The effect of the combination therapy of licorice cyclopamine and total flavonoids of fragrant orchid on pulmonary fibrosis mice
Pulmonary fibrosis (PF) is a chronic progressive lung disease with obvious histopathological patterns, including a large number of chronic respiratory system lesions, accompanied by the growth of connective tissue in various pulmonary compartments. Among them, interstitial lung disease and idiopathic pulmonary fibrosis (IPF) are the most serious and irreversible diseases of pulmonary parenchymal progressive fibrosis. PF is caused by abnormal tissue repair and is associated with sustained severe tissue damage and cellular stress. This abnormal property involves insufficient repair of the epithelial cell barrier, accompanied by impaired regulation of fibroblasts, leading to profound changes in alveolar epithelial cells and fibroblasts, resulting in uncontrolled fibrosis characterized by thickening of fibrotic alveolar walls, impaired gas transmission, limited ventilation patterns, and ultimately respiratory failure. In recent years, PF disease has seriously endangered human health in the field of modern medicine. Due to the continuous progress of diagnostic methods and the aging of the population, the mortality and incidence rate of PF continue to rise. The pathogenesis of this disease is complex, and the pathological mechanism is still unclear. The clinical manifestations of patients are progressive decline in lung tissue compliance, decreased diffusion function, and imbalance of ventilation/blood flow, leading to patients with refractory dyspnea, and ultimately respiratory failure and death.
The Hedgehog (Hh) pathway plays an important role in embryonic development and tissue remodeling. The Hh signaling pathway is mainly composed of signaling molecules (SHH, IHH, DHH), transmembrane receptors (Ptch, SMO), and downstream transcription factors (Gli1 protein family). After activation, the Hh pathway mainly exerts its effects through the Gli1 protein family. It has been confirmed in IPF that the activity of the Hh pathway increases, and significant expression of SHH and its signaling effectors has been detected in the alveolar epithelium and submucosal fibroblasts in the area undergoing fibrosis remodeling. In the lungs, SHH treatment increased fibroblast proliferation, survival, migration, and extracellular matrix (ECM) production, but did not increase the expression of α – smooth muscle actin (α – SMA). In the PF model induced by bleomycin, SHH expression is elevated in airway and alveolar epithelial cells. In the bleomycin model, inhibiting Hh signaling does not prevent fibrosis, but overexpression of SHH during fibrosis exacerbates PF. Therefore, although SHH signaling is crucial for normal lung development and maintaining interstitial homeostasis, it is also chronically activated in PF.
Cyclopamine (Cyc) is a steroid alkaloid and an inhibitor of the Hh signaling pathway. By changing the spatial conformation of SMO, the activity of SMO is inhibited to suppress downstream signal transduction of the Hh signaling pathway, thereby putting the Hh pathway in an inhibited state. Cyc can inhibit the transcription and expression of tumor genes, intervene in the SHH mediated Hh signaling pathway, exert anti-tumor and anti PF effects, and thereby inhibit the progression of related diseases. Related studies have shown that by binding to SMO proteins in the Hh signaling pathway and affecting the downstream signal transduction of SMO proteins, the expression of α – SMA and collagen type 1 (Col-1) proteins can be reduced, thereby weakening or even preventing this change in high-sensitivity C-reactive protein and improving portal hypertension. In addition, drugs such as Cyc that interfere with Hh signaling may help prevent androgen resistance and disease progression in prostate cancer cells. The Hh signaling pathway affects the occurrence and development of diseases, however, the role of Cyc in PF related diseases is still unclear.
Dracocephalum moldevica L. is a plant of Labiatae, which is used as medicine in the whole grassland. Its main chemical components are volatile oil, polysaccharide, terpenoids, flavonoids, amino acids, and trace elements. It has a variety of pharmacological effects such as protecting cardiovascular and cerebrovascular vessels, anti atherosclerosis, anti asthma, clearing heat and detoxification, promoting blood circulation and unblocking collaterals, antioxidant, regulating dyslipidemia, as well as wind and heat clearing, cold and fever, cough and asthma. Research has found that Xiangqinglan contains a total of 41 flavonoids, and the medicinal properties of Xiangqinglan mainly come from flavonoids. Previous studies have shown that TFDM, as one of the main active ingredients of Xiangqinglan, has a very effective effect on relieving cough and asthma. It can significantly slow down the high reactivity inside the airways of mice, thereby repairing lung function damage. The release of interleukin-6 (IL-6), interleukin-17 (IL-17), and tumor necrosis factor – α (TNF – α) in mice significantly reduced the ratio of interleukin-4 (IL-4) to interferon – γ (IFN – γ), thereby adjusting the Th1/Th2 immune imbalance. Among them, TFDM can control the expression of NOD like receptor thermal protein domain associated protein 3 (NL-RP3) by reducing the content of Col-1 in lung tissue, ultimately achieving the goal of alleviating pulmonary fibrosis. And previous studies have shown that total flavonoids from Dracocephalum moldavica L. (TFDM) can effectively alleviate PF caused by bleomycin by downregulating or upregulating important regulatory proteins such as SHH, Ptch1, SMO, SUFU, Gli1 in the Hh signaling pathway. Therefore, this article studies the mechanism of action of Cyc and TFDM on PF based on the Hh signaling pathway.

A large amount of research was conducted on Xiangqinglan in the early stage, and the optimal extraction process was determined. The Xiangqinglan extract was purified using macroporous adsorption resin to obtain the effective parts of Xiangqinglan. The effective parts of Xiangqinglan were separated and structurally analyzed for identification. Prior to this, the content of effective parts of Xiangqinglan was determined, and based on the clinical dosage, it was converted to the animal’s in vivo dosage according to relevant literature. And in previous studies, the mechanism of action of TFDM (90, 180, 360mg/kg) related doses in PF mouse models was explored based on the Hh signaling pathway. It was found that the Hh signaling pathway was activated in the PF mouse model, and TFDM could improve PF by intervening in important regulatory proteins of the Hh signaling pathway, with a more significant effect observed at high doses of TFDM (360mg/kg).
Therefore, based on previous research, this article focuses on exploring the inhibition of the Hh signaling pathway by Cyc and the related effects of TFDM (360mg/kg) in bleomycin induced PF mice. A PF mouse model was established by intratracheal instillation of bleomycin. On the second day of model construction, mice were orally administered with related drugs such as Cyc and TFDM. Due to the insolubility of Cyc and TFDM drugs, 0.5% carboxymethyl cellulose sodium was prepared as the corresponding suspension and administered by gavage.
In the early stage of constructing the PF model, the diseased mice showed symptoms such as weight loss, loss of appetite, messy fur color, and poor mental condition, and a large number of deaths. At the same time, based on the results of HE and Masson stained lung tissue pathological sections, it was concluded that the construction of the PF model was successful. After continuous drug intervention, various symptoms of mice were significantly improved in the later stage of PF model construction. The results of lung tissue pathological sections showed that the lung tissue structure of Cyc group and TFDM group was normal, and inflammatory cells and fibrotic tissue were significantly reduced. Subsequently, the content of HYP was measured, and the research results showed that the HYP content in the model group increased, while the Cyc group and TFDM group could significantly reduce the content of HYP. The clinical manifestations of PF are persistent dry cough and acute respiratory distress, accompanied by worsening lung injury, resulting in a continuous decline in lung function and ultimately leading to death. PF can cause loss of lung function, characterized by progressive respiratory distress, leading to gas exchange disorders and restrictive ventilation problems. Therefore, this study conducted pulmonary function tests and found that the Cyc group and TFDM group can improve PF airflow limitation and alleviate restrictive ventilation dysfunction by reducing RL, RE, and FEV0.1/FVC, while increasing Cdyn, FVC, and PEF, effectively alleviating symptoms of bleomycin induced PF.
The Hh pathway plays an important role in the PF process, such as ECM secretion and metabolism, to control PF. The Hh signaling pathway is initiated by the secretion of ligands, and Ptch eliminates the inhibitory effect on SMO, thereby promoting the activation of transcription factor Gli1 and nuclear translocation, leading to the expression of Hh target genes such as Gli1 and SMO. Therefore, in the current study, the SMO inhibitor Cyc was used to inhibit the Hh pathway to determine the role of TFDM in PF mice. This study detected related proteins such as Col-1, α – SMA, FN1, SHH, SMO, SUFU, Gli1, etc. Cyc and TFDM inhibition of the Hh pathway can inhibit ECM production and angiogenesis, which may be achieved by downregulating related proteins such as Col-1, α – SMA, FN1, SHH, SMO, Gli1 and upregulating SUFU protein. This further confirms that PF is related to the inhibition of the Hh signaling pathway. Both the Cyc group and TFDM group were able to repair lung tissue damage, improve collagen fiber content, downregulate or upregulate Hh signaling pathway related protein content, thereby alleviating the progression of lung tissue fibrosis. Moreover, the combination of Cyc and TFDM showed better efficacy.
In summary, this article further elaborates on the occurrence and development of PF, and verifies that TFDM has a certain therapeutic effect. It also delves into the mechanism of action of the Hh signaling pathway related to PF, providing a good research foundation for the study of PF’s related mechanisms of action.