Periplaneta americana (Insecta: Blattodea) and organ fibrosis: A mini review

Fibrosis is the end stage of many chronic inflammatory diseases and eventually leads to organ failure. Periplaneta americana (P. americana) is referred to as “the product of flesh and blood” in traditional Chinese medicine and has a wide range of therapeutic effects. Owing to the growing interest in this insect for its application in the treatment of tissue injury-healing disorders that induce organ fibrosis, it has attracted the interest of researchers. A literature search was performed using core collections of electronic databases, such as PubMed, Web of Science, China National Knowledge Infrastructure, and Wanfang, using the keywords given below and terms such as pharmacological and biochemical details of this insect. P. americana extracts presented a wide range of therapeutic and biological activities, including antifibrotic, antiinflammatory, antioxidative, and tissue repair activities. Emerging evidence suggests that P. americana extracts may improve scarring, pulmonary fibrosis, liver fibrosis, and kidney fibrosis through the regulation of fibroblast activation, cytokine secretion, and deposition of fibrin, indicating the potential role of P. americana as a therapeutic option for organ fibrosis. P. americana is a potential therapeutic agent for treating fibrosis. Further studies are required for a more in-depth characterization of the antifibrogenic mechanism of P. americana prior to its clinical application in the treatment of organ fibrosis. (Fig. 1).


Introduction
Fibrosis refers to the hardening of organs caused by the excessive deposition of extracellular matrix (ECM) proteins during various chronic inflammatory diseases, resulting in organ failure. The mortality rate from fibrotic diseases has shown an increasing trend over the years, accounting for approximately 45% of the total mortality in developed countries; however, there are no approved antifibrotic therapies. [1,2] Research on developing therapeutic strategies against fibrosis is a key imperative.
Periplaneta americana (P. americana) (Insecta: Blattodea) L. is a valuable Chinese medicine material, although it is a notorious pest widely distributed in human habitats. [3] In traditional Chinese medicine (TCM) has been used in the treatment of severe qi-and blood stagnation-induced diseases and various traumas for thousands of years, including tissue sclerosis, abdominal distension, gastric ulcer, malnutrition, and snakebite. [4] Studies have shown that P. americana contains a wide range of active ingredients, including amino acids, polysaccharides, nucleosides, and peptides, of which amino acids account for the highest proportion, with a total of 43.17% of free amino acids and 35.37% of essential amino acids after acid hydration. [5][6][7][8][9] Studies have demonstrated a diverse range of therapeutic effects of P. americana, such as antifibrotic, [10,11] anti-inflammatory, [12] antioxidation, [13] antitumor, [5,14,15] immunoregulatory effects, [16] and XZ and MY contributed equally to this work.

This work was supported by Sichuan Provincial Department of Science and Technology Planning Projects (2019YFS0163 and 2020YFS0376).
The authors have no conflicts of interest to disclose.
promotion of tissue repair. [17] In recent years, a variety of TCM preparations containing extracts of P. americana as the main component have been used in clinical practice, including Kangfu-xin liquid (KFX), Xin-mai-long injection, Gan-long capsule, and Xiao-zheng-yi-gan tablets (Table 1).
Recently, the antifibrotic activity of P. americana has gradually attracted the attention of researchers and has shown good application prospects in the treatment of organ fibrosis. Continuous exposure to an inflammatory microenvironment contributes to an imbalance in tissue injury healing mechanisms that induce fibrotic products. Numerous studies have demonstrated the antioxidant and anti-inflammatory effects of P. americana in the setting of organ inflammation, such as in the skin, [18] lung, [19] liver, [20] and stomach. [21] P. americana is referred to as "the product of flesh and blood" (a natural animal drug with nutritional support) in TCM, as it helps in restoring the balance of injury-healing mechanisms that induce fibrotic products, mainly breaking accumulation, promoting blood circulation, nourishing yin, and strengthening muscles by inducing potential therapeutic responses. [17,22,23] These functions are considered as the primitive application of the antifibrosis activity of this insect. [24,25] Emerging evidence suggests that P. americana can alleviate organ fibrosis, including scarring, in the lungs, liver, and kidney by regulating fibroblast activation, cytokine secretion and ECM deposition function. [20,[26][27][28] Thus, P. americana is a potential candidate for an antifibrotic agent. This review focuses on the antifibrotic effects of P. americana and the underlying mechanisms.

Search strategy
To collect relevant in vitro and ex vivo experiments, we searched electronic databases including 2 English databases [PubMed, Web of Science (SCI)] and 2 Chinese databases [China National Knowledge Infrastructure and Wanfang]. The search keywords used were "Periplaneta americana," "fibrosis," "mechanisms," "biology," and "pharmacology." The initial search data were March 25, 2022, and an updated search was conducted on Table 1 Overview of clinical application of relevant preparations of P. americana.

Name
Character Efficacy Attending disease Adjuvant therapy for primary liver cancer Figure 1. Graphical abstract. An inflammatory microenvironment contributes to an imbalance in the tissue injury healing mechanism, which induces organ fibrosis. P. americana is a potential candidate as an antifibrotic agent that acts through the regulation of fibroblast activation, cytokine secretion, and fibrin deposition.
June 22, 2022. A database search was conducted by a single researcher (XZ). All studies published before the date of the search were considered.

Inclusion criteria
Relevant study selection was based on the following inclusion criteria: studies on organ fibrosis; intervention for study: extract or active ingredient of P. americana; studies performed in vitro or ex vivo, including primary cells and/or cell lines; and animal experiments without restrictions to species. Two researchers (XZ and MY) independently assessed the eligibility of the searched articles, and any disagreements were resolved through discussion under the arbitration of a third reviewer (JJ).

Exclusion criteria
Relevant study selection was based on the following exclusion criteria: duplicate studies and overlapping criteria; title and/or abstract of the study failed to meet the inclusion criteria; and editorials, letters, conference abstracts, comments, and opinions. [29] The selected articles were evaluated for information on the dosage form of P. americana, route of administration, experimental model, potential mechanism, results, and discussion. After applying inclusion and exclusion criteria, 38 articles were selected (Fig. 2).

Pathogenesis of organ fibrosis
The pathophysiology of fibrosis involves the activation of profibrotic fibroblasts, secretion of cytokines, and excessive production and deposition of ECM proteins, such as collagen, elastin, proteoglycan, and glycoprotein. [30] The factors causing fibrosis include wound healing disorders caused by the destruction of the skin ultrastructure (such as cellular mitochondria, endoplasmic reticulum, and desmosomes), oxidative stress response caused by smoke stimulation, persistent infection, repeated exposure to toxins, obesity, genetic factors, and immune diseases. Moreover, the pathological process of fibrosis is complex and involves a cascade of cytokines and transducing signals in response to diverse fibrotic triggers. More specifically, chronic lesions in various organs, such as the skin, lung, liver, and kidney, lead to dyshomeostasis in the injury-healing mechanism that induces fibrotic products. Although there are fibrosis-specific initiators in organs, the molecular mechanisms of fibrosis signaling, such as fibroblast activation, cytokine secretion, integrin receptor activation, upregulation of the transforming growth factor-β (TGF-β) signaling pathway, and ECM deposition, are highly conserved (Fig. 3).
Continuous exposure to various harmful stimuli, including toxins, infectious pathogens, autoimmune reactions, and mechanical stress, induces an inflammatory microenvironment that leads to the infiltration of macrophages and immune cells. [2,31,32] Inflammatory areas have been shown to have prominent infiltrates of lymphocytes, neutrophils, eosinophils, monocytes, and macrophages. [33,34] Lymphocytes, are at the core of the immune response. Stimulation by antigens leads to lymphocyte activation. Neutrophils contain large numbers of lysosomes in the cytoplasm, which activate the phagocytic and digestive functions of cells in the context of tissue damage, acute infection, and malignancy. Eosinophils play an important role in immune response and allergic reactions. These promote inflammatory progression by releasing the granule contents. Macrophages and monocytes are both phagocytes. When monocytes extravasate from blood vessels into connective tissue, they gradually differentiate into macrophages, which are characterized by an increased size, proliferation of endoplasmic reticulum and mitochondria, and increased number of lysosomes with more powerful phagocytic function. The inflammatory microenvironment is an important trigger of regeneration and fibrosis.
The activation of immune cells is presumed to result in the release of early acting cytokines, such as nuclear factor kappa-B (NF-κB), tumor necrosis factor (TNF), and interleukin-6. [35] Subsequent activation and possible phenotypic alteration of structural cells induce the release of other growth factors, including TGF, vascular endothelial growth factor (VEGF), basic fibroblast growth factor, connective tissue growth factor, and platelet-derived growth factor (PDGF). [36] Upon entry into the tissue, these cytokines become further activated and may modulate the differentiation and function of fibroblasts via diverse molecular mechanisms and regulate fibrogenesis. Immune cells are substrates of myeloperoxidase reactions. Peroxidase production is closely related to immune cell infiltration. Reactive oxygen species and proteolytic enzymes not only aggravate the cytotoxic damage response to hypochlorous acid, but also stimulate cytokine production that mediates fibroproliferative responses, including nuclear factor erythroid 2-related factor 2 (Nrf2), NF-κB, TNF-α, and TGF-β. [37,38] TGF-β is a prototypical member of the TGF-β family of growth and differentiation factors. TGF-β is critical for the regulation of the healing process, has an impact on almost every cell type involved, and tips the balance from regulated physiological repair to excessive pathological repair. [39,40] Integrins are transmembrane heterodimeric cell adhesion molecules composed of an α chain and a β chain, in which the α chain is antigen-specific and the β chain is related to signal transduction. Integrins promote cell adhesion and migration but also control the local activation of latent TGF-β contained in the ECM or cell surface reservoirs. Accumulating evidence suggests that the integrin-dependent activation of TGF-β is a key mechanism by which tissue-propagating cells directly circulate and resident immune cells. [41][42][43] Thus, inflammatory factors, reactive oxygen species, and integrins act as activators of TGF-β receptors. Latencyassociated peptide and latent TGF-β-binding protein form large latent complexes. Subsequently, large latent complex cells bind noncovalently to transmembrane protein receptor − integrins on the surface of various cells (including fibroblasts, interstitial cells, and epithelial cells), mediating cell-cell and cell-ECM adhesion and signal transmission. TGF-β ligands bind to TGF-β receptor I (TβR 1) and II (TβR 2) on the cell surface, and activated TβR 1 upregulates Smad2/3 phosphorylation levels and forms a complex with co-mediated Smad4 to induce nuclear translocation via the Smad-dependent classical transduction pathway. [44,45] Alternatively, activated TGF-β can activate the mitogen-activated protein kinase (MAPK) signaling pathways. [46][47][48][49] For example, the activator-binding domain of the Ras protein is catalyzed to convert to Ras-GTP, then binds to C-Raf-1 and phosphorylates MAPK kinase (MEK/MKK). This triggers extracellular regulated protein kinase 1/2 (ERK1/2), further upregulating Smad expression. MKK4 and MKK3/6 catalyze the downstream substrates c-Jun N-terminal kinase and p38, respectively, thereby promoting fibroblast activation and collagen deposition.
Moreover, in response to cytokine activation, fibroblast cells undergo further proliferation, and may begin to release their own autocrine factors and "feed back" some fibrotic signals similar to ECM proteins, such as the overexpression of alphasmooth muscle actin (α-SMA), laminin (LN), fibronectin (FN), and collagen, which triggers the conversion of fibroblasts into a myofibroblast phenotype, leading to further excessive deposition of ECM. [50][51][52][53] Matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) play a pivotal role in both fibrolysis and fibrogenesis. [54] TIMP has been traditionally thought to control ECM proteolysis through direct inhibition of MMP-dependent ECM proteolysis, which suggests that increased TIMP levels result in ECM accumulation, whereas loss of TIMP leads to enhanced matrix proteolysis. [55,56] Therefore, targeting myofibroblast proliferation, regulating cytokine secretion, and reducing ECM deposition are potential strategies to alleviate fibrosis.

Characteristics of P. americana
Traditional Chinese medicine "cockroach" was first published in Shennong's Herbal Classic and has a long history of use. According to TCM, cockroaches are salty in taste, cold in nature, and act on the meridians of the liver, spleen, and kidney. The book also mentioned a wide range of therapeutic effects of cockroaches, such as breaking accumulation, promoting blood circulation, nourishing yin and strengthening muscles, and relieving swelling and detoxification. [4] The administration of cockroaches involves the consumption of a soup that has been boiled with the dry powder of the whole insect or directly as a poultice applied to the surface of a patient's wound, [23] and has been used to alleviate severe qi-and blood stagnation-induced diseases and various traumas, including tissue sclerosis, abdominal distension, gastric ulcer, malnutrition, and snakebite. Even today, people in Southwest China still treat wounds with mashed cockroaches to promote wound healing and inhibit scarring. [57] Currently, nutritional interference is considered an option for improving fibrosis, [30] which somewhat coincides with the viewpoint of TCM, because cockroaches, also known as "the product of flesh and blood" (a natural animal drug with nutritional support), can break the accumulation and generate muscle, thereby promoting the quality of tissue repair. [23] P. americana belongs to the phylum Arthropoda, class Insecta, order Cockrobiales, and family Cockrobiaceae. P. americana may act on fibroblast activation, cytokine signaling, and fibrin component deposition. Evidence has shown that P. americana may accelerate acute wounds by increasing the number of blood vessels and regulating the levels of angiogenesis-related cytokines (α-SMA, VEGF, and CD31). [58] Jie and Li et al demonstrated that P. americana extracts regulate fibroblast migration and proliferation. [11,59] Moreover, P. americana alleviates the deposition of ECM in the late stage of tissue healing in a way that promotes tissue repair and avoids fibrosis formation. [20,60] P. americana has great potential for application in full-thickness wound healing. KFX promotes the quality of wound healing by promoting the ultrastructural repair of dermal cells, such as the mitochondria, endoplasmic reticulum, and desmosomes. [61] The colonic mucosa epithelium layer, crypt, muscle layer mucosa, and submucosa were also well repaired after KFX treatment in ulcerative colitis mice. [62] Collectively, these lines of evidence indicate the potential role of P. americana as a therapeutic agent for organ fibrosis.

Antifibrogenesis properties of P. americana
Owing to the strong anti-inflammatory and antioxidative effects of P. americana, some studies have investigated its antifibrogenic effects. P. americana has been shown to exert antifibrotic effects through TGF-β/Smad signaling, Shh (Sonic Hedgehog) signaling, Wnt/β-catenin signaling, NF-κB signaling, and the Nrf2/HO-1 (heme oxygenase-1) cascade, which are related to its anti-inflammatory, antioxidant, apoptosis-inductive, [81,82] and gene regulatory effects. [83] They have been shown to inhibit fibroblast activation, cytokine secretion, and deposition of fibrin components. The effects and major mechanisms of P. americana extract on organ fibrosis are summarized in Table 3.

P. americana and hypertrophic scars
Severe trauma, endocrine disorders, and genetic factors can impair skin repair, leading to hypertrophy. Scar tissue is not a perfect replacement for preinjury tissue because scar tissue exhibits reduced tension and impaired nutrient metabolism. [84] Many studies have confirmed that excessive proliferation of myofibroblasts, disorganization of collagen, and destruction of the ultrastructure of skin cells (such as the mitochondria, endoplasmic reticulum, and desmosomes) are the main pathological features of scars. [61,85,86] These are all associated with the abnormal function of fibroblasts. Thus, improving fibroblast function is a potential mechanism for inhibiting scar formation. P. americana extracts Ento-A, Ento-B, Ento-C, Ento-D, and Ento-E inhibited the proliferation of CCC-ESF-1 (human embryonic skin fibroblasts) in a time-dependent manner. [85] P. americana extract affects wound repair via a variety of mechanisms, including TNF, NF-κB signaling, MAPK, and AMPK signaling. [87] TGF-β1 plays a key role in fibroblast proliferation, differentiation, and migration. Yang et al found that the aqueous extract of P. americana promotes the repair of burn wounds while inhibiting scarring owing to its interaction with TGF-β1 release at various stages of the repair process. [88] Another ethanol extract, PAE, reduced collagen fiber content by downregulating CD 68, VEGF, and basic fibroblast growth factor. [86] Moreover, lncRNAs play an important biological role in wound repair. It may regulate the expression of inflammatory factors, such as TNF-α and IL-6, through lncRNAH19 to promote tissue repair and minimize scar formation. [89] KFX, a single preparation of an ethanol extract of P. americana, has been found to improve the quality of wound healing. In refractory healing wounds, KFX improves the quality of wound healing by promoting the ultrastructure of dermal cells, such as mitochondria, endoplasmic reticulum, and desmosomes. [61] Moreover, Smads are classical downstream transduction molecules of TGF-β1 signaling that regulate inflammatory signaling in dermal cells, [90] and KFX accelerates the wound inflammatory response by downregulating Smad6 and upregulating Smad9 expression. [61] KFX can inhibit ECM deposition in the late stage of wound healing by regulating the expression of collagen production to promote repair and avoid scar formation. [60] Moreover, KFX inhibits the proliferation of fibroblasts in hypertrophic scars by inhibiting Shh signaling, which is an essential signal transduction pathway in long-term Wntactivated cells. [91,92] In further studies, Chen et al found that the KFX-treated group had high expression levels of Ereg, Gli2, and Tgm3, as assessed by RNA sequencing. [93] Collectively, these lines of evidence suggest that P. americana has the potential to ameliorate scarring.

P. americana and pulmonary fibrosis
Pulmonary fibrosis is the end-stage of most chronic lung diseases and is characterized by the initiation of fibroblast recruitment, resulting in excessive ECM deposition. [94] Notably, inflammatory mediators are catalysts that continuously advance the process of pulmonary fibrosis. [95] Therefore, the inhibition of inflammatory mediators and downregulation of specific fibrotic factors may be an effective therapeutic strategy.
In an in vitro experiment, P. americana extract effectively inhibited the activation of MRC-5 while reducing specific fibrosis factors, including α-SMA, LN, FN, connective tissue growth factor, and Col I/III. [96] The researchers then used primary lung fibroblasts and pointed out that P. americana extract may regulate the dynamic balance between MMPs and TIMPs, such as MMP1, MMP3, MMP9, and TIMP-1, which results in ECM degradation and deposition. In addition, it can affect the cycle of primary lung fibroblasts by inhibiting cyclin D1 and promoting cyclin-dependent kinase inhibitors. [97] In in vivo experiments, an extract of P. americana (ML-HB) ameliorated bleomycin-induced pulmonary fibrosis by inhibiting the expression of TNF-α, TGF-β1, and hydroxyproline. [98,99] TNF-α is a proinflammatory cytokine that promotes collagen production in addition to stimulating neutrophil and eosinophil infiltration to release lysosomal enzymes. [100] Hyp is a unique amino acid in collagen, accounting for approximately 13% of the total collagen amino acids, and can be used as an indicator of connective tissue degradation. [101] In addition, some extracts exhibited the ability ameliorated the oxidative stress response in the lungs by regulating oxidative stress markers, for example, by downregulating malondialdehyde and nitric oxide and upregulating glutathione peroxidase. [102] These data illustrate multiple mechanisms by which P. americana may improve pulmonary fibrosis.
potential treatments for hepatic fibrosis is a key research imperative. ML-HB showed a good inhibitory effect on the proliferation of rat hepatic stellate cells (HSC-T6). [103] The sticky sugar glycine, an extract of P. americana, protects against liver function damage in immune liver fibrosis and inhibits fibrosis. [104] In addition, there may be a link between its mechanism and downregulation of TGF-β1/TIMP-1 signaling, as well as inhibition of the release of inflammatory factors, including NF-κB, IL-6, and intercellular cell adhesion molecule-1. Intercellular cell adhesion molecule-1 is a transmembrane glycoprotein that plays key roles in leukocyte migration and activation. [105] In a study by Gao et al, KFX was found to inhibit the expression of liver fibrosis markers, including hyaluronic acid, type III procollagen amino peptide, FN, α-SMA, and Col III/IV, by regulating TGF-β1/Smad and Wnt/β-catenin signaling and, more specifically, by downregulating the expression of β-catenin and upregulating the expression of Axin and glycogen synthase kinase 3β. [106] These results suggest that KFX may regulate the "crosstalk" between multiple signaling pathways during the development of liver fibrosis. Professor Xiao's research team conducted a series of studies on P. americana extract PA-B, which is thought to specifically resist liver fibrosis. PA-B significantly inhibited the proliferation and migration of HSC-T6 cells in vitro by downregulating TGF-β1/Smad signaling. PA-B reduced collagen deposition in the liver interstitium by inhibiting the expression of Col I, Col III, and α-SMA. In addition, PA-B may induce apoptosis in HSC-T6 cells by lowering mitochondrial membrane potential. Alternatively, decreased B-cell lymphoma-2 (Bcl-2) expression, and conversely, increased Bcl-2 associated X protein and caspase-9 expression, also indicate that PA-B activates the apoptosis of HSC-T6 cells. [107] Second, PA-B was found to ameliorate the oxidative stress response in the liver. Nrf-2/HO-1 cascade signaling-related factors are potential targets of PA-B, including Nrf2, NADH quinone oxidoreductase 1, HO-1, and Kelch-like ECH-associated protein 1, [108] which protect against hepatic injury via anti-inflammatory, antioxidant, antiendoplasmic reticulum stress, and antiapoptotic properties. [109] The study also found that PA-B modulated the expression of hyaluronic acid, LN, procollagen III (PC-III), Col-IV, and malondialdehyde while increasing glutathione peroxidase, catalase, and total superoxide dismutase. [108] Finally, in the process of experimental liver fibrosis, PA-B was found to inhibit collagen synthesis and secretion by regulating TGF-β1/Smad and Bel-2/Bcl-2-associated X protein signaling. [110] In addition, they conducted an in vivo experiment to compare the efficacy of PA-B after gastric and intestinal administrations. The results suggest that both administration methods significantly improved experimental liver fibrosis, but there was no significant difference in efficacy. [111] This is the first study to explore the efficacy of different formulations of PA-B. These findings suggested that P. americana alleviates hepatic fibrosis.

P. americana and renal fibrosis
Renal fibrosis is an irreversible condition that results from all chronic kidney diseases. Studies investigating the effect of P. americana on renal fibrosis are in their preliminary stages. However, KFX exhibited a renoprotective effect by alleviating renal fibrosis induced by unilateral ureteral ligation in a dose-dependent manner, and the mechanism was related to the regulation of the TGF-β1/Smad signaling pathway transducers, including TGF-β1, Smad3, and Smad7. [112] Subsequently, Huang et al conducted a preliminary exploration that suggested that upregulation of hepatocyte growth factor and MMP9 and downregulation of TIMP-1, which reduces nephritic cell infiltration and fibrous tissue deposition, may be potential targets of KFX in renal fibrosis. [113] 5.5. P. americana and fibrosis in other organs KFX was found to ameliorate chronic inflammation-induced intestinal fibrosis by regulating TGF-β1/Smad signaling and, more specifically, by reducing TGF-β1, Smad2/3, and phospho-Smad2/3 while promoting Smad7. [114] Abnormal proliferation of fibroblasts is a feature of the pathological scars in gastric ulcers. In a previous study, KFX was shown to optimize fibroblast function by inhibiting growth hormone secretion and Hyp synthesis, thereby improving the pathological scar of gastric ulcers. [115] 6. Conclusions P. americana has a wide range of therapeutic effects in TCM. Advances in modern pharmacological research will help to unravel the potential targets of P. americana. Available evidence suggests that P. americana extracts can alleviate fibrosis in multiple organs by regulating fibroblast activation, cytokine signaling, and fibrin component deposition. Thus, P. americana is a potential therapeutic agent for fibrosis. However, current research on the effect of P. americana in improving organ fibrosis is at an experimental stage. More importantly, there is a paucity of studies on its kinetics, metabolism, and toxicity. Therefore, further studies are required for a more in-depth characterization of the antifibrogenic mechanism of P. americana prior to its clinical application in the treatment of organ fibrosis.

Author contributions
XZ (xinzreserch@126.com) and MY (ym1993928@126.com) wrote the main manuscript and prepared the figures and tables; JJ (jinjings1981@126.com) offered professional advices for the article; ZL (lizhi-swmu@126.com) and JC (jiechen0115@sohu. com) coordinated the writing and made the overall revisions to the article. All authors have read and approved the final manuscript.