Tag: rat study

Effect of transcutaneous electrical acupoint stimulation on learning and memory ability of chronic fatigue syndrome rats and its mechanisms

Abstract:

in English, Chinese

Objective: To observe the effect of transcutaneous electrical acupoint stimulation (TEAS) on the histomorphological manifestations of hippocampal CA1 region and the expression of extracellular regulatory protein kinase (ERK), cyclic adenosine response element binding protein (CREB) and brain-derived neurotrophic factor (BDNF) in chronic fatigue syndrome (CFS) rats, so as to explore the mechanisms of TEAS in improving the learning and memory abilities of CFS rats.

Methods: Forty male Wistar rats were randomly divided into normal group (10 rats) and modeling group (30 rats); then after modeling, they were selected and randomly divided into model group (10 rats) and TEAS group (10 rats). CFS rats model was prepared by sleep deprivation combined with weight-bearing swimming. Rats in the TEAS group were stimulated with Han’s acupoint nerve stimulator at bilateral “Zusanli” (ST36) and “Shenshu” (BL23) (2 Hz/15 Hz, 1-2 mA), 20 min each time, once a day for 4 weeks with 1 d rest every 6 d. The score of general conditions of rats was evaluated. The learning and memory ability was tested with Morris water maze. The morphology and ultrastructure of hippocampal CA1 region were observed by HE staining and transmission electron microscopy. The expression levels of ERK, CREB and BDNF mRNAs and proteins in hippocampus were detected by real time quantitative PCR and Western blot, respectively.

Results: Compared with the normal group, the score of general condition was increased (P<0.01); the escape latency was prolonged (P<0.05, P<0.01) and the times of crossing the original platform was decreased (P<0.05); the expression levels of ERK, CREB and BDNF mRNAs and proteins in hippocampus were decreased (P<0.05, P<0.01) in the model group. Compared with the model group, the scores of general condition on the 42nd and 49th day were decreased (P<0.05, P<0.01); the escape latency was shortened (P<0.01, P<0.05)and the times of crossing the original platform were increased (P<0.05); the expression levels of ERK, CREB and BDNF mRNAs and proteins in hippocampus were increased (P<0.01, P<0.05) in the TEAS group. The morphology of neurons in hippocampal CA1 region was normal in the normal group. In the model group, the number of neurons in hippocampal CA1 region decreased, the arrangement of nerve cells was scattered, the number of apoptotic cells increased, some nuclear structures disappeared, nuclear heterochromatin increased, the cell membrane wrinkled, the chromatin appeared empty bright area, and the crista was incomplete. Compared with the model group, the nerve cells morphology in hippocampal CA1 region was more regular, the number of apoptotic cells decreased, the chromatin and the cytoplasm were uniformly distributed, and the crista was relatively intact in the TEAS group.

Conclusion: TEAS can improve the learning and memory ability of CFS rats, the mechanisms may be related to improving the neural structure of hippocampal CA1 region and up-regulating the expression levels of ERK/CREB/BDNF.

Source: Zhong XL, Tong BY, Yang YH, Zeng HL, Lin C, Jing Y, He LL, You SJ. [Effect of transcutaneous electrical acupoint stimulation on learning and memory ability of chronic fatigue syndrome rats and its mechanisms]. Zhen Ci Yan Jiu. 2023 Apr 25;48(4):317-24. Chinese. doi: 10.13702/j.1000-0607.20221032. PMID: 37186194. https://pubmed.ncbi.nlm.nih.gov/37186194/

Ginsenoside Rg1 can reverse fatigue behavior in CFS rats by regulating EGFR and affecting Taurine and Mannose 6-phosphate metabolism

Abstract:

Background: Chronic fatigue syndrome (CFS) is characterized by significant and persistent fatigue. Ginseng is a traditional anti-fatigue Chinese medicine with a long history in Asia, as demonstrated by clinical and experimental studies. Ginsenoside Rg1 is mainly derived from ginseng, and its anti-fatigue metabolic mechanism has not been thoroughly explored.

Methods: We performed non-targeted metabolomics of rat serum using LC-MS and multivariate data analysis to identify potential biomarkers and metabolic pathways. In addition, we implemented network pharmacological analysis to reveal the potential target of ginsenoside Rg1 in CFS rats. The expression levels of target proteins were measured by PCR and Western blotting.

Results: Metabolomics analysis confirmed metabolic disorders in the serum of CFS rats. Ginsenoside Rg1 can regulate metabolic pathways to reverse metabolic biases in CFS rats. We found a total of 34 biomarkers, including key markers Taurine and Mannose 6-phosphate. AKT1, VEGFA and EGFR were identified as anti-fatigue targets of ginsenoside Rg1 using network pharmacological analysis. Finally, biological analysis showed that ginsenoside Rg1 was able to down-regulate the expression of EGFR.

Conclusion: Our results suggest ginsenoside Rg1 has an anti-fatigue effect, impacting the metabolism of Taurine and Mannose 6-phosphate through EGFR regulation. This demonstrates ginsenoside Rg1 is a promising alternative treatment for patients presenting with chronic fatigue syndrome.

Source: Lei C, Chen J, Huang Z, Men Y, Qian Y, Yu M, Xu X, Li L, Zhao X, Jiang Y, Liu Y. Ginsenoside Rg1 can reverse fatigue behavior in CFS rats by regulating EGFR and affecting Taurine and Mannose 6-phosphate metabolism. Front Pharmacol. 2023 Apr 10;14:1163638. doi: 10.3389/fphar.2023.1163638. PMID: 37101547; PMCID: PMC10123289. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10123289/ (Full text)

Panax ginseng improves physical recovery and energy utilization on chronic fatigue in rats through the PI3K/AKT/mTOR signalling pathway

Abstract:

Context: Panax ginseng C. A. Meyer (Araliaceae) is a tonic herb used in ancient Asia.

Objective: This study investigated the antifatigue effect of P. ginseng on chronic fatigue rats.

Materials and methods: Sprague-Dawley rats were divided into control, model and EEP (ethanol extraction of P. ginseng roots) (50, 100 and 200 mg/kg) groups (n = 8). The rats were subcutaneously handled with loaded swimming once daily for 26 days, except for the control group. The animals were intragastrically treated with EEP from the 15th day. On day 30, serum, liver and muscles were collected, and the PI3K/Akt/mTOR signalling pathway was evaluated.

Results: The swimming times to exhaust of the rats with EEP were significantly longer than that without it. EEP spared the amount of muscle glycogen, hepatic glycogen and blood sugar under the chronic state. In addition, EEP significantly (p < 0.05) decreased serum triglycerides (1.24 ± 0.17, 1.29 ± 0.04 and 1.20 ± 0.21 vs. 1.58 ± 0.13 mmol/L) and total cholesterol (1.64 ± 0.36, 1.70 ± 0.15 and 1.41 ± 0.19 vs. 2.22 ± 0.19 mmol/L) compared to the model group. Regarding the regulation of energy, EEP had a positive impact on promoting ATPase activities and relative protein expression of the PI3K/Akt/mTOR pathway.

Conclusions: Our results suggested that EEP effectively relieved chronic fatigue, providing evidence that P. ginseng could be a potential dietary supplement to accelerate recovery from fatigue.

Source: Zhang G, Lu B, Wang E, Wang W, Li Z, Jiao L, Li H, Wu W. Panax ginseng improves physical recovery and energy utilization on chronic fatigue in rats through the PI3K/AKT/mTOR signalling pathway. Pharm Biol. 2023 Dec;61(1):316-323. doi: 10.1080/13880209.2023.2169719. PMID: 36695132; PMCID: PMC9879180. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9879180/ (Full text)

Low molecular weight cytotoxic components (DAMPs) form the post-COVID-19 syndrome

Abstract:

We studied the role of cytotoxic components (DAMPs) formed in the body of patients with COVID-19 in ensuring the long-term preservation of post-COVID-19 manifestations and the possibility of creating an experimental model by transferring DAMPs to rats. In patients with post-COVID-19 syndrome (PCS) 2 months after SARS-CoV-2 infection we determined the presence of cytotoxic components in the blood serum (Terasaki test, Dunaliella viridis test and content of DAMPs).

In post-COVID-19 syndrome patients with a high content of serum cytotoxic oligopeptide fraction (selective group, n = 16) we determined the number of leukocytes, lymphocytes, neutrophil granulocytes and monocytes in the blood, the content of C-reactive protein (CRP), the concentration of C3 and C4 complement components and circulating immune complexes, the serum content of IL-6, IL -10, IL-18, TNF-α, phagocytic activity of neutrophils, presence of neutrophil traps and autoantibodies ANA.

It has been shown that in patients with PCS, there are components with cytotoxicity in the blood serum, form specific immunopathological patterns, which are characterized by: an increased content of CRP, complement system components C3 and C4 and cytokines (TNF-α, IL-6, IL-10, IL-18) activation, the formation of a wide range of autoantibodies ANA, the low efficiency of endocytosis in oxygen-independent phagocytosis; their phagocytic activity reaches its functional limit, and against this background, activation of neutrophil traps occurs, which can contribute to further induction of DAMPs. This self-sustaining cell-killing activation provided long-term preservation of PCS symptoms.

The transfer of blood serum components from selective group patients with PCS to rats was accompanied by the appearance of cytotoxic components in them which induced sensitization and immunopathological reactions. Preventive administration of a biologically active substance with polyfunctional properties MF to experimental animals “corrected” the initial functional state of the body’s immune-metabolic system and eliminated or facilitated immuno-inflammatory reactions.

Source: Klimova EM, Bozhkov AI, Lavinska OV, Drozdova LA, Kurhuzova NI. Low molecular weight cytotoxic components (DAMPs) form the post-COVID-19 syndrome. Immunobiology. 2023 Jan;228(1):152316. doi: 10.1016/j.imbio.2022.152316. Epub 2022 Dec 20. PMID: 36565610; PMCID: PMC9764760. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9764760/ (Full text)

Effect of herbal cake-separated moxibustion on behavioral stress reactions and blood lactic acid level and muscular AMPK/PGC-1α signaling in rats with chronic fatigue syndrome

Abstract:

in English, Chinese

Objective: To observe the effect of herbal cake-separated moxibustion (HCSM) on serum lactic acid (BLA) level and AMPK/PGC-1α signaling pathway in the quadriceps femoris in chronic fatigue syndrome (CFS) rats, so as to explore its mechanisms underlying improvement of CFS.

Methods: According to the random number table, 50 SD rats were divided into blank control, model, HCSM, sham HCSM and medication (herbal medicine gavage) groups, with 10 rats in each group. The CFS model was established by using chronic restraint and exhaustive swimming, alternately, once daily for 21 days. The herbal cake was made of Xiaoyao Powder (Mental Ease Powder, composed of [Danggui (Radix Angelicae Sinensis), Baishao (Radix Paeoniae Alba), Chaihu (Radix Bupleuri), Fuling (Poria), Baizhu (Rhizoma Atractylodis, Macrocephalae), etc.]. The HCSM was applied to “Shenque” (CV8), “Guanyuan “(CV4), bilateral “Zusanli” (ST36) and “Qimen” (LR14), 5 moxa-cones for each acupoint, once daily for 10 days. For sham HCSM, the excipient was instead of herbal cake, and the same 5 moxa-cones was given as the HCSM group. Rats of the medication group received gavage of Xiaoyao Powder suspension (60 mg·kg-1), once daily for 10 days. The open field test and tail suspension test were conducted for determining the animals’ locomotor activity. The blood sample was taken from the abdominal aorta under anesthesia for assaying the levels of serum BLA, chemokine ligand CXCL9 and β-endorphin (EP) by ELISA. Bilateral quadriceps femoris were sampled for observing histopathological changes after staining with conventional H.E. technique, and for detecting the expression levels of phosphorylated AMP-activated protein kinase (p-AMPK) and peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) by using immunohistochemistry.

Results: Compared with the blank control group, the number of rearing and horizontal grid-crossing times, struggling times of tail suspension test were significantly decreased (P<0.05), and the immobility time was obviously prolonged (P<0.05) in the model group. Compared with the model group, both HCSM and medication groups had a significant increase of rearing, horizontal grid-crossing times and struggling times (P<0.05), and the immobility time had a significant decrease (P<0.05). But there were no significant differences in the total movement distance among the 5 groups (P>0.05), and in the 5 indexes of behavioral measurements between the HCSM and medication groups (P>0.05). The sham HCSM could also evidently increase the struggling times and reduce the immobility time (P<0.05). The contents of serum BLA, CXCL9 and β-EP were obviously higher in the model group than in the blank control group (P<0.05), as well as remarkably lower in the HCSM and medication groups than in the model group (P<0.05). Whereas the expression levels of muscular p-AMPK and PGC-1α were considerably lower in the model group than in the blank control group (P<0.05), and significantly increased in both HCSM and medication groups relevant to the model group (P<0.05). Compared with the sham HCSM group, the contents of BLA, CXCL9 and β-EP in serum of the HCSM group and contents of CXCL9, β-EP in medication group were significantly decreased (P<0.05), and the protein expressions of p-AMPK and PGC-1α in quadriceps femoris in both HCSM and medication groups were significantly increased (P<0.05). H.E. staining showed smaller intercellular space, uneven cytoplasmic staining in some muscle fibers, nucleus pyknosis and condensation, and inflammatory cell infiltration in the model group, which was milder in both HCSM and medication groups.

Conclusion: HCSM can mitigate the stress behavioral state in CFS rats, which may be related with its functions in lowering the levels of serum BLA, CXCL9 and β-EP, and activating AMPK/PGC-1α signaling pathway (balancing energy metabolism) in the quadriceps femoris.

Source: Xu XS, Ma W, Xiong LJ, Zhai CT, Li W, Tian YF. [Effect of herbal cake-separated moxibustion on behavioral stress reactions and blood lactic acid level and muscular AMPK/PGC-1α signaling in rats with chronic fatigue syndrome]. Zhen Ci Yan Jiu. 2022 Oct 25;47(10):878-84. Chinese. doi: 10.13702/j.1000-0607.20220017. PMID: 36301164. https://pubmed.ncbi.nlm.nih.gov/36301164/

Metabolomics study of the effect of Danggui Buxue Tang on rats with chronic fatigue syndrome

Abstract:

Danggui Buxue Tang (DBT), a traditional Chinese medicine formula for “invigorating qi and enriching blood”, has been reported to produce a good effect on chronic fatigue syndrome (CFS). However, the related mechanism remains largely unresolved. In this study, a metabolomics approach with gas chromatography coupled to mass spectrometry combined with pattern recognition was devised to estimate the extent to which DBT alleviated CFS induced by food restriction and force swimming in rats.

After four weeks of treatment, the endurance capability of rats was significantly better and the motionless time was significantly shorter in the DBT group than in CFS model group. Moreover, the activities of SOD and GSH-Px were increased, while the levels of MDA, IL-6 and TNF-α were decreased in the DBT treatment group. Fifteen significantly changed metabolites were observed in the serum of rats with CFS, which was reversed markedly by DBT treatment. Metabolic pathway analysis showed that DBT could possibly alleviate CFS in rats by regulating phenylalanine, tyrosine and tryptophan biosynthesis, glycine, serine and the metabolism of threonine, glycerolipid, glyoxylate, dicarboxylate and tyrosine. It was observed that the metabolism of glycine, serine and threonine was most closely related to the improvement of CFS by DBT treatment. This study showed that DBT could improve CFS effectively and metabolomics was a powerful means to gain insights into the traditional Chinese medicine formulas against CFS.

Source: Miao X, Li S, Xiao B, Yang J, Huang R. Metabolomics study of the effect of Danggui Buxue Tang on rats with chronic fatigue syndrome. Biomed Chromatogr. 2022 Apr 4:e5379. doi: 10.1002/bmc.5379. Epub ahead of print. PMID: 35373377.  https://pubmed.ncbi.nlm.nih.gov/35373377/

Changes in TCA cycle and TCA cycle-related metabolites in plasma upon citric acid administration in rats

Abstract:

Recent studies have reported that plasma levels of tricarboxylic acid (TCA) cycle metabolites and TCA cycle-related metabolite change in patients with chronic fatigue syndrome (CFS) and in healthy humans after exercise. Exogenous dietary citric acid has been reported to alleviate fatigue during daily activities and after exercise. However, it is unknown whether dietary citric acid affects the plasma levels of these metabolites. Therefore, the present study aimed to investigate the effects of exogenously administered citric acid on TCA cycle metabolites and TCA cycle-related metabolites in plasma.

Sprague-Dawley rats were divided into control and citric acid groups. We evaluated the effect of exogenous dietary citric acid on the plasma TCA cycle and TCA cycle-related metabolites by metabolome analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS). TCA cycle metabolites, including plasma citrate, cis-aconitate, and isocitrate, were significantly elevated after exogenous administration of citric acid. Anaplerotic amino acids, which are converted to TCA cycle metabolites, such as serine, glycine, tryptophan, lysine, leucine, histidine, glutamine, arginine, isoleucine, methionine, valine, and phenylalanine, also showed significantly elevated levels.

Citric acid administration significantly increased the levels of initial TCA cycle metabolites in the plasma. This increase after administration of citric acid was shown to be opposite to the metabolic changes observed in patients with CFS. These results contribute novel insight into the fatigue alleviation mechanism of citric acid.

Source: Hara Y, Kume S, Kataoka Y, Watanabe N. Changes in TCA cycle and TCA cycle-related metabolites in plasma upon citric acid administration in rats. Heliyon. 2021 Dec 4;7(12):e08501. doi: 10.1016/j.heliyon.2021.e08501. PMID: 34934832; PMCID: PMC8654791. https://pubmed.ncbi.nlm.nih.gov/34934832/

Electroacupuncture improves cognitive function by inhibiting NF-κB activity in rats with chronic fatigue syndrome

Abstract:

in English, Chinese

Objective: To observe the effect of electroacupuncture (EA) on the expression of NF-κB p65 in hippocampus and the morphology of hippocampus in rats with chronic fatigue syndrome (CFS), so as to explore its mechanism in improving cognitive dysfunction of CFS.

Methods: Forty-eight SD rats were randomly divided into control, model, EA and inhibitor groups (n=12 in each group). The CFS model was established by multi-factor compound stress stimulation method. Rats of the EA group received EA (50 Hz, 1 mA) at “Baihui” (GV20), Emotional Area I and bilateral Sensory Area for 30 min, once daily for 15 days. For rats in the inhibitor group, pyrrolidine dithiocarbamate (100 mg·kg-1·d-1) was injected intraperitoneally, once a day for 15 days. Learning and memory ability was evaluated by Morris water maze test. HE staining was used to observe the morphology of hippocampus. Western blot was used to determine the expression level of NF-κB p65 in hippocampus.

Results: After mode-ling, the general status score was increased （P<0.01）, the escape latency was prolonged(P<0.01), the times of crossing the platform was decreased(P<0.01), and the expression level of NF-κB p65 in hippocampus tissue was significantly increased (P<0.05) in the model group compared with the control group. Compared with the model group, the general status score was decreased （P<0.01）, the escape latency was shortened(P<0.01), the times of crossing the platform was increased(P<0.01), and the expression level of NF-κB p65 in hippocampus tissue was significantly decreased (P<0.05) in the EA and inhibitor groups. HE staining showed that in the model group, the hippocampal nerve cells were arranged disorderly, the structure was loose, and the number of apoptotic bodies and inflammatory cells was significantly increased. The degree of tissue damage of the EA and inhibitor groups was milder than that of the model group.

Conclusion: EA can improve the cognitive function in CFS rats, which may be associated with its effect in inhibiting the expression of NF-κB and reducing the inflammation response in hippocampus.

Source: Feng CW, Qu YY, Sun ZR, Wang YL, Zhang P, Wang QY, Lin WJ, Zhang L, Yang TS. [Electroacupuncture improves cognitive function by inhibiting NF-κB activity in rats with chronic fatigue syndrome]. Zhen Ci Yan Jiu. 2021 Sep 25;46(9):775-81. Chinese. doi: 10.13702/j.1000-0607.200827. PMID: 34558244. https://pubmed.ncbi.nlm.nih.gov/34558244/ [Article in Chinese]

Acupuncture of the Beishu acupoint participates in regulatory effects of ginsenoside Rg1 on T cell subsets of rats with chronic fatigue syndrome

Abstract:

Background: There are close relationships between the spleen and limb muscles and thoughts. The study aims to test the effects of ginsenoside Rg1 in combination with acupuncture of the Beishu acupoint on T cell subsets of rats with chronic fatigue syndrome (CFS).

Methods: The model was set up by combining forced cold-water swimming with chronic restraint. The rats were randomly divided into blank control, model, ginsenoside, acupuncture, and ginsenoside plus acupuncture groups (n=10). For the acupuncture group, the Beishu acupoint was acupunctured on the 2nd day after modeling. For the ginsenoside group, the ginsenoside Rg1 solution was injected into the tail vein on the 2nd day after modeling. For the combination group, both processes were conducted. These groups were compared regarding exhausted swimming time, number of struggles, resting time, serum levels of IgA, IgG, IgM, IFN-α, IFN-β, and IFN-γ, lymphocyte transformation rate, T cell subsets, and skeletal muscle activities of malondialdehyde (MDA), total antioxidative capacity (T-AOC) and acetylcholinesterase (Ache).

Results: The exhausted swimming time, number of struggles, and resting time of combination group surpassed those in the ginsenoside and acupuncture groups significantly (P<0.05). The serum levels of IgA, IgG, IgM, IFN-β, IFN-γ, T-AOC, and Ache, together with CD3+ and CD8+ T cell percentages of combination groups, were significantly higher than those of ginsenoside and acupuncture groups. However, the IFN-α level, MDA activity, and CD4+ T cell percentage were significantly lower (P<0.05). Compared with the model group, the CD4+/CD8+ T cell ratios of acupuncture, ginsenoside, and combination groups decreased significantly (P<0.05). Compared with the combination group, the ratio of the ginsenoside group increased significantly (P<0.05).

Conclusions: Both acupuncture of the Beishu acupoint and intravenous injection of ginsenoside Rg1 have anti-fatigue effects, and their combination works synergistically. This study supplies an experimental basis for joint therapy using acupuncture and drugs to combat fatigue synergistically.

Source: He J, Yu Q, Wu C, Sun Z, Wu X, Liu R, Zhang H. Acupuncture of the Beishu acupoint participates in regulatory effects of ginsenoside Rg1 on T cell subsets of rats with chronic fatigue syndrome. Ann Palliat Med. 2020 Sep;9(5):3436-3446. doi: 10.21037/apm-20-1714. PMID: 33065794. http://apm.amegroups.com/article/view/52609/html (Full text)

Therapeutic Effect and Metabolic Mechanism of A Selenium-Polysaccharide from Ziyang Green Tea on Chronic Fatigue Syndrome

Abstract:

Ziyang green tea was considered a medicine food homology plant to improve chronic fatigue Ssyndrome (CFS) in China. The aim of this research was to study the therapeutic effect of selenium-polysaccharides (Se-TP) from Ziyang green tea on CFS and explore its metabolic mechanism.

A CFS-rats model was established in the present research and Se-TP was administrated to evaluate the therapeutic effect on CFS. Some serum metabolites including blood urea nitrogen (BUN), blood lactate acid (BLA), corticosterone (CORT), and aldosterone (ALD) were checked. Urine metabolites were analyzed via gas chromatography-mass spectrometry (GC-MS). Multivariate statistical analysis was also used to check the data. The results selected biomarkers that were entered into the MetPA database to analyze their corresponding metabolic pathways.

The results demonstrated that Se-TP markedly improved the level of BUN and CORT in CFS rats. A total of eight differential metabolites were detected in GC-MS analysis, which were benzoic acid, itaconic acid, glutaric acid, 4-acetamidobutyric acid, creatine, 2-hydroxy-3-isopropylbutanedioic acid, l-dopa, and 21-hydroxypregnenolone. These differential metabolites were entered into the MetPA database to search for the corresponding metabolic pathways and three related metabolic pathways were screened out. The first pathway was steroid hormone biosynthesis. The second was tyrosine metabolism, and the third was arginine-proline metabolism. The 21-hydroxypregnenolone level of rats in the CFS group markedly increased after the Se-TP administration.

In conclusion, Se-TP treatments on CFS rats improved their condition. Its metabolic mechanism was closely related to that which regulates the steroid hormone biosynthesis.

Source: Shao C, Song J, Zhao S, Jiang H, Wang B, Chi A. Therapeutic Effect and Metabolic Mechanism of A Selenium-Polysaccharide from Ziyang Green Tea on Chronic Fatigue Syndrome. Polymers (Basel). 2018 Nov 15;10(11). pii: E1269. doi: 10.3390/polym10111269. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6401680/ (Full article)