection of a bit of fat; OVX group underwent bilateral ovariectomy; E2 group was treated with 17β-Estradiol based on OVX and ICI group was treated with 17β-Estradiol and were also pretreated with ICI182780 (inhibitor of the estrogen receptor) based on OVX (Mean ± SD; n = 10). HE: hematoxylin and eosin; E2: 17β-Estradiol. *p < .05.

Figure 5. Protein level of caspase-3, MMP-3, MMP-13 and collagen Type II. Forty, three-month-old female Sprague Dawley rats were randomly divided into four groups: Sham, OVX, E2 and ICI. Sham group only underwent the resection of a bit of fat; OVX group underwent bilateral ovariectomy; E2 group was treated with 17β-Estradiol based on OVX; ICI group was treated with 17β-Estradiol and were also pretreated with ICI182780 (inhibitor of the estrogen receptor) based on OVX (Mean ± SD; n = 10). MMP-3: matrix metalloproteinase-3; MMP-13: matrix metalloproteinase-13; E2: 17β-Estradiol. *p < .05.

Figure 6. Quantitative real-time PCR (qRT-PCR) analysis. The mRNA level of caspase-3, MMP-3, MMP-13 and collagen Type II. Forty, three-month-old female Sprague Dawley rats were randomly divided into four groups: Sham, OVX, E2 and ICI. Sham group only underwent the resection of a bit of fat; OVX group underwent bilateral ovariectomy; E2 group was treated with 17β-Estradiol based on OVX and ICI group was treated with 17β-Estradiol and were also pretreated with ICI182780 (inhibitor of the estrogen receptor) based on OVX (Mean ± SD; n = 10). MMP-3: matrix metalloproteinase-3; MMP-13: matrix metalloproteinase-13; E2: 17β-Estradiol. *p < .05.

Figure 7. Immunohistochemistry was performed to detect the expression of caspase-17, MMP-3 and MMP-13. Forty 3-month-old female Sprague Dawley rats were randomly divided into four groups: Sham, OVX, E2, ICI. Sham group only underwent the resection of a bit of fat; OVX group underwent bilateral ovariectomy; E2 group was treated with 17β-Estradiol based on OVX; ICI group was treated with 17β-Estradiol and pretreated ICI182780 (inhibitor of the estrogen receptor) based on OVX ((Mean ± SD; n = 10). MMP-3: matrix metalloproteinase-3; MMP-13: matrix metalloproteinase-13; E2: 17β-Estradiol. *p < .05).