**4. Discussion**

In the present study, we demonstrated that LF suppresses myopia in a mouse LIM model. This was concomitant with the inhibition of an LIM-induced IL-6 increase, MMP-2 activation, and a decrease in collagen 1A1 in the sclera and choroid.

Scleral ECM remodeling reportedly plays an essential role in myopia development. Furthermore, MMP-2 is a key player in scleral remodeling and myopia development. In myopic schlera, the expression of MMP-2 was increased when compared with control eyes in mice, chicks, tree shrews, and guinea pigs [27–29]. An increase in MMP-2 levels in the human aqueous humor was positively correlated with axial length [27]. Forced expression of MMP-2 in the sclera was sufficient to induce a myopic shift in refraction. Moreover, an injection of Adeno-associated virus 8 packaging with shRNA to MMP-2 suppressed the form of deprivation-induced increases in the latter's expression and in myopia development [29]. We demonstrated that while LIM enhanced MMP-2 activity, oral administration of LF reversed this effect. Therefore, LF suppressed LIM-induced MMP-2 activation. Inoculation of *Escherichia coli* enhanced MMP-2, -3, and -9 activities in cervical tissue. In contrast, pre-treatment with LF before *E. coli* inoculation suppressed the increase [30]. Apolactoferrin inhibited the catalytic domain of MMP-2 in the metal-free forms of LF in vitro [31]. Our results sugges<sup>t</sup> that orally administered LF inhibited MMP-2 activation during LIM, followed by a suppression of myopia development.

Inflammation is a critical factor in myopia onset and development [6]. Consistent with previous studies, IL-6 expression was increased by myopia-inducing stimuli. We also found that the increase did not occur in the LF group. The changes in MMP2 activity and expression levels with myopia induction and LF administration were consistent with this IL-6 expression pattern, and IL-6 is a positive regulator of MMP-2 expression [25,26], suggesting that LF suppresses the activation of MMP-2 by myopia induction through its anti-inflammatory e ffects, including IL-6 expression.

MMP-2 is an ECM degradation enzyme. Furthermore, its activation induces collagen degradation, followed by a weakening of scleral sti ffness. Myopia development is concomitant with an increase in MMP-2 expression and a decrease in collagen 1A1 content [5,8,32,33]. We reproduced a decrease in collagen 1A1 by LIM. Moreover, oral administration of LF could prevent this decrease. LF reportedly has a suppressive e ffect on MMPs [30,31] that is consistent with the present study (Figure 3). Furthermore, LF stimulates *COL1A1* transcription and collagen I synthesis in human dermal fibroblasts [18]. Fibroblasts are the dominant cell population in the sclera. Thus, LF can counteract myopia development by suppressing collagen I degradation by MMP-2 inhibition, and by enhancing collagen I synthesis in scleral fibroblasts.

In conclusion, oral administration of LF can prevent negative-lens-induced myopia in mice by suppressing the IL-6–MMP-2 axis and collagen 1a1 degradation. Hence, LF is considered beneficial to prevent myopia development in humans.
