**3. Results**

#### *3.1. Oral LF Supplementation Suppressed Minus-Lens-Induced Myopia Development in C57BL*/*6J Mice*

There was no impact of LF administration on body weight. A −30 D lens induced refractive shift and axial elongation, thus indicating successful myopia development in the control group (Figure 2a,b). In contrast, LIM failed to induce axial elongation and refractive shift in the LF group (Figure 2a,b). Therefore, LF supplementation suppressed lens-induced myopia in C57BL6J mice.

**Figure 2.** Scleral ER stress suppression or induction was sufficient to modulate axial elongation. (**a**) lens-induced myopia (LIM)-induced axial elongation was inhibited by oral administration of LF (lactoferrin) for 3 weeks (*n* = 4 per group); \* *p* < 0.05. (**b**) LIM-induced myopic shift in refraction was inhibited by oral LF administration for 3 weeks (*n* = 4 per group); \* *p* < 0.05.

#### *3.2. While LIM Increased Active MMP-2 Activity and IL-6 Expression in the Choroid and Sclera, LF Administration Reversed This E*ff*ect*

Axial length elongation was accompanied by scleral remodeling, such as a suppression of collagen production and an increase in the activity of collagen degradative enzymes. Thus, we assessed pro-MMP-9, pro-MMP-2, and active MMP activity by gelatin zymography from the choroid and sclera (Figure 3a). The gelatin digesting activity of pro-MMP-9 and active-MMP-2 was enhanced in LIM in the right eyes of the control group. In contrast, the MMP activity was comparable between LIM and control eyes in the LF-treated group (Figure 3b). Consistent with the results of zymography, the expression level of MMP-2 protein was also higher in the myopia-induced group than in the control group, and there was no difference in the expression level of MMP-2 protein between the control and myopia-induced groups in mice treated with LF (Figure 3c,d). Thus, LF administration inhibited LIM-induced proteolytic activity of MMP-2 and MMP-9, followed by the suppression of myopia development.

**Figure 3.** LIM activated MMP-2 activity and oral LF administration reversed this effect. (**a**) Gel image of gelatin zymography. (**b**) Quantified data of pro-MMP-2, active MMP-2, and pro-MMP-9; \* *p* < 0.05. (**c**) Immunoblots showing that LIM increased IL-6 and MMP-2 expression and that LF suppressed them; representative blots from 3 independent experiments are shown. (**d**) The densitometry quantitation of MMP-2 (upper) and IL-6 (lower) blots; the Ringer's–no lens (NL) group was assigned a value of 1.0; all other values are expressed relative to this value; \* *p* < 0.05.

LF has anti-inflammatory properties and its administration decreased the expression level of inflammatory cytokines, such as tumor necrosis factor-α and IL-6 [13]. As IL-6 is a positive regulator of MMP2 expression and is increased in myopic conditions [25,26], we hypothesized that IL-6 is an upstream factor in the increased MMP-2 activity in myopic eyes and examined the effect of LIM and LF on IL-6 expression level. As shown in Figure 3c,d, IL-6 expression level was higher in −30 D lens-wearing eyes in the Ringer's group, however, the expression level of IL-6 was comparable between the no-lens (NL) and −30 D eyes in the LF-treated group. Taken together, it is suggested that myopic stimuli induced IL-6 expression, followed by enhanced MMP-2 expression/activity, and that LF can reverse it.

#### *3.3. LIM Decreased the Content of Collagen 1A1 Protein in Choroid and Sclera, and LF Administration Reversed This E*ff*ect*

Collagen 1A1 is the most abundant structural collagen in the sclera. Furthermore, its content is decreased by form-deprived and minus-lens-induced myopia, which reduces *COL1A1* expression and increases its degradation by MMP. Therefore, we assessed the effects of LIM and orally administered LF on the protein expression of collagen 1A1. LIM decreased the level of its expression in the choroid and sclera (but not statistically significantly; *p* = 0.062). In contrast, there was an increase in the expression level in the LIM sclera of the LF-treated group (Figure 4a,b), suggesting the protective effect of LF against LIM-induced collagen 1A1 degradation.

**Figure 4.** LIM decreased collagen 1A1 expression and oral administration of LF reversed its effect. (**a**) Immunoblotting showed a decrease in collagen 1A1 expression in the right eye in the control group; this decrease was absent in the LF group. (**b**) The densitometry quantitation of blots: the Ringer's–NL group was assigned a value of 1.0; all other values are expressed relative to this value; \* *p* < 0.05.
