Anti-aging Functional Factors And Mechanism Of Cistanche Deserticola Ⅱ

3. Result

3.1 Potential active ingredients and targets of Cistanche deserticola

By reviewing the literature and combining with the previous research of this research group, 12 chemical components including phenylethanol glycosides, iridoids and lignans were selected as target compounds for network pharmacology research. The specific information is shown in Table 3. After removing duplicates from the database, a total of 86 targets for 12 potential active ingredients were obtained.

High-quality cistanche for anti-aging

3.2 Aging-related targets

7753 and 295 aging-related targets were collected in the GeneCards and OMIM databases respectively.

3. 3 Common targets of drugs and diseases and construction of PPI network

The intersection of drug targets and disease targets resulted in 75 intersection targets, which were imported into the STRING network analysis platform, unconnected nodes in the network were hidden, and a PPI network was constructed. See enhanced publication materials. There are a total of 45 nodes and 59 connections in the PPI network. Eight core targets were screened out through topological analysis, as shown in Table 4.

3.4 GO enrichment analysis and KEGG enrichment analysis

Through the OmicsBean database, core targets were subjected to GO functional enrichment and KEGG pathway analysis. GO analysis includes three aspects: cellular component, molecular function and biological process. The top 10 items with the smallest P value are shown in the enhanced publication material. The results show that biological processes mainly involve regulating reactive oxygen metabolism, positive regulation of immune responses, protein localization to organelles, polysaccharide digestion, etc.; cellular components mainly include endomembrane system, extracellular region, plasma membrane, and membrane-bound cells.

Organs, extracellular secretory bodies, Golgi apparatus, etc.; molecular functions are mainly related to glucosidase activity, nitric oxide synthase regulatory activity, carbohydrate binding, β-fructose furanosidase activity, etc. A total of 27 aging-related pathways were obtained through KEGG enrichment screening, and the top 20 pathways were drawn into a pathway bubble diagram (see enhanced publication materials). Cistanche deserticola delays aging.

Table 3 Active ingredients of Cistanche deserticola

Table 4: Core targets of Cistanche deserticola to delay aging

It mainly involves cancer pathways, neurosecretory systems and signal transduction pathways. Cancer pathways mainly include prostate cancer, breast cancer and other pathways; the neuroendocrine system involves the estrogen signaling pathway, GnRH signaling pathway, and insulin signaling pathway (insulin signaling pathway) and so on; the signaling pathways include phosphatidylinositol 3-kinase-protein kinase B (PI3K Akt) signaling pathway and phospholipase D signaling pathway (phospholipase D signaling pathway).

3.5 Medicinal materials-components-targets-pathway network construction

The active ingredients, targets, and pathways of Cistanche deserticola that delay aging were mapped into the medicinal material-ingredient-target-pathway network through Cytoscape 3.8.0 software. There are a total of 114 nodes and 218 lines in this network (see enhanced publication material).

3.6 Effect of Cistanche deserticola on the life span of nematodes

The toxicity test results are shown in Figure 1. Compared with the control group, Cistanche deserticola 25 and 50 μg·mL–1

It can extend the lifespan of nematodes. 100 μg·mL–1 has no obvious effect. 200 and 500 μg·mL–1

It has certain toxicity to nematodes and shortens their life span. Based on the toxicity test results, select

Cistanche deserticola was administered at mass concentrations of 25, 50, 75, and 100 μg·mL–1 to determine the lifespan of N2 nematodes. The results are shown in Table 5. Compared with the control group, Cistanche deserticola 25, 50, 75 μg·mL–1

The life span of nematodes in the group was significantly extended, so subsequent experiments were conducted at 25, 50, and 75 μg·mL–1.

Figure 1 Survival curve of nematodes after administration of different mass concentrations of Cistanche deserticola

• Legend:

• Red: Control Group
• Blue: Cistanche 25 µg·mL⁻¹ Group
• Green: Cistanche 50 µg·mL⁻¹ Group
• Purple: Cistanche 100 µg·mL⁻¹ Group
• Orange: Cistanche 200 µg·mL⁻¹ Group
• Brown: Cistanche 500 µg·mL⁻¹ Group

• Axes:

• Y-axis: Survival Rate (%)

• X-axis: Time (t/d)

  
  

3.7 Effect of Cistanche deserticola on the stress ability of nematodes

Under H2O2-induced oxidative stress conditions, compared with the control group, the lifespan of nematodes in the Cistanche deserticola treatment group was extended (Figure 2A). The average lifespan of nematodes in the control group was (1.44±0.07) h, and Cistanche deserticola 75 μg·mL– The average life span of nematodes in group 1 was (2. 02±0. 05) h, and the life span was significantly extended (P<0. 001). Under heat stress conditions, compared with the control group, the heat shock resistance of nematodes in the Cistanche deserticola treatment group was improved and their lifespan was extended (Figure 2B). The average lifespan of nematodes in the control group was (19.25±0.20) h, and that of Cistanche deserticola was 75 The average lifespan of nematodes in the μg·mL–1 group was (22.80±0.08) h, and compared with the control group, the lifespan extension effect was significant (P<0.001).

Table 5 Effect of different mass concentrations of Cistanche deserticola on the life span of nematodes (xˉ±s, n=100)

Note: Compared with the control group, *P<0.05, **P<0.01, ***P<0.001.

3. 8 Effect of Cistanche deserticola on lipofuscin accumulation and body bending ability of nematodes

Lipofuscin gradually accumulates with age. Compared with the control group, the blue fluorescence levels of nematodes in the Cistanche deserticola 25, 50, and 75 μg·mL–1 groups decreased by 15. 09%, 28. 7%, and 39. 86% respectively on the 7th day (Figure 3A~B); On the 11th day, the blue fluorescence levels decreased by 8. 55%, 22. 36%, and 36. 84% respectively, indicating that Cistanche deserticola inhibited the accumulation of lipofuscin.

The effect of Cistanche deserticola on the body bending ability of nematodes is shown in Figure 3C. Compared with the control group, the number of body bending times of nematodes in the Cistanche deserticola 75 μg·mL–1 group increased by 25% and 36% within 30 s on days 3 and 7, respectively. 17%, indicating that Cistanche deserticola can promote the body bending of nematodes.

3.9 Effect of Cistanche deserticola on the swallowing ability and reproductive ability of nematodes

On days 3 and 7, compared with the control group, there was no statistically significant difference in the pharyngeal pumping rate (number of pharyngeal pumps within 60 s) of the nematodes in the Cistanche deserticola administration group (Figure 4A), indicating that Cistanche deserticola did not change the pharyngeal pumping rate of N2 nematodes. Food intake, Cistanche deserticola-mediated longevity effects are not dependent on dietary restriction. There were no significant changes in the number of eggs (Figure 4B) and offspring number (Figure 4C) of nematodes treated with the drug compared with the control group, indicating that Cistanche deserticola did not affect the reproductive ability of nematodes.

3.10 Effect of Cistanche deserticola on ROS, MDA content and antioxidant enzyme activity

Acute stress toxicity results in increased ROS levels. As shown in Figures 5A and 5B, compared with the control group, the ROS accumulation level of nematodes in the Cistanche deserticola group was significantly reduced on the 7th day of administration (P<0.05, P<0.01, P<0.001). In order to explore the effect of Cistanche deserticola on the endogenous antioxidant defense ability of nematodes, the activity of antioxidant enzymes in nematodes was measured. Compared with the control group, the SOD, CAT, and GSH-Px activities of nematodes in the Cistanche deserticola 25, 50, and 75 μg·mL–1 groups were significantly increased (Figure 5C~E). MDA is an important biomarker of lipid peroxidation. Compared with the control group, the MDA level in nematodes in the Cistanche deserticola 75 μg·mL–1 group was significantly reduced (P<0.05, Figure 5F). The above results show that Cistanche deserticola can effectively alleviate oxidative stress in nematodes.

• Legend:

• Red: Control Group
• Blue: Cistanche 25 µg·mL⁻¹ Group
• Green: Cistanche 50 µg·mL⁻¹ Group
• Purple: Cistanche 100 µg·mL⁻¹ Group
• Orange: Cistanche 200 µg·mL⁻¹ Group
• Brown: Cistanche 500 µg·mL⁻¹ Group

3.11 Effect of Cistanche deserticola on the mRNA expression of aging-related genes

The insulin (IIS) signaling pathway among the top 20 pathways analyzed by KEGG is closely related to C. elegans aging, and the mechanism research is relatively mature [10], thus verifying the expression of aging-related genes on the insulin signaling pathway. Insulin-like peptides (ILPs) released in C. elegans bind to their receptor DAF-2 and activate advanced glycation end product-1 (AGE-1), which further activates protein kinase B-1/2 (Akt-1/2). , phosphorylating the transcription factor DAF-16, resulting in DAF-16 activity and

Its migration to the nucleus is inhibited [11]. As shown in Figure 6, compared with the control group, the expression levels of DAF-2, AGE-1, and Akt-1 mRNA in nematodes in the Cistanche deserticola 75 μg·mL–1 group were significantly reduced (P<0.05, P<0.01) , the expression level of DAF-16 mRNA was significantly increased (P<

0.01). It was shown that the life-extending effect mediated by Cistanche deserticola depends on the IIS signaling pathway. The sir-2.1 gene plays an important role in physiological activities such as nematode aging, stress and apoptosis. Up-regulating the expression of the sir-2.1 gene will extend the lifespan of nematodes [12], but there is no effect after drug administration. Significantly changed the expression level of SIR-2. 1 mRNA (Figure 6). HSF-1 and SKN-1 play an important role in regulating oxidative stress and aging in C. elegans [11]. The results showed that compared with the control group, Cistanche deserticola 75 μg·mL–1

The expression levels of HSF-1, HSP-16. 1, HSP-16. 2 and SKN-1 mRNA in C. elegans group were significantly increased (Figure 6). The transcription factors DAF-16/forkhead box protein (FOXO) and SKN-1/nuclear transcription factor E2-related factor 2 (Nrf2) are activated and can regulate many genes encoding antioxidant-related proteins [13]. Compared with the control group, the expression levels of SOD-3, GST-4, CTL-1, CTL-2, and MTL-1 mRNA of nematodes in the Cistanche deserticola 75 μg·mL–1 group were significantly increased, indicating that Cistanche deserticola may enhance antioxidant capacity. extend your life. Dietary restriction can improve mitochondrial function and extend lifespan by limiting food intake [14]. There was no statistically significant difference in the expression of EAT-2 mRNA in nematodes in each group, indicating that the life-extending effect mediated by Cistanche deserticola does not occur through the dietary restriction pathway (Figure 6).

Figure 4 Effect of Cistanche deserticola on the swallowing ability and reproductive ability of nematodes (xˉ±s, n=5)

Legend:

• 对照组: Control group
• 肉苁蓉 25 μg·mL-1组: Cistanche 25 μg/mL group
• 肉苁蓉 50 μg·mL-1组: Cistanche 50 μg/mL group
• 肉苁蓉 75 μg·mL-1组: Cistanche 75 μg/mL group

Graph B:

• 30 s内水平游泳距离/次: Horizontal swimming distance in 30 seconds / time
• 第3天: Day 3
• 第7天: Day 7

Graph C:

• 相关水平的百分率: Percentage of the corresponding level
• 第7天: Day 7
• 第11天: Day 11

Graph A:

• 60 s内爬壁次数: Number of wall climbs in 60 seconds
• 第0天: Day 0
• 第3天: Day 3
• 第7天: Day 7

Graph B:

• 广泛测试得分: Open field test score
• 第1天: Day 1
• 第2天: Day 2
• 第3天: Day 3
• 第4天: Day 4
• 第5天: Day 5

Graph C:

• 千元迷宫次数: Y-maze errors
• 第1天: Day 1
• 第2天: Day 2
• 第3天: Day 3
• 第4天: Day 4
• 第5天: Day 5

3. 12 Effect of Cistanche deserticola on the lifespan of nematodes with related gene mutants

Compared with the control group, the lifespan curve of daf-16, skn-1, and hsf-1 gene mutant nematodes in the Cistanche deserticola 75 μg·mL–1 group did not shift significantly to the right after treatment (Figure 7A~C). The average lifespan of eat -2 gene mutant nematodes increased by 16. 45% (P < 0. 001, Figure 7D).

The results showed that the life-extending effect mediated by Cistanche deserticola was related to the daf-16, hsf-1 and skn-1 genes, but not to the eat-2 gene.

3. 13 Effect of Cistanche deserticola on nuclear translocation of DAF-16 and SKN-1 proteins

Representative pictures of DAF-16 protein expression in the cytoplasm, intercellular space, and nucleus are shown in Figure 8A. Compared with the control group, the nuclear localization ratio of DAF-16 protein in nematodes was significantly increased and the cytoplasmic localization ratio was significantly reduced after Cistanche deserticola administration (P<0.05, P<0.01, P<0.001, Figure 8C). Representative pictures of SKN-1 protein expression in the cytoplasm and nucleus are shown in Figure 8B. Compared with the control group, the proportion of SKN-1 protein localized in the cytoplasm of nematodes in the Cistanche deserticola 75 μg·mL–1 group was significantly reduced, while the proportion of nuclear localized protein was significantly increased (P<0.05, Figure 8D).

Figure 5 Effects of Cistanche deserticola on ROS, MDA content and antioxidant enzyme activity in nematodes (xˉ±s, n=100)

Legend:

• 对照组: Control group
• 肉苁蓉 25 μg·mL-1组: Cistanche 25 μg/mL group
• 肉苁蓉 50 μg·mL-1组: Cistanche 50 μg/mL group
• 肉苁蓉 75 μg·mL-1组: Cistanche 75 μg/mL group

Graph B:

• 30 s内水平游泳距离/次: Horizontal swimming distance in 30 seconds / time
• 第3天: Day 3
• 第7天: Day 7

Graph C:

• 相关水平的百分率: Percentage of the corresponding level
• 第7天: Day 7
• 第11天: Day 11

Graph A:

• 60 s内爬壁次数: Number of wall climbs in 60 seconds
• 第0天: Day 0
• 第3天: Day 3
• 第7天: Day 7

Graph B:

• 广泛测试得分: Open field test score
• 第1天: Day 1
• 第2天: Day 2
• 第3天: Day 3
• 第4天: Day 4
• 第5天: Day 5

Graph C:

• 千元迷宫次数: Y-maze errors
• 第1天: Day 1
• 第2天: Day 2
• 第3天: Day 3
• 第4天: Day 4

• 第5天: Day 5

  
  

3. 14 Effect of Cistanche deserticola on protein expression of SOD-3, GST-4 and HSP-16.2 in nematodes

SOD-3, GST-4, and HSP-16.2 protein expression levels were visualized and quantified using CF1553, CL2166, and TJ375 transgenic nematodes, respectively (Figure 9). Compared with the control group, the protein expression levels of SOD-3 and GST-4 in nematodes in each mass concentration group of Cistanche deserticola were significantly increased (P<0.05, P<0.01, P<0.001). The HSP-16.2 protein expression level of nematodes in Cistanche deserticola 50 and 75 μg·mL–1 groups was significantly increased (P<0.01, P<0.001).