The Iridoid Glycoside Loganin Modulates Autophagic Flux Following Chronic Constriction Injury-Induced Neuropathic Pain

Autophagy facilitates the degradation of organelles and cytoplasmic proteins in a lysosome-dependent manner. It also plays a crucial role in cell damage. Whether loganin affects autophagy in chronic constriction injury (CCI)-induced neuropathic pain remains unclear. We investigated the neuroprotective effect of loganin on the autophagic–lysosomal pathway in the rat CCI model. Sprague–Dawley rats were divided into sham, CCI, sham + loganin, and CCI + loganin. Loganin (5 mg/kg/day) was intraperitoneally injected once daily, and rats were sacrificed on day 7 after CCI. This study focused on the mechanism by which loganin modulates autophagic flux after CCI. CCI enhanced the autophagic marker LC3B-II in the ipsilateral spinal cord. The ubiquitin-binding protein p62 binds to LC3B-II and integrates into autophagosomes, which are degraded by autophagy. CCI caused the accumulation of p62, indicating the interruption of autophagosome turnover. Loganin significantly attenuated the expression of Beclin-1, LC3B-II, and p62. Double immunofluorescence staining was used to confirm that LC3B-II and p62 were reduced by loganin in the spinal microglia and astrocytes. Loganin also lessened the CCI-increased colocalization of both proteins. Enhanced lysosome-associated membrane protein 2 (LAMP2) and pro-cathepsin D (pro-CTSD) in CCI rats were also attenuated by loganin, suggesting that loganin improves impaired lysosomal function and autophagic flux. Loganin also attenuated the CCI-increased apoptosis protein Bax and cleaved caspase-3. Loganin prevents CCI-induced neuropathic pain, which could be attributed to the regulation of neuroinflammation, neuronal autophagy, and associated cell death. These data suggest autophagy could be a potential target for preventing neuropathic pain.


Introduction
Neuropathic pain, which involves neuroinflammation in the spinal cord, is a severely debilitating condition that adversely affects the quality of life. Various nerve injury stimuli in the peripheral or central nervous system can result in neuropathic pain with characteristic spontaneous pain, hyperalgesia, and allodynia [1,2]. Suitable therapies to attenuate Int. J. Mol. Sci. 2022, 23, 15873 2 of 12 neuropathic pain are limited because the mechanism of pathophysiology causing this is not completely understood.
Autophagy involves pathological conditions, including inflammatory diseases, cancer, and neurodegenerative diseases [11]. It maintains cellular homeostasis by clearing damaged organelles and proteins [12]. Research has also shown that autophagy regulates neuropathic pain. One report found that autophagy in GABAergic interneurons affects neuropathic pain [13]. In contrast, another showed that autophagy modulates neuropathic pain via the mammalian target of the rapamycin (mTOR) signaling pathway [14]. Previous articles demonstrated that autophagy is blocked in the spinal dorsal horn after CCI or spinal nerve injury, which can trigger neuropathic pain [15,16]. Based on those findings, we hypothesized that autophagy has an essential role in the maintenance of neuropathic pain. Modulation of autophagy may be a potential therapeutic target in human diseases.
The pathogenesis of neuropathic pain is not entirely understood. We further aimed to investigate the possible effects of loganin on neuropathic pain-associated autophagy using the CCI rat model. We found that autophagy impairment in the spinal cord led to an enhancement of neuropathic pain. In contrast, pharmacological agents stimulating autophagy could reduce inflammation and sustained pain. In this study, we suggest that loganin might improve neuropathic pain by stimulating autophagy and inhibiting apoptosis in the spinal cord of CCI rats.

Loganin Promoted Autophagy in CCI Rats
Since the effects of loganin on pain behaviors have been published previously [10], these specimens were from the same batch of rats to reduce unnecessary sacrifices and ensure ethical treatments. To explore the mechanisms of autophagy after CCI, we evaluated the expression of autophagy in the spinal cord proteins by Western blot. The regulatory protein Beclin1 is involved in initiating autophagy and autophagosome formation [17]. Beclin-1 levels were significantly elevated on day 7 after CCI, compared with the sham group ( Figure 1A,B), suggesting that autophagy is activated after CCI.
The conversion of LC3B-I protein into LC3B-II is a reliable marker of the autophagy mechanism [18]. LC3B-II levels increased on day 7 after CCI compared with the sham group ( Figure 1A,B). However, enhanced LC3B-II levels can reflect either the inhibition of autophagy or the failure to clean up the autophagosomes. The adaptor protein p62/SQSTM1 delivers ubiquitinated cargo to autophagosomes and is degraded by autophagy [19]. Levels of p62 significantly increased on day 7 after CCI and reverted in the loganin-treated group ( Figure 1A,B). Loganin reduced p62 to a level comparable to that of the sham group. These findings were consistent with the abnormal p62 protein degradation after CCI, including decreased autophagic flux. Sham-operated rats were the subjects of the same procedure without nerve manipulation. These data represent the mean ± SE for 6 rats per group. # p < 0.05, ## p < 0.01, ### p < 0.001 compared with the sham group at the corresponding time points; * p < 0.05, ** p < 0.01 compared with the CCI group.

Loganin-Induced Autophagy Occurred in Neuronal and Microglial Cells in CCI Rats
Autophagy is the dynamic process of sequestering organelles or cytoplasmic proteins into lytic components. To further investigate the cell-type specificity of autophagosome accumulation, we co-immunostained the spinal cord sections with LC3B and different cell-type markers (Figure 2A-C). The total number of LC3B-positive cells significantly increased in the spinal dorsal horn after CCI ( Figure 2D). In the spinal dorsal horn, LC3B showed a high level of colocalization with NeuN (neuron marker) and Iba1 (microglia marker) but not GFAP (astrocyte marker) in the CCI group. Those effects were attenuated in the loganin-treated group ( Figure 2E). These results indicated that loganin improved the impairment of autophagy in neurons and microglia but not astrocytes. Sham-operated rats were the subjects of the same procedure without nerve manipulation. These data represent the mean ± SE for 6 rats per group. # p < 0.05, ## p < 0.01, ### p < 0.001 compared with the sham group at the corresponding time points; * p < 0.05, ** p < 0.01 compared with the CCI group.

Loganin-Induced Autophagy Occurred in Neuronal and Microglial Cells in CCI Rats
Autophagy is the dynamic process of sequestering organelles or cytoplasmic proteins into lytic components. To further investigate the cell-type specificity of autophagosome accumulation, we co-immunostained the spinal cord sections with LC3B and different cell-type markers (Figure 2A-C). The total number of LC3B-positive cells significantly increased in the spinal dorsal horn after CCI ( Figure 2D). In the spinal dorsal horn, LC3B showed a high level of colocalization with NeuN (neuron marker) and Iba1 (microglia marker) but not GFAP (astrocyte marker) in the CCI group. Those effects were attenuated in the loganin-treated group ( Figure 2E). These results indicated that loganin improved the impairment of autophagy in neurons and microglia but not astrocytes.

Autophagosomes Accumulate after CCI Because of Impaired Autophagic Flux
Disruption of autophagic flux is related to neuropathic pain [15]. To investigate autophagic flux, it is necessary to determine the degradation of the ubiquitin-binding protein p62/SQSTM1, an autophagic substrate, to monitor autophagic activity. Since p62 is degraded by autophagy along with its cargo, the accumulation of p62 suggests disruption of autophagic degradation. CCI promotes ubiquitin-and LC3B-binding protein p62 accumulation in the ipsilateral spinal cord. This result inversely correlates with autophagic activity, indicating the blockage of autophagosome turnover ( Figure 3A,B). CCI increased the colocalization of LC3B and p62, and the effect was inhibited by loganin ( Figure 3A,C). Accumulation of autophagosomes in CCI rats could be attributed to the impairment of autophagic flux. This impaired autophagy can be reversed by loganin. The pathological accumulation of autophagosomes is predominant in neurons associated with signs of impaired lysosomes.

Lysosomal Dysfunction Contributed to the Disruption of Autophagic Flux
Lysosomes fuse with autophagosomes to permit the degradation of autophagosomal cargo by lysosomal hydrolases [20]. The lysosome-associated membrane protein 2 (LAMP2) was immunostained to estimate the lysosomal defects after CCI. We found increased LAMP2 in CCI rats, which was significantly attenuated on day 7 after loganin treatment ( Figure 4A,B). CCI also exhibited increases in LC3B-II, indicating autophagosome cleaning failure. Furthermore, cathepsin D (CTSD) is an essential protease regulating lysosome proteolytic activity in neurons [21]. Accumulating immature CTSD forms (pro-CTSD) is used as a marker of lysosomal impairment. The expression of the pro-CTSD protein was increased in CCI rats, which was decreased following loganin treatment ( Figure 4C,D). Altogether, these findings suggested that loganin prevents CCI-induced lysosomal dysfunction and autophagosome accumulation. . Sham-operated rats were the subjects of the same procedure without nerve manipulation. These data represent the mean ± SE for 6 rats per group. ## p < 0.01, ### p < 0.001 compared with sham group at the corresponding time points; * p < 0.05, ** p < 0.01, *** p < 0.001 compared with CCI group. . Sham-operated rats were the subjects of the same procedure without nerve manipulation. These data represent the mean ± SE for 6 rats per group. ## p < 0.01, ### p < 0.001 compared with sham group at the corresponding time points; * p < 0.05, ** p < 0.01, *** p < 0.001 compared with CCI group. mulation in the ipsilateral spinal cord. This result inversely correlates with autophagic activity, indicating the blockage of autophagosome turnover ( Figure 3A,B). CCI increased the colocalization of LC3B and p62, and the effect was inhibited by loganin ( Figure 3A,C). Accumulation of autophagosomes in CCI rats could be attributed to the impairment of autophagic flux. This impaired autophagy can be reversed by loganin. The pathological accumulation of autophagosomes is predominant in neurons associated with signs of impaired lysosomes. (C) Pearson's correlation coefficients quantified the degree of colocalization between p62 and LC3B-II. Sham-operated rats were the subjects of the same procedure without nerve manipulation. These data represent the mean ± SE for 6 rats per group. # p < 0.05, ## p < 0.01, ### p < 0.001 compared with sham group at the corresponding time points; * p < 0.05, *** p < 0.001 compared with CCI group. (C) Pearson's correlation coefficients quantified the degree of colocalization between p62 and LC3B-II. Sham-operated rats were the subjects of the same procedure without nerve manipulation. These data represent the mean ± SE for 6 rats per group. # p < 0.05, ## p < 0.01, ### p < 0.001 compared with sham group at the corresponding time points; * p < 0.05, *** p < 0.001 compared with CCI group.

Autophagic Flux Disruption Is Associated with Neuronal Cell Death
We next tested whether the disruption of autophagosome degradation caused neuronal cell death after CCI. The expression of apoptosis-related proteins (Bax and cleaved caspase3) was measured. Loganin significantly diminished the increasing cleaved caspase-3 and proapoptotic protein Bax levels caused by CCI ( Figure 5A,B). We used double immunofluorescence staining to characterize the aforementioned defects for cleaved caspase3 and NeuN (neuronal marker). We observed that cleaved caspase3 was greatly increased and colocalized with neurons after CCI in the spinal dorsal horn ( Figure 5C-E). In particular, loganin treatment significantly prevented CCI-induced apoptosis. some cleaning failure. Furthermore, cathepsin D (CTSD) is an essential protease reg ing lysosome proteolytic activity in neurons [21]. Accumulating immature CTSD fo (pro-CTSD) is used as a marker of lysosomal impairment. The expression of the pro-C protein was increased in CCI rats, which was decreased following loganin treatment ure 4C,D). Altogether, these findings suggested that loganin prevents CCI-induced somal dysfunction and autophagosome accumulation. . Sham-operated rats were the subjects of the procedure without nerve manipulation. These data represent the mean ± SE for 6 rats per gro p < 0.05, ### p < 0.001 compared with the sham group at the corresponding time points; * p < 0.0 p < 0.001 compared with the CCI group.

Autophagic Flux Disruption Is Associated with Neuronal Cell Death
We next tested whether the disruption of autophagosome degradation caused ronal cell death after CCI. The expression of apoptosis-related proteins (Bax and cle caspase3) was measured. Loganin significantly diminished the increasing cle caspase-3 and proapoptotic protein Bax levels caused by CCI ( Figure 5A,B). We used ble immunofluorescence staining to characterize the aforementioned defects for cle (D) Quantification of Western blot data in (C). Sham-operated rats were the subjects of the same procedure without nerve manipulation. These data represent the mean ± SE for 6 rats per group. # p < 0.05, ### p < 0.001 compared with the sham group at the corresponding time points; * p < 0.05, *** p < 0.001 compared with the CCI group. caspase3 and NeuN (neuronal marker). We observed that cleaved caspase3 was greatly increased and colocalized with neurons after CCI in the spinal dorsal horn ( Figure 5C-E).
In particular, loganin treatment significantly prevented CCI-induced apoptosis.  (E) Pearson's correlation coefficients quantified the degree of colocalization between C-caspase3 and NeuN. Sham-operated rats were the subjects of the same procedure without nerve manipulation. These data represent the mean ± SE for 6 rats per group. # p < 0.05, ## p < 0.01, ### p < 0.001 compared with the sham group at the corresponding time points; * p< 0.05, ** p < 0.01, *** p < 0.001 compared with the CCI group. (E) Pearson's correlation coefficients quantified the degree of colocalization between C-caspase3 and NeuN. Sham-operated rats were the subjects of the same procedure without nerve manipulation. These data represent the mean ± SE for 6 rats per group. # p < 0.05, ## p < 0.01, ### p < 0.001 compared with the sham group at the corresponding time points; * p< 0.05, ** p < 0.01, *** p < 0.001 compared with the CCI group.

Discussion
This study shows the correlation between autophagy, analgesic effects, and the antineuroinflammation effects [10] of loganin on CCI-induced neuropathic pain. Loganin significantly affected the increases of Beclin-1, LC3B-II, p62, LAMP2, and pro-CTSD in the ipsilateral spinal cord of CCI rats. In the CCI group, increased LC3B-II was paralleled by p62 accumulation in the spinal cord and spinal glial cells, suggesting a block in autophagic flux. Loganin also reduced the CCI-enhanced apoptosis-related proteins (Bax and cleaved caspase-3). These results indicated that loganin's analgesic and anti-neuroinflammatory effects and neuroprotection could be attributed in part to facilitating autophagic flux in rats with CCI-induced neuropathic pain.
The pathophysiological mechanism of neuropathic pain is complex. Autophagy is described by the formation of autophagosomes, which fuse with the lysosome to deliver the contents into the autolysosomes, where they are degraded [12]. Although the precise mechanism of autophagy's contribution to neuropathic pain is not well understood, several neuropathic pain models have demonstrated changes in autophagic flux. Autophagy was disrupted in the mice's spinal cords that underwent spinal nerve ligation (SNL). The accumulation of the ubiquitin-and LC3-binding protein p62 likely results from a block of autophagosome turnover [15]. In spare nerve injury (SNI) models, dysfunction of spinal autophagy could be relevant for altered nociceptive processing [22]. Enhancing autophagy through pharmacological approaches can slow neuropathic pain [23]. Conversion of LC3Bl to LC3B-ll by adding phosphatidylethanolamine (PE) is essential for the formation of autophagosomes and is a marker of autophagosome formation and accumulation. Moreover, the adaptor protein p62/SQSTM1 mediates the transport of ubiquitinated cargo to autophagosomes. Accumulation of p62 inversely correlates with autophagic activity. Our results show that CCI leads to LC3B-ll accumulation in neurons and a block of autophagic flux. Loganin decreases the colocalization of LC3B-II and p62, indicating that loganin promotes autophagic flux in neurons. Recently, autophagy was shown to be a cell-protective mechanism after injury under various circumstances, including oxidative stress [24], inflammatory response [25], acute liver injury [26], and cerebral ischemia [27]. Induced by various stresses, autophagy is upregulated to participate in cell adaption and survival. However, when the autophagic flux is impaired, damaged organelles accumulate, and protein aggregates interfere with cellular functions and may eventually result in cell death.
Elevated autophagic flux appears because of autophagosome degradation. This process requires the fusion of autophagosomes and lysosomes [28]. Our results suggested that autophagic flux is significantly disturbed after CCI. We then examined whether the lysosomal function has significance for CCI-induced neuropathic pain. LAMP2 is a heavily glycosylated membrane protein protecting lysosomal membranes from self-digestion and CTSD is a necessary protease that regulates lysosome proteolytic activity. Lysosome integrity is affected by disruption in disease states [29]. We found impaired lysosomal protease on day 7 after CCI. However, autophagy can also contribute to cell death when the lysosomal function is compromised. We then sought to understand the possible interplay between apoptosis and autophagy. Caspase3, the major executioner caspase, is cleaved to an active form in apoptosis. This study shows that CCI-induced lysosomal defects resulting from autophagosome accumulation in neurons occur when autophagy is blocked, which likely contributes to neuronal cell death.
In our previous report, CCI-enhanced mechanical allodynia and thermal hyperalgesia were significantly inhibited by loganin. Loganin also decreased proinflammatory cytokine levels (such as TNF-α and IL-1β) by reducing astrocyte and microglia activation [9,10]. The induction of microglial autophagy decreases cytokine production by inhibiting inflammasome formation, which could reduce neuropathic pain [30]. Our findings extend previous findings in the literature by demonstrating that loganin alleviated neuropathic pain after CCI and restored autophagic flux.
Overall, our data provide evidence that loganin could be an effective agent following peripheral nerve injury, enhancing cell-protective autophagy while attenuating neuropathic pain. These results suggest that the regulation of autophagy opens novel therapeutic avenues to improve the patient's quality of life and reduce suffering from neuropathic pain. Revealing novel cellular and molecular mechanisms behind neuroinflammation and neuropathic pain are indispensable for developing potential targets of pharmacotherapy.

Experimental Animals
Six-week-old male Sprague-Dawley (SD) rats (n = 72) (BioLASCO Taiwan Co., Ltd., Taipei, Taiwan) were used in all experiments. Animals were adapted to standard laboratory conditions for at least one week, and food and water were available ad libitum. They were divided into sham, CCI, sham + loganin, and CCI + loganin groups [10]. All protocols were approved by the Animal Care and Use Committee (IACUC approval No. 105112, 1 August 2017 to 31 July 2020) of Kaohsiung Medical University and adhered to the National Institute of Health guidelines for the use of the experimental animals.

CCI Model Preparation
The CCI model is considered an appropriate model of neuropathic pain [31]. Unilateral CCI surgery was performed as described [31][32][33][34], and the ipsilateral spinal cord was collected for the following experiments. Briefly, SD rats were anesthetized with pentobarbital sodium (40 mg/kg) intraperitoneally. The sciatic nerve was exposed and freed, and then 3 ligatures of 4-0 chromic catgut were placed around the sciatic nerve [32][33][34][35]. Three ligatures were tied loosely to the rat's hind limb, causing a brief twitch. The same surgical procedures were executed in sham-operated rats, but no ligatures were placed.

Western Blot
Animals were sacrificed on day 7 after CCI surgery, and the lumbar spinal segment (L4-L5) was rapidly removed. Total protein in the ipsilateral part of the rats' spinal cord was extracted. The protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a PVDF membrane. After blocking, the membranes were incubated overnight (4

Statistical Analysis
Experimental data in bar graphs were expressed as the mean ± SE (n = 6) with each experiment conducted in triplicate. Pearson's correlation coefficients are represented for the colocalization analysis. One-way analysis of variance (ANOVA) was used to analyze the data and followed by a posthoc test Tukey-Kramer pairwise comparison. p < 0.05 was indicated as statistically significant.

Conclusions
Enhanced LC3B-II and p62 show that impaired autophagy in CCI-induced neuropathic pain may result from blocking the late degradation phase. Increased LAMP2 further confirmed the late phase of lysosomal dysfunction and autophagosome accumulation. Loganin mitigated CCI-increased LC3B-II, p62, and LAMP2, suggesting that it can modulate the autophagic flux in CCI-induced pain and neuroinflammation. Loganin has also been shown to inhibit apoptosis and protect spinal neuronal cells ( Figure 6). Thus, loganin may help provide neuroprotection and control of neuropathic pain. the colocalization analysis. One-way analysis of variance (ANOVA) was used to analyze the data and followed by a posthoc test Tukey-Kramer pairwise comparison. p < 0.05 was indicated as statistically significant.

Conclusions
Enhanced LC3B-II and p62 show that impaired autophagy in CCI-induced neuropathic pain may result from blocking the late degradation phase. Increased LAMP2 further confirmed the late phase of lysosomal dysfunction and autophagosome accumulation. Loganin mitigated CCI-increased LC3B-II, p62, and LAMP2, suggesting that it can modulate the autophagic flux in CCI-induced pain and neuroinflammation. Loganin has also been shown to inhibit apoptosis and protect spinal neuronal cells ( Figure 6). Thus, loganin may help provide neuroprotection and control of neuropathic pain. Figure 6. Proposed iridoid glycoside loganin mechanism in a CCI rat model. Loganin attenuates neuropathic pain by stimulating autophagy, inhibiting astrocyte/microglia excess activation, and reducing neuroinflammation and apoptosis.

Study Limitations
Autophagy is a highly dynamic event, and it is important to study its progress at different times after peripheral nerve injury. We decided to study the CCI rat model on day 7 since the most severe neuropathic pain occurred on this day. This study did not clarify whether loganin could stimulate autophagy through other potential molecular Figure 6. Proposed iridoid glycoside loganin mechanism in a CCI rat model. Loganin attenuates neuropathic pain by stimulating autophagy, inhibiting astrocyte/microglia excess activation, and reducing neuroinflammation and apoptosis.

Study Limitations
Autophagy is a highly dynamic event, and it is important to study its progress at different times after peripheral nerve injury. We decided to study the CCI rat model on day 7 since the most severe neuropathic pain occurred on this day. This study did not clarify whether loganin could stimulate autophagy through other potential molecular mechanisms in the CCI rat model. In-depth studies need to be conducted to investigate other potential signaling pathways of loganin in autophagic flux after CCI.  Institutional Review Board Statement: The animal study protocol was approved by the Institutional Animal Care and Use Committee at Kaohsiung Medical University (approval IACUC code: KMU105112, execution date: 1 August 2017-31 July 2020).

Informed Consent Statement: Not applicable.
Data Availability Statement: The corresponding author will provide the data supporting this study's findings, which are available upon reasonable request.