Ginsenoside Rg1 Improves Inflammation and Autophagy of the Pancreas and Spleen in Streptozotocin-Induced Type 1 Diabetic Mice

Background Ginsenoside Rg1 (Rg1) is one of the key bioactive components of the precious Traditional Chinese Medicine that has been used to treat diabetes in China. Ginsenosides have been reported to protect diabetics from tissue damage, inflammation, and insulin resistance. Type 1 diabetes (T1D) is an organ-specific autoimmune disease that occurred frequently among adolescents over the world, its development was related to inflammation and β-cells immunodeficiency. The aim of this study is to explore the biological mechanism of Rg1 on inflammation and autophagy of β-cells in T1D and its therapeutic potential. Methods The model of T1D mice was established by injecting Streptozotocin (STZ) (55 mg/kg) or citric acids once a day for 5 days and from the fourth day of injection, mice were administered with Rg1 (20 mg/kg) or saline by gavage once a day for 12 days. Hematoxylin-eosin staining, immunofluorescence, ELISA, quantitative real-time PCR, and Western blot were used to observe the histopathological changes, inflammatory factor levels, and autophagy markers after administration of ginsenoside Rg1. Results Compared to the T1D mice, Rg1 improved the weight (p < 0.05) and blood glucose (p < 0.01) of mice, advanced the injury and apoptosis of β-cells in islets (p < 0.01), and markedly inhibited the protein expression degrees of CD45, CXCL16, ox-LDL, and TF in the pancreas and spleens (p < 0.01), also activated the degrees of insulin in serum (p < 0.01). Besides, in T1D mice' pancreas and spleen, Rg1 markedly repressed the IL-1β, TNF-α, and NOS2 mRNA levels (p < 0.05 or p < 0.01), inhibited the CXCL16, NF-κB, and TF proteins (p < 0.05 or p < 0.01), while elevating the ratio of LC3 II/I (p < 0.01) and P62 (p < 0.05) protein level. Conclusions This study proved that Rg1 protected mice against T1D possibly by improving islet injury and tissue inflammation, raising serum insulin, and tissue autophagy marker.


Introduction
Type 1 diabetes (T1D) is an organ-specifc autoimmune disease with selective destruction of β-cells in islets and dysfunction of insulin secretion, afecting more than 490 thousand of the world's children [1]. Epidemiological studies suggest that childhood obesity, eating habits, virus infection, and other environmental factors are related to the onset of T1D [2]. Additionally, the destruction of the β-cell is one of the reasons to result in the reduction of insulin, the uncontrolled production of glucose, and hyperglycemia [3]. Inhibition of hyperglycemia has always been the focus of T1D treatment [4]. Additionally, modern science focused on preventing or postponing β-cell loss in T1D [5,6]. Scientists reported that the autoimmune response in T1D resulted in an infammatory state in β-cells [7]. Clinical evaluation of children with high genetic risk found that infammation, cytotoxicity, angiogenesis, and antigen-presenting cell activity increased in children progressing towards islet autoimmunity [8]. Also, Park's team noticed that Alpha-1 antitrypsin with anti-infammatory properties favorably impacted the development of T1D in mice [9]. It suggests that the anti-infammatory treatment of T1D may be efective.
Scientists found that many plant extracts have the potential to improve diabetes [10][11][12]. Extracts of Olea europaea treatment reduced fasting blood glucose in diabetes animals [13]. Scientists reported that the extract of Malva neglecta Wallr inhibited the level of oxidative stress in diabetes animals [14,15]. Ginsenoside Rg1 (Rg1) is one of the key bioactive components of Panax ginseng C. A. Mey (family Araliaceae), a precious Traditional Chinese Medicine that has been used to treat diabetes in China [16,17]. Rg1 is a triterpenoid saponin containing a protopanaxatriol structure [18]. In recent years, scientists have discovered that ginsenosides can protect tissues such as the heart, pancreas, and spleen, antagonize infammation, and improve insulin resistance in diabetes [19][20][21]. Research pointed out that Rg1 inhibited the IL-1β and IL-18 levels in T1D mice [22]. Similarly, Yu and colleagues studied that Rg1 could prevent the high glucose-/palmitate-induced damage in H9C2 cells via the AKT-GSK-3beta-Nrf2 pathway [23]. And Luo and colleagues reported that Rg1 blocked the pro-infammatory efects of lipopolysaccharide on neonatal rat cardiomyocytes [24]. In addition, the regulation of autophagy in β-cells is important to maintain the stability of insulin [25]. A study reported that inhibiting autophagy promoted the IL-1β level [26]. And extracts of ginsenoside can induce protective autophagy in HepG2 cells [27] and regulate autophagy in acute liver injury [28]. Modern research argues that infammation and autophagy play crucial roles in the treatment of diabetes [29,30]. However, there was still a lack of research on the biological efect of Rg1 on T1D. To explore the biological mechanism of Rg1 on infammation in T1D and study its therapeutic potential, this study constructed the T1D mouse model by injecting STZ and proposed the possibility that Rg1 ameliorates infammation in T1D mice via elevating autophagy. . A temperature-, humidity-, and light-controlled animal room (20°C, 60% humidity, and a 12 h light/dark cycle) was used to house all mice with free access to food and water for 7 days. Te mice were divided into 4 groups with 6 mice in each group: the mice in the DM group and the DM + Rg1 group were injected intraperitoneally with Streptozotocin (STZ) (55 mg/kg) (98%, S817944, Mackin, Shanghai, CHN) that was dissolved in citric acids (CA, 50 mM, pH4.5) once a day for 5 days; the mice in the control group and Rg1 group were injected intraperitoneally with CA for 5 days, whose volume was equal to the amount of STZ in the DM group. Additionally, the mice in the Rg1 group and DM + Rg1 group were further administered with Rg1 (20 mg/kg) (≥98%, G909436, Mackin, Shanghai, CHN) by gavage once a day for 12 days since the fourth day of STZ/CA injection. Te time axis of animal treatments is shown in Figure 1(a). Te dose of Rg1 was set according to previous studies on Rg1 [22,31].

Evaluation of Type 1 Diabetic Mice Modeling.
On the 7th day after the last STZ was injected, the tail vein blood of mice was randomly taken to measure blood glucose for 3 days, and the weight of the mice was recorded. Te level of blood glucose was greater than or equal to 16.7 mmol/L for 3 consecutive days, indicating the success of the T1D model.

Collection of Serum and Tissue Samples.
On the 10 th day after the last injection of STZ, the mice were anesthetized with ether and euthanasia with carbon dioxide. Te pancreas and spleen were collected and kept in 10% saline formalin. Te serum was separated by centrifugation at 4000 rpm for 15 min from the blood that was collected from the retroorbital venous plexus by small capillary tubes before euthanasia. Tissue and serum were stored at −80°C for subsequent experiments.

Detection of ox-LDL and Insulin (INS) in Serum.
According to the instructions of the ox-LDL (MM-0908M1, Meimian, Jiangsu, CHN) and INS (MM-0579M1, Meimian, Jiangsu, CHN) ELISA kits, serum samples were added to the enzyme label plate to incubate at 37°C for 60 min. After washing the plate, chromogenic agents A and B that were added to the plate were well-mixed and stood in the dark for 15 min, before being fnally added to be termination solution to terminate the reaction. Te absorbance (expressed as an OD value) was detected at a wavelength of 450 nm, and there were 6 parallel setups in each group.

Hematoxylin-Eosin (HE) Staining of the Pancreas.
Pancreases have been fxed, dehydrated with an alcohol gradient, made transparent with xylene and alcohol, and then embedded in parafn. Te tissues with parafn wrapping were made into 5 μm sections and stained by HE staining kits (G1003, ServiceBio, Wuhan, CHN). An optical microscope was used to observe the tissues. Te histology of the pancreas was evaluated according to the islet volume and the regular edges of the islet, and the score was proportional to the degree of injury. 50, sc-373785, SCBS, US) were used for incubating with the sections overnight at 4°C, and then after washing, the tissue that was incubated with the goat antirabbit IgG H&L (ab150078, Abcam, US) that was incubated in dark for 50 min. Following PBS washing, the nuclei were counterstained with DAPI (G1012, ServiceBio, CHN). Tirdly, after being washed, the sections were quenched by the antifuorescence quenching sealing reagent (G1401, Servicebio, CHN) for 5 min and rinsed with running water for 10 min. Between every two steps, the sections were washed 3 times with PBS (pH � 7.4) on the shaking table. Finally, a NIKON eclipse upright microscope was used to observe and image the fuorescence (the nucleus is blue, and the positive expression is red or green).

Quantitative Real-Time PCR Analysis (qRT-PCR) of Infammatory Factors in the Pancreas and Spleen.
Te tissue was lysed with the Total RNA Extractor (B511311, Sangon Biotech, CHN) and centrifuged to remove the precipitation, then the supernatant was dissolved in chloroform and centrifuged again to separate the supernatant. Te supernatant was soaked in isopropanol for 15 minutes to obtain precipitation, and then the precipitation was soaked in 75% absolute ethanol. Finally, the dried precipitate was dissolved 5 15 day Intraperitoneal injection of citric acids or Streptozotocin

Statistical
Analyses. Data analysis by SPSS 16.0. Te comparisons of multiple groups were analyzed by one-way ANOVA followed by Tukey, and the t-test was used to analyze the diferences between every two groups. If it is a multigroup comparison with a nonnormal distribution or uneven variance, the Kruskal−Wallis H-Test was used. All data were expressed as mean ± standard deviation (mean ± SD), p < 0.05 meaning that the diference was statistically signifcant.

Rg1 Improved the Blood Glucose and Body Weight in T1D
Mice. On day 7 after the frst intraperitoneal injection of STZ, the blood glucose of mice ≥16.7 lasted for 3 days, which means that the T1D models in the DM group and the DM + Rg1 group were successfully constructed (Figure 1(b)). At the end of the 15th day of Rg1 gavage, the blood glucose in the DM group was remarkably higher than that in the control group. While the blood glucose in the DM + Rg1 group was remarkably lower than that in the DM group (Figures 1(b) and 1(d)). Te weight of mice in the DM group was lower than that in the control group, while the weight of mice in the DM + Rg1 group was higher than that in the DM group on day 15 (Figures 1(c) and 1(e)).

Rg1 Ameliorated the Pathological Damage to the Pancreas in T1D
Mice. Te type 1 diabetic mouse model was built successfully, as shown in Figure 2, the results of pancreatic HE staining. In the control and Rg1 groups, the islet was plump, and the edges of islet cells were evenly regular (Figures 2(a) and 2(b)). While in the DM group, the size and edges of the islets were uneven (Figure 2(c)). And Rg1 treatment could improve the histology of the injury (Figure 2(d)). Compared with the control group, the score of the DM group was notably higher (p < 0.01). And the score of the DM + Rg1 group was immensely lower (Figure 2(e)).

Rg1
Regulated the Expressions of CXCL16, CD45, and INSR in the Pancreas. Te colocalization of CD45 or INSR protein with CXCL16 in the pancreas was observed by immunofuorescence (Figures 3(a)-3(f )). Te CD45 positive expression was obviously detected in the DM group, which was notably higher than that in the control group (p < 0.01), and the expression of the colocated CXCL16 was also higher in the DM group too (p < 0.01). Ten it was obvious that the expressions of CD45 and CXCL16 proteins decreased enormously by Rg1 treatment in DM mice (p < 0.01) (Figures 3(b) and 3(c)). By analyzing the level of INSR expression, it was found that there were no statistical differences in it among the four groups (p > 0.05). Ten the expression of CXCL16 was signifcantly upregulated in the DM group compared to the control group, while the CXCL16 expression was signifcantly reduced by Rg1 treatment (p < 0.01) (Figures 3(d)-3(f )).

Rg1 Regulates the Expression of CXCL16 and CD45
Proteins in the Spleen. Trough an immunofuorescence assay, we observed the colocalization of CXCL16 and CD45 in the spleen (Figures 4(a)-4(c)). Te levels of CD45 and CXCL16 proteins were markedly increased in the DM group compared to the control group (p < 0.01). While in the DM + Rg1 group, the levels of CD45 and CXCL16 were both lower than that in the DM group (p < 0.01).

Rg1 Regulates the Expression of ox-LDL an TF Proteins in the Pancreas and Spleen.
Te degrees of ox-LDL and TF proteins in the pancreas and spleen of T1D mice were observed by immunofuorescence (Figures 5 and 6). In the pancreas and spleen, compared with the control group, the degrees of ox-LDL and TF proteins in DM mice in the Rg1 group were not signifcant (p > 0.05), the expression of these two proteins in the DM group was signifcantly upregulated (p < 0.01). Te ox-LDL and TF proteins levels of the pancreas and spleen in the DM + Rg1 group compared with the DM group was markedly reduced (p < 0.01 and p < 0.05). As Figures 7(a) and 7(b) show, according to the results of the ELISA, we noticed that Rg1 treatment inhibited the levels of ox-LDL and advanced the expression degree of INS in the serum of T1DM. In the DM group, the level of ox-LDL was immensely upregulated (p < 0.01), and the INS level was repressed markedly more than that in the control group (p < 0.01). While in the DM + Rg1 group, compared with the DM group, the degree of ox-LDL was immensely inhibited (p < 0.05), and the INS level was signifcantly increased (p < 0.01).

Rg1 Restrained the Transcription Level of Infammatory
Factors in the Pancreas and Spleens. Te transcription degrees of IL-1β, TNF-α, and NOS2 were semiquantifed by qRT-PCR. Te results are shown in Figures 7(c)-7(h), in the DM group, the degrees of IL-1β, TNF-α, and NOS2 mRNA in the pancreas and spleen were notably superior to those in the control group (p < 0.01). And in the DM + Rg1 group, the transcription degrees of IL-1β, TNF-α, and NOS2 in the pancreas and spleens were inhibited compared with those in the DM group (p < 0.05) (Figures 7(c) and 7(h)).

Rg1 Suppresses the Expression of CXCL16, NF-κB, TF, and Activated LC3 and P62 Proteins in the Pancreas and Spleen of DM Mice.
By WB analyzing, in the DM group, the expression of CXCL16, NF-κB, and TF proteins in the pancreas and spleen were raised prominently compared to the control group (p < 0.01), and the degrees of CXCL16, NF-κB, and TF proteins were also huge down-regulated in the Rg1 treatment T1DM mice (p < 0.05) (Figures 8(a)-8(c) and 8(g)-8(i)).
On the contrary, the LC3 and P62 protein levels in the DM group were inhibited signifcantly compared with the control group (p < 0.01) (Figures 8(d), 8(e), 8(j) and 8(k)). And compared with the DM group, the expression degrees of LC3 protein in the pancreas and spleen were elevated enormously in the Rg1 treatment T1DM mice (p < 0.01) (Figures 8(d) and 8(j)). As well as the level of P62 protein in the pancreas and spleen was raised markedly in the DM + Rg1 group (p < 0.05) (Figures 8(e) and 8(k)).

Discussion
Te β-cells in islets are the main sites to regulate INS. Islet atrophy and apoptosis of β-cells in T1D rats is increased.
Research reported that improved the apoptosis of β-cells and the morphology of islets were able to reduce blood glucose levels in the model rats [32]. Tis report discovered that Rg1 treatment on DM mice advanced the mice's weight,  International Journal of Endocrinology improved the number and morphology of islets, and inhibited the blood glucose level. Also, Rg1 treatment raised the serum INS level. It reminded that Rg1 can improve the function of the islet in T1D mice. Besides, a piece of evidence displayed an increased ox-LDL level in T1DM patients [33]. Te increase of ox-LDL induced lipid accumulation and an infammatory response, which caused damage to tissues [34,35]. And activation of ox-LDL could induce the infammatory response and promote the secretion of infammatory factors, such as TF, the infammatory biomarker, which would be activated through the CXCL16/ ox-LDL pathway in β-cell [36,37]. CXCL16, a special chemokine, can combine with immune cells to drive into the infammatory part, and some can internalize ox-LDL [38,39]. Tis study observed the colocalized expression of INSR or CD45 with CXCL16 by immunofuorescence and found there was infammatory infltration in the pancreas of T1D mice, and Rg1 treatment could signifcantly improve this situation. Te above experimental results indicated that Rg1 had the potential to improve T1D mice by adjusting ox-LDL. Furthermore, this study found IL-1β and TNF-α levels were repressed signifcantly by Rg1 treatment in the pancreas of DM model mice. A study reported that in macrophages of diabetics, the IL-1β and TNF-α levels increased signifcantly [40][41][42]. Additionally, scientists have proved that β-cell injury in T1D was accompanied by the upregulation of infammatory factors, NF-κB and iNOS, while the expression levels of them were decreased after improving the injury [43]. Dampening NF-κB-iNOS-NO pathway negatively regulated the infammation and apoptosis of β-cells [44]. Trough qRT-PCR and WB, this study found the expression of NF-κB and iNOS was antagonized by Rg1 treatment. In short, it is suggested that Rg1 can antagonize the infammation of the pancreas in T1D mice via inhibiting the NF-κB-iNOS signaling pathways.
Additionally, scientists have proven that immune defciency in diabetes is associated with the damage of the spleen [45]. Moreover, splenocytes from a diabetic animal can transfer the T1D to healthy recipients [46]. Terefore, this study focused on the efect of Rg1 on the spleen of T1D mice and found it could improve the infammation in the spleen. Studies have reported IL-1β, TNF-α, and NOS2 were closely related to macrophages polarization and macrophages played an important role in the development of infammation [47]. Te results of this study indicated that the levels of macrophage polarization-related cytokines were enormously upregulated in the spleen of T1D mice, and Rg1 treatment was able to reduce them in the spleen. Rg1 treatment could signifcantly inhibit macrophage polarization and improve infammation in the spleen of T1D mice. Moreover, this study also analyzed the levels of autophagy markers, LC3 and P62, in the pancreas and spleen [44]. Additionally, the immune-related signaling molecules can regulate autophagy, which is essential to improving immune disorders and the infammatory response [48]. In recent years, scientists have proposed that autophagy disorder of islets of T1D patients, and autophagy can reduce the harmful efects of ER stress and DNA damage by delaying apoptosis of β-cells [49,50]. A study about metformin reported that increasing autophagy can improve T17 infammation [51]. Tis study discovered that Rg1 stimulated the expression of LC3 and P62 proteins suggesting that Rg1 has a promoting efect on the autophagy of β-cells and spleen tissues. Tis report found that the efect of Rg1 on autophagy in T1D mice may be related to CXCL16 and NF-κB. A study reported that the incretion of ox-LDL sensitized adipocytes to the lower insulin-induced glucose uptake and increased the levels of NF-κB and the marker of apoptosis and autophagy (Bnip3), suggesting the ox-LDL was related to infammation, apoptosis, and autophagy [52]. Te efect of Rg1 on autophagy in T1D deserves further study. Additionally, this study has some shortcomings. If multiple dose groups of Rg1 can be set, the results would be more convincing in this study, and this study will further explore the efects of multi-dose Rg1 in the future. In summary, this paper studied the pharmacology efcacy of the mechanism of Rg1 improving T1D, which indicated that the protective impact of Rg1 on T1D might be associated with antagonizing infammation and improving autophagy disorder. Tis team has planned to further explorate the relevant targets in the follow-up research.

Conclusion
Te study successfully constructed T1D mice and revealed the biological action of Rg1 on improving T1D by antagonizing infammation and improving autophagy disorders. It proved that the Rg1 ameliorated the blood glucose, body weight, pancreas histological damage. Also, this study proved that Rg1 decreased the INS level in serum and pancreas, and reduced the levels of CD45, CXCL16, INS, ox-LDL, and TF in the spleen and pancreas. And the inhibition of Rg1 on the mRNA levels of IL-1β, NOS2, and TNF-α in the spleen and pancreas suggested that Rg1 ameliorates T1D of mice by inhibiting infammation. Interestingly, this study proved that the Rg1 treatment on T1D mice could raise the ratio of LC3 II protein to LC3 I protein which suggesting that Rg1 has potential to advance the autophagy to treatment of T1D. Tis study provides a scientifc basis for the Rg1 treatment for T1D and a new direction for investigation of its biological mechanism.

Data Availability
Te data supporting the fndings of this study are available from the corresponding author, G. X., upon reasonable request.

Disclosure
Yi Zong and Weihua Yu contributed equally to this study and should be considered co-frst authors.